Structurally conserved water molecules in ribonuclease T1.

PubMed

Malin, R; Zielenkiewicz, P; Saenger, W

1991-03-15

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

NASA Astrophysics Data System (ADS)

Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

2015-10-01

The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

Genetic code expansion and live cell imaging reveal that Thr308 phosphorylation is irreplaceable and sufficient for Akt1 activity.

PubMed

Balasuriya, Nileeka; Kunkel, Maya T; Liu, Xuguang; Biggar, Kyle K; Li, Shawn S-C; Newton, Alexandra C; O'Donoghue, Patrick

2018-05-17

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr308, Ser473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 in order to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to un-phosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr308. Live imaging of COS7 cells confirmed that phosphorylation of Thr308, but not Ser473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr308 function cannot be mimicked with acidic residues nor could unphosphorylated Thr308 be mimicked by an Ala mutation. An Akt1 variant with pSer308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr308 and Cys310 in the Akt1 active site. Thus, pThr308 is necessary and sufficient to stimulate Akt signaling in cells and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr308 should be regarded as the primary diagnostic marker of Akt activity. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Mutational analysis of the active site flap (20s loop) of mandelate racemase.

PubMed

Bourque, Jennifer R; Bearne, Stephen L

2008-01-15

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

PubMed Central

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2.

PubMed

Gárriz, Andrés; Qiu, Hongfang; Dey, Madhusudan; Seo, Eun-Joo; Dever, Thomas E; Hinnebusch, Alan G

2009-03-01

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

Structural insights into translational recoding by frameshift suppressor tRNA SufJ

DOE PAGES

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; ...

2014-10-28

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA SufJ contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL SufJ ormore » tRNA SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL SufJ and ASL Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.« less

Intestinal fatty acid binding protein Ala54Thr polymorphism is associated with peripheral atherosclerosis combined with type 2 diabetes mellitus.

PubMed

Khattab, Salma A; Abo-Elmatty, Dina M; Ghattas, Maivel H; Mesbah, Noha M; Mehanna, Eman T

2017-09-01

Intestinal fatty acid-binding protein 2 (FABP2) is expressed in enterocytes and binds saturated and unsaturated long-chain fatty acids. The FABP2 Ala54Thr polymorphism has been reported to effect lipid metabolism. The aim of the present study was to assess the relationship between this polymorphism and peripheral atherosclerosis combined with type 2 diabetes mellitus (T2DM) in an Egyptian population. The study was performed on 100 T2DM patients with peripheral atherosclerosis and 100 control subjects. The Ala54Thr polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism, whereas serum FABP2 levels were determined using ELISA. Fasting blood glucose, fasting serum insulin concentrations, HbA1c, lipid profile, body mass index (BMI) and systolic and diastolic blood pressure (SBP and DBP, respectively) were determined. There was a higher frequency of the Thr54 allele among the patient group (P = 0.002). In Ala54/Thr54 heterozygotes and carriers of the rare Thr54/Thr54 genotype, there were significant increases in BMI and FABP2. Those with the Thr54/Thr54 genotype had significantly decreased high-density lipoprotein cholesterol (HDL-C) concentrations; in addition, those with the Thr54/Thr54 genotype had significantly higher SBP and DBP than subjects with the Ala54/Ala54 and Ala54/Thr54 genotypes. There was a positive correlation between FABP2 levels and BMI, SBP and DBP, and a negative correlation with HDL-C. The Thr54 allele of the FABP2 Ala54Thr polymorphism was associated with an increased incidence of peripheral atherosclerosis combined with T2DM in the population studied. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

A Network of Hydrophobic Residues Impeding Helix αC Rotation Maintains Latency of Kinase Gcn2, Which Phosphorylates the α Subunit of Translation Initiation Factor 2▿

PubMed Central

Gárriz, Andrés; Qiu, Hongfang; Dey, Madhusudan; Seo, Eun-Joo; Dever, Thomas E.; Hinnebusch, Alan G.

2009-01-01

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the α subunit of translation initiation factor 2 (eIF2α). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix αC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2α phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of αC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of αC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation. PMID:19114556

Sequence composition and environment effects on residue fluctuations in protein structures

NASA Astrophysics Data System (ADS)

Ruvinsky, Anatoly M.; Vakser, Ilya A.

2010-10-01

Structure fluctuations in proteins affect a broad range of cell phenomena, including stability of proteins and their fragments, allosteric transitions, and energy transfer. This study presents a statistical-thermodynamic analysis of relationship between the sequence composition and the distribution of residue fluctuations in protein-protein complexes. A one-node-per-residue elastic network model accounting for the nonhomogeneous protein mass distribution and the interatomic interactions through the renormalized inter-residue potential is developed. Two factors, a protein mass distribution and a residue environment, were found to determine the scale of residue fluctuations. Surface residues undergo larger fluctuations than core residues in agreement with experimental observations. Ranking residues over the normalized scale of fluctuations yields a distinct classification of amino acids into three groups: (i) highly fluctuating-Gly, Ala, Ser, Pro, and Asp, (ii) moderately fluctuating-Thr, Asn, Gln, Lys, Glu, Arg, Val, and Cys, and (iii) weakly fluctuating-Ile, Leu, Met, Phe, Tyr, Trp, and His. The structural instability in proteins possibly relates to the high content of the highly fluctuating residues and a deficiency of the weakly fluctuating residues in irregular secondary structure elements (loops), chameleon sequences, and disordered proteins. Strong correlation between residue fluctuations and the sequence composition of protein loops supports this hypothesis. Comparing fluctuations of binding site residues (interface residues) with other surface residues shows that, on average, the interface is more rigid than the rest of the protein surface and Gly, Ala, Ser, Cys, Leu, and Trp have a propensity to form more stable docking patches on the interface. The findings have broad implications for understanding mechanisms of protein association and stability of protein structures.

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

PubMed Central

Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

2015-01-01

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

Proline Restricts Loop I Conformation of the High Affinity WW Domain from Human Nedd4-1 to a Ligand Binding-Competent Type I β-Turn.

PubMed

Schulte, Marianne; Panwalkar, Vineet; Freischem, Stefan; Willbold, Dieter; Dingley, Andrew J

2018-04-19

Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I β-turn interact with the PPxY motif of the human epithelial Na + channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the ( i + 1) position found in β-turns. In this report, molecular dynamics simulations and experimental data were combined to characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Ω loop rather than the type I β-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I β-turn. Thus, proline in loop I ensures a stable peptide binding-competent β-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.

A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

PubMed

Kuryavyi, V; Majumdar, A; Shallop, A; Chernichenko, N; Skripkin, E; Jones, R; Patel, D J

2001-06-29

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher

The Thr505 and Ser557 residues of the AGT1-encoded alpha-glucoside transporter are critical for maltotriose transport in Saccharomyces cerevisiae.

PubMed

Smit, A; Moses, S G; Pretorius, I S; Cordero Otero, R R

2008-04-01

The main objective of this study was to identify amino acid residues in the AGT1-encoded alpha-glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae. The sequences of two AGT1-encoded alpha-glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1p(WH310) and Agt1p(WH314)) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1-encoded alpha-glucoside transporters. A 23-amino-acid C-terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport. The amino acids Thr(505) and Ser(557), which are respectively located in the transmembrane (TM) segment TM(11) and on the intracellular segment after TM(12) of the AGT1-encoded alpha-glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae. Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch-efficient fermentation systems, and offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whisky industries.

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

PubMed Central

Huang, Xuhui; Wang, Dong; Weiss, Dahlia R.; Bushnell, David A.; Kornberg, Roger D.; Levitt, Michael

2010-01-01

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the β-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the “closed” conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity. PMID:20798057

The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment.

PubMed

Pizzitutti, Francesco; Giansanti, Andrea; Ballario, Paola; Ornaghi, Prisca; Torreri, Paola; Ciccotti, Giovanni; Filetici, Patrizia

2006-01-01

Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain. Copyright 2005 John Wiley & Sons, Ltd.

Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases.

PubMed

Borgford, T J; Gray, T E; Brand, N J; Fersht, A R

1987-11-17

Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.

Two M-T hook residues greatly improve the antiviral activity and resistance profile of the HIV-1 fusion inhibitor SC29EK

PubMed Central

2014-01-01

Background Peptides derived from the C-terminal heptad repeat (CHR) of HIV-1 gp41 such as T20 (Enfuvirtide) and C34 are potent viral fusion inhibitors. We have recently found that two N-terminal residues (Met115 and Thr116) of CHR peptides form a unique M-T hook structure that can greatly enhance the binding and anti-HIV activity of inhibitors. Here, we applied two M-T hook residues to optimize SC29EK, an electrostatically constrained peptide inhibitor with a potent anti-HIV activity. Results The resulting peptide MT-SC29EK showed a dramatically increased binding affinity and could block the six-helical bundle (6-HB) formation more efficiently. As expected, MT-SC29EK potently inhibited HIV-1 entry and infection, especially against those T20- and SC29EK-resistant HIV-1 variants. More importantly, MT-SC29EK and its short form (MT-SC22EK) suffered from the difficulty to induce HIV-1 resistance during the in vitro selection, suggesting their high genetic barriers to the development of resistance. Conclusions Our studies have verified the M-T hook structure as a vital strategy to design novel HIV-1 fusion inhibitors and offered an ideal candidate for clinical development. PMID:24884671

Mitochondrial tRNA(Thr) A15951G mutation may not be associated with Leber's Hereditary Optic Neuropathy.

PubMed

Zhang, Xi; Yu, Shuaishuai; Tu, Yunhai; Huang, Wenjie

2016-07-01

Mutation in mitochondrial DNA (mtDNA) has been found to play an important role in the pathogenesis of Leber's Hereditary Optic Neuropathy (LHON). Three primary mutations, the ND4 G11778A, ND6 T14484C, and ND1 G3460A, have been found to account more than 90% of LHON patients in many families worldwide. In addition to the mutations in genes encoding the respiratory chain complex I, reports concerning the mt-tRNA gene mutations associated with LHON have increased, some pathogenic mutations caused the failure in mt-tRNA metabolism, thereby worsened the mitochondrial dysfunction that is responsible for LHON. Recently, the A15951G mutation in mt-tRNA(Thr) gene has been reported to be a "modified" factor in increasing the penetrance and expressivity of LHON-associated ND4 G11778A mutation in three Chinese families. However, evolutionary conservation analysis of this mutation suggested a poor conservation index and the pathogenicity scoring system showed that this mutation was a neutral polymorphism.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

PubMed Central

Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

2015-01-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

Comparative assessment of the efficacy of closed helical loop and T-loop for space closure in lingual orthodontics-a finite element study.

PubMed

Chacko, Ajay; Tikku, Tripti; Khanna, Rohit; Maurya, Rana Pratap; Srivastava, Kamna

2018-05-28

Retraction in lingual orthodontics has biomechanical differences when compared to labial orthodontics, which is not yet established. Thus, we have intended to compare the biomechanical characteristics of closed helical loop and T-loop on 1 mm activation with 30° of compensatory curvatures during retraction in lingual orthodontics. STb lingual brackets were indirectly bonded to maxillary typhodont model that was scanned to obtain FEM model. Closed helical loop (2 × 7 mm) and T-loop (6 × 2 × 7 mm) of 0.016″ × 0.016″ TMA wire were modeled without preactivation bends. Preactivation bends at 30° were given in the software. Boundary conditions were set. The force (F) and moment (M) of both the loops were determined on 1 mm activation, using ANSYS software. M/F ratio was also calculated for both the loops. T-loop exerted less force, thus increased M/F ratio as compared to closed helical loop on 1 mm activation. When torque has to be preserved in the anterior segment during retraction in lingual orthodontics, T-loop can be preferred over closed helical loop.

Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops.

PubMed

Kar, Anirban; Willcox, Smaranda; Griffith, Jack D

2016-11-02

The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

PubMed

Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

2016-10-04

Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

The Thr92Ala 5′ Type 2 Deiodinase Gene Polymorphism Is Associated with a Delayed Triiodothyronine Secretion in Response to the Thyrotropin-Releasing Hormone–Stimulation Test: A Pharmacogenomic Study

PubMed Central

Butler, Peter W.; Smith, Sheila M.; Linderman, Joyce D.; Brychta, Robert J.; Alberobello, Anna Teresa; Dubaz, Ornella M.; Luzon, Javier A.; Skarulis, Monica C.; Cochran, Craig S.; Wesley, Robert A.; Pucino, Frank

2010-01-01

Background The common Thr92Ala D2 polymorphism has been associated with changes in pituitary–thyroid axis homeostasis, but published results are conflicting. To investigate the effects of the Thr92Ala polymorphism on intrathyroidal thyroxine (T4) to triiodothyronine (T3) conversion, we designed prospective pharmacogenomic intervention aimed to detect differences in T3 levels after thyrotropin (TSH)-releasing hormone (TRH)–mediated TSH stimulation of the thyroid gland. Methods Eighty-three healthy volunteers were screened and genotyped for the Thr92Ala polymorphism. Fifteen volunteers of each genotype (Thr/Thr, Thr/Ala, and Ala/Ala) underwent a 500 mcg intravenous TRH stimulation test with serial measurements of serum total T3 (TT3), free T4, and TSH over 180 minutes. Results No differences in baseline thyroid hormone levels were seen among the study groups. Compared to the Thr/Thr group, the Ala/Ala group showed a significantly lower TRH-stimulated increase in serum TT3 at 60 minutes (12.07 ± 2.67 vs. 21.07 ± 2.86 ng/dL, p = 0.029). Thr/Ala subjects showed an intermediate response. Compared to Thr/Thr subjects, the Ala/Ala group showed a blunted rate of rise in serum TT3 as measured by mean time to 50% maximum delta serum TT3 (88.42 ± 6.84 vs. 69.56 ± 6.06 minutes, p = 0.028). Subjects attained similar maximal (180 minutes) TRH-stimulated TT3 levels. TRH-stimulated TSH and free T4 levels were not significantly different among the three genotype groups. Conclusions The commonly occurring Thr92Ala D2 variant is associated with a decreased rate of acute TSH-stimulated T3 release from the thyroid consistent with a decrease in intrathyroidal deiodination. These data provide a proof of concept that the Thr92Ala polymorphism is associated with subtle changes in thyroid hormone homeostasis. PMID:21054208

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

PubMed

Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

2018-05-30

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Characterization of cDNAs and genomic DNAs for human threonyl- and cysteinyl-tRNA synthetases

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

Cruzen, M.E.

1993-01-01

Techniques of molecular biology were used to clone, sequence and map two human aminoacyl-tRNA synthetase (aaRS) cDNAs: threonyl-tRNA synthetase (ThrRS) a class II enzyme and cysteinyl-tRNA synthetase (CysRS) a class I enzyme. The predicted protein sequence of human ThrRS is highly homologous to that of lower eukaryotic and prokaryotic ThRSs, particularly in the regions containing the three structural motifs common to all class II synthetases. Signature regions 1 and 2, which characterize the class IIa subgroup (SerRS, ThrRS and HisRS) are highly conserved from bacteria to human. Structural predictions for human ThrRS based on the known structure of the closelymore » related SerRS from E.coli implicate strongly conserved residues in the signature sequences to be important in substrate binding. The amino terminal 100 residues of the deduced amino acid sequence of ThrRS shares structural similarity to SerRS consistent with forming an antiparallel helix implicated in tRNA binding. The 5' untranslated sequence of the human ThrRS gene shares short stretches of common sequence with the gene for hamster HisRS including a binding site for the promoter specific transcription factor sp-1. The deduced amino acid sequence of human CysRS has a high degree of sequence identify to E. coli CysRS. Human CysRS possesses the classic characteristics of a class I synthetase and is most closely related to the MetRS subgroup. The amino terminal half of human CysRS can be modeled as a nucleotide binding fold and shares significant sequence and structural similarity to the other enzymes in this subgroup. The CysRS structural gene (CARS) was mapped to human chromosome 11p15.5 by fluorescent in situ hybridization. CARS is the first aaRS gene to be mapped to chromosome 11. The steady state of both CysRS and ThrRs mRNA were quantitated in several human tissues. Message levels for these enzymes appear to be subjected to differential regulation in different cell types.« less

Genetic polymorphisms in extracellular superoxide dismutase Leu53Leu, Arg213Gly, and Ala40Thr and susceptibility to type 2 diabetes mellitus.

PubMed

Yang, Y M; Xie, X R; Jin, A L

2016-12-02

The most common type of endocrine disease is type 2 diabetes mellitus (T2DM); genetic factors contribute to the development to T2DM. In this study, we investigated the role of the Leu53Leu, Arg213Gly, and Ala40Thr polymorphisms in extracellular superoxide dismutase (EC-SOD) gene in the development of T2DM in a Chinese population. DNA was extracted from peripheral blood samples obtained from 256 T2DM patients and 324 control subjects recruited from our hospital between January 2013 and March 2015. DNA was genotyped by polymerase chain reaction-restriction fragment length polymorphism. The obtained data was then statistically analyzed. The chi-square test revealed a statistically significant difference in the genotype frequencies of EC-SOD Ala40Thr (χ 2 = 13.26, P = 0.001) between the patients and controls. Unconditional regression analysis indicated that the GA and AA genotypes of EC-SOD Ala40Thr were associated with an increased risk of T2DM compared to the GG genotype {adjusted odds ratio (OR) [95% confidence interval (CI)] = 1.46 (1.01-2.11) and 2.67 (1.48-4.85), respectively}. In the dominant model, the GA+AA genotype of EC-SOD Ala40Thr was correlated with a higher risk of T2DM, in comparison with the GG genotype (OR = 1.64, 95%CI = 1.16-2.33). In the recessive model, AA of EC-SOD Ala40Thr showed a 2.19-fold higher risk of developing T2DM than the GG+GA genotype. In conclusion, people with the Ala40Thr polymorphism in EC-SOD are at a higher risk of developing T2DM; therefore, this may be utilized as a biomarker for early screening of T2DM in a Chinese population.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

PubMed

Chan, Tung O; Zhang, Jin; Tiegs, Brian C; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M; Armen, Roger S; Rodeck, Ulrich; Penn, Raymond B

2015-10-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. © 2015 Authors; published by Portland Press Limited.

Influence of phosphorylation of THR-3, SER-11, and SER-15 on deoxycytidine kinase activity and stability.

PubMed

Smal, C; Ntamashimikiro, S; Arts, A; Van Den Neste, E; Bontemps, F

2010-06-01

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.

Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship.

PubMed

Wang, Qing; Bao, Luyao; Jia, Chenjun; Li, Mei; Li, Jian-Jun; Lu, Xuefeng

2017-03-16

Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k cat value below 1 min -1 , which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required. The amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k cat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%. Some important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k cat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.

The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription.

PubMed Central

Ke, N; Gao, X; Keeney, J B; Boeke, J D; Voytas, D F

1999-01-01

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated. PMID:10411136

Improvement of the activity of the anti-HIV-1 integrase aptamer T30175 by introducing a modified thymidine into the loops.

PubMed

Virgilio, Antonella; Amato, Teresa; Petraccone, Luigi; Esposito, Francesca; Grandi, Nicole; Tramontano, Enzo; Romero, Raquel; Haider, Shozeb; Gomez-Monterrey, Isabel; Novellino, Ettore; Mayol, Luciano; Esposito, Veronica; Galeone, Aldo

2018-05-10

In this paper, we report our investigations on analogues of the anti-human immunodeficiency virus type 1 (HIV-1) integrase (IN) aptamer T30175 in which the individual thymidines forming the loops were replaced by 5-hydroxymethyl-2'-deoxyuridine residues (H). Circular dichroism, nuclear magnetic resonance and gel electrophoresis investigations clearly indicated that all the modified aptamers preserve the ability to form the original 5'-5' end-stacked head-to-head dimeric G-quadruplex structure, in which each G-quadruplex adopts a parallel arrangement and is characterized by three G-tetrads, three propeller loops and one bulge-loop. All the modified aptamers were tested in an IN inhibition LEDGF-independent assay. While the modified aptamers INTB-H13 and INTB-H17 showed IC 50 values comparable with that of the parent aptamer (INTB-nat), analogues INTB-H2, INTB-H5 and, to a lesser extent, INTB-H9 showed a higher ability to inhibit the HIV IN than the unmodified aptamer. Molecular modelling studies evaluating the aptamer/HIV IN interaction highlighted the ability of the modified thymidines to establish several contacts with the target protein. All the data point to the importance of loops in the aptamer/target interaction and suggest that the site-specific replacement of loop residues with commercially available analogues can be considered a straightforward strategy to improve the biological activities of several G-quadruplex aptamers.

The diversity of H3 loops determines the antigen-binding tendencies of antibody CDR loops.

PubMed

Tsuchiya, Yuko; Mizuguchi, Kenji

2016-04-01

Of the complementarity-determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen-binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen-binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen-binding tendencies of all the CDR loops. © 2016 The Protein Society.

Loop engineering reveals the importance of active-site-decorating loops and gating residue in substrate affinity modulation of arginine deiminase (an anti-tumor enzyme).

PubMed

Cheng, Feng; Yang, Jianhua; Bocola, Marco; Schwaneberg, Ulrich; Zhu, Leilei

2018-05-05

Protein engineering of enzyme loop regions is an effective strategy to improve enzymatic properties. Previous studies that aimed to boost the activity of PpADI (an arginine deiminase from Pseudomonas plecoglossicida) under physiological conditions yielded several significantly improved variants that harbor substitutions predominantly located in active-site-decorating loops. A multi-site saturation mutagenesis at four positions in loop 1 (37, 38, 42, and 43) and three positions in loop 4 (402, 403, and 404) was performed to elucidate the importance of these loops in modulating the substrate affinity of PpADI. The identified "best" variant (M6-L1-4) showed a decreased S 0.5 ('K M ') of 0.48 mM compared with the parent M6 (0.81 mM). Subsequently, a rational design to recombine beneficial substitutions within loops 1 and 4 yielded variant L6 with a substantially decreased S 0.5 value (0.17 mM). A comprehensive simulation analysis resulted in a conclusion that high loop flexibility (especially the gating residue Arg400) is beneficial for substrate affinity due to less efficient blocking of the active site. Copyright © 2018 Elsevier Inc. All rights reserved.

Maple Syrup Urine Disease in Cypriot Families: Identification of Three Novel Mutations and Biochemical Characterization of the p.Thr211Met Mutation in the E1α Subunit

PubMed Central

Georgiou, Theodoros; Chuang, Jacinta L.; Wynn, R. Max; Stylianidou, Goula; Korson, Mark; Chuang, David T.

2009-01-01

We report five mutations, three of them novel, responsible for maple syrup urine disease in four unrelated Cypriot families. The five children studied are the first cases of classic maple syrup urine disease to be reported among Cypriots. The first novel mutation identified is a single-base deletion in exon 6 of the Elα gene (c.718delG), which leads to a frameshift after Ala240 and to a stop codon 89 residues further downstream. The other two novel mutations identified are in the Elβ subunit: a two-base deletion in exon 6, c.662_663delCC, which leads to a frameshift after Ala221 and creates a stop codon 17 residues further downstream, as well as a splice mutation, IVS3[+3]delA, which results in the skipping of exon 3. The two known mutations identified are in the Elα gene: the G > C transversion at the 3′-splice acceptor site, (IVS5-1G > C), which results in the deletion of the entire exon 6, and the missense mutation in exon 5 (c.632C > T), which corresponds to a p.Thr211Met substitution. The p.Thr211Met substitution is located in a potassium-ion pocket in the E1 component required for stability of the bound cofactor thiamine diphosphate. The mutant E1 protein harboring the p.Thr211Met substitution was shown unable to bind thiamine diphosphate, leading to undetectable E1 activity. PMID:19715473

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

PubMed

Sarkar, Koustav; Han, Seong-Su; Wen, Kuo-Kuang; Ochs, Hans D; Dupré, Loïc; Seidman, Michael M; Vyas, Yatin M

2017-12-15

Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. We sought to define how dysfunctional gene transcription is causally linked to the degree of T H cell deficiency and genomic instability in the XLT/WAS clinical spectrum. In human T H 1- or T H 2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in T H 1 cells relative to T H 2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (T H 1 genes) in T H 1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (T H 2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. Transcriptional R-loop imbalance is a novel molecular defect causative in T H 1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum

Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21.

PubMed

Jabbur, James R; Tabor, Amy D; Cheng, Xiaodong; Wang, Hua; Uesugi, Motonari; Lozano, Guillermina; Zhang, Wei

2002-10-10

Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21(Waf1/Cip1) (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha-helical conformation within the transactivation domain. Mutagenesis-derived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF(II)31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF(II)31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF(II)31. Consistently, p53-null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF(II)31 recruitment.

Surface EEG-Transcranial Direct Current Stimulation (tDCS) Closed-Loop System.

PubMed

Leite, Jorge; Morales-Quezada, Leon; Carvalho, Sandra; Thibaut, Aurore; Doruk, Deniz; Chen, Chiun-Fan; Schachter, Steven C; Rotenberg, Alexander; Fregni, Felipe

2017-09-01

Conventional transcranial direct current stimulation (tDCS) protocols rely on applying electrical current at a fixed intensity and duration without using surrogate markers to direct the interventions. This has led to some mixed results; especially because tDCS induced effects may vary depending on the ongoing level of brain activity. Therefore, the objective of this preliminary study was to assess the feasibility of an EEG-triggered tDCS system based on EEG online analysis of its frequency bands. Six healthy volunteers were randomized to participate in a double-blind sham-controlled crossover design to receive a single session of 10[Formula: see text]min 2[Formula: see text]mA cathodal and sham tDCS. tDCS trigger controller was based upon an algorithm designed to detect an increase in the relative beta power of more than 200%, accompanied by a decrease of 50% or more in the relative alpha power, based on baseline EEG recordings. EEG-tDCS closed-loop-system was able to detect the predefined EEG magnitude deviation and successfully triggered the stimulation in all participants. This preliminary study represents a proof-of-concept for the development of an EEG-tDCS closed-loop system in humans. We discuss and review here different methods of closed loop system that can be considered and potential clinical applications of such system.

Arg-Pro-X-Ser/Thr is a Consensus Phosphoacceptor Sequence for the Meiosis-Specific Ime2 Protein Kinase in Saccharomyces cerevisiae†

PubMed Central

Moore, Michael; Shin, Marcus; Bruning, Adrian; Schindler, Karen; Vershon, Andrew; Winter, Edward

2008-01-01

Ime2 is a meiosis-specific protein kinase in Saccharomyces cerevisiae that is functionally related to cyclin-dependent kinase. Although Ime2 regulates multiple steps in meiosis, only a few of its substrates have been identified. Here we show that Ime2 phosphorylates Sum1, a repressor of meiotic gene transcription, on Thr-306. Ime2 protein kinase assays on Sum1 mutants and synthetic peptides define a consensus motif Arg-Pro-X-Ser/Thr that is required for efficient phosphorylation by Ime2. The carboxyl residue adjacent to the phosphoacceptor (+1 position) also influences the efficiency of Ime2 phosphorylation with alanine being a preferred residue. This information has predictive value in identifying new potential Ime2 targets as shown by the ability of Ime2 to phosphorylate Sgs1 and Gip1 in vitro, and could be important in differentiating mitotic and meiotic regulatory pathways. PMID:17198398

Molecular dynamics simulations of the Bcl-2 protein to predict the structure of its unordered flexible loop domain.

PubMed

Raghav, Pawan Kumar; Verma, Yogesh Kumar; Gangenahalli, Gurudutta U

2012-05-01

B-cell lymphoma (Bcl-2) protein is an anti-apoptotic member of the Bcl-2 family. It is functionally demarcated into four Bcl-2 homology (BH) domains: BH1, BH2, BH3, BH4, one flexible loop domain (FLD), a transmembrane domain (TM), and an X domain. Bcl-2's BH domains have clearly been elucidated from a structural perspective, whereas the conformation of FLD has not yet been predicted, despite its important role in regulating apoptosis through its interactions with JNK-1, PKC, PP2A phosphatase, caspase 3, MAP kinase, ubiquitin, PS1, and FKBP38. Many important residues that regulate Bcl-2 anti-apoptotic activity are present in this domain, for example Asp34, Thr56, Thr69, Ser70, Thr74, and Ser87. The structural elucidation of the FLD would likely help in attempts to accurately predict the effect of mutating these residues on the overall structure of the protein and the interactions of other proteins in this domain. Therefore, we have generated an increased quality model of the Bcl-2 protein including the FLD through modeling. Further, molecular dynamics (MD) simulations were used for FLD optimization, to predict the flexibility, and to determine the stability of the folded FLD. In addition, essential dynamics (ED) was used to predict the collective motions and the essential subspace relevant to Bcl-2 protein function. The predicted average structure and ensemble of MD-simulated structures were submitted to the Protein Model Database (PMDB), and the Bcl-2 structures obtained exhibited enhanced quality. This study should help to elucidate the structural basis for Bcl-2 anti-apoptotic activity regulation through its binding to other proteins via the FLD.

Chromatin looping defines expression of TAL1, its flanking genes, and regulation in T-ALL.

PubMed

Zhou, Yan; Kurukuti, Sreenivasulu; Saffrey, Peter; Vukovic, Milica; Michie, Alison M; Strogantsev, Ruslan; West, Adam G; Vetrie, David

2013-12-19

TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

PubMed

Stephen, Preyesh; Ye, Sheng; Zhou, Ming; Song, Jian; Zhang, Rongguang; Wang, En-Duo; Giegé, Richard; Lin, Sheng-Xiang

2018-05-25

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNA Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identification of an evolutionarily conserved extracellular threonine residue critical for surface expression and its potential coupling of adjacent voltage-sensing and gating domains in voltage-gated potassium channels.

PubMed

Mckeown, Lynn; Burnham, Matthew P; Hodson, Charlotte; Jones, Owen T

2008-10-31

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding

PubMed Central

Sarek, Grzegorz; Vannier, Jean-Baptiste; Panier, Stephanie; Petrini, John H.J.; Boulton, Simon J.

2015-01-01

Summary The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1R1264H. Conversely, we define a TRF2I124D substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1R1264H mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. PMID:25620558

TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding.

PubMed

Sarek, Grzegorz; Vannier, Jean-Baptiste; Panier, Stephanie; Petrini, John H J; Boulton, Simon J

2015-02-19

The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

PubMed

Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

2017-01-01

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Simple "TRS" Auxiliary tube for retraction of anterior segment using segmental T loop mechanics.

PubMed

Shyagali, Tarulatha R; Rajpara, Yagnesh; Trivedi, Kalyani

2014-01-01

Segmental T loop is the most popular frictionless mechanics so far. This biomechanically sound system was designed for the Burstone's canine bracket, which can be extra inventory for the clinicians who want to practice the segmental T loop routinely. The present manuscript provides the alternate to Burstones canine bracket for the retraction of the anterior segment.

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop.

PubMed Central

van Dongen, M J; Mooren, M M; Willems, E F; van der Marel, G A; van Boom, J H; Wijmenga, S S; Hilbers, C W

1997-01-01

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair. PMID:9092659

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity.

PubMed

Tran, Tam N T; Shelton, Jennifer; Brown, Susan; Durrett, Timothy P

2017-10-01

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl-TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

PubMed Central

Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

2016-01-01

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

49 CFR 178.45 - Specification 3T seamless steel cylinder.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Section 178.45 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS... limits. Permanent closures formed by spinning are prohibited. (b) Material, steel. Only open hearth...

Suppression of Akt-mediated HDAC3 expression and CDK2 T39 phosphorylation by a bichalcone analog contributes to S phase retardation of cancer cells.

PubMed

Hung, Kuang-Chen; Lin, Meng-Liang; Hsu, Shih-Wei; Lee, Chuan-Chun; Huang, Ren-Yu; Wu, Tian-Shung; Chen, Shih-Shun

2018-06-15

Targeting cell cycle regulators has been a suggested mechanism for therapeutic cancer strategies. We report here that the bichalcone analog TSWU-CD4 induces S phase arrest of human cancer cells by inhibiting the formation of cyclin A-phospho (p)-cyclin-dependent kinase 2 (CDK2, threonine [Thr] 39) complexes, independent of mutant p53 expression. Ectopic expression of CDK2 (T39E), which mimics phosphorylation of the Thr 39 residue of CDK2, partially rescues the cells from TSWU-CD4-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TSWU-CD4-triggered S phase arrest. Decreased histone deacetylase 3 (HDAC3) expression after TSWU-CD4 treatment was demonstrated, and TSWU-CD4 induced S phase arrest and inhibitory effects on cyclin A expression and CDK2 Thr 39 phosphorylation, while cyclin A-p-CDK2 (Thr 39) complex formation was suppressed by ectopic wild-type HDAC3 expression. The co-transfection of CDK2 (T39E) along with HDAC3 completely restored cyclin A expression, Thr 39-phosphorylated CDK2, cyclin A-p-CDK2 (Thr 39) complex formation, and the S phase population to normal levels. Protein kinase B (Akt) inactivation was required for TSWU-CD4-induced S phase cell cycle arrest, because constitutively active Akt1 blocks the induction of S phase arrest and the suppression of cyclin A and HDAC3 expression, CDK2 Thr 39 phosphorylation, and cyclin A-p-CDK2 (Thr 39) complex formation by TSWU-CD4. Taken together, our results indicate that TSWU-CD4 induces S phase arrest by inhibiting Akt-mediated HDAC3 expression and CDK2 Thr 39 phosphorylation to suppress the formation of cyclin A-p-CDK2 (Thr 39) complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.

PubMed

Brennan, S O; Myles, T; Peach, R J; Donaldson, D; George, P M

1990-01-01

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

Human METTL20 Methylates Lysine Residues Adjacent to the Recognition Loop of the Electron Transfer Flavoprotein in Mitochondria*

PubMed Central

Rhein, Virginie F.; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

2014-01-01

In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. PMID:25023281

Human METTL20 methylates lysine residues adjacent to the recognition loop of the electron transfer flavoprotein in mitochondria.

PubMed

Rhein, Virginie F; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M; Walker, John E

2014-08-29

In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates.

PubMed

Molle, Virginie; Leiba, Jade; Zanella-Cléon, Isabelle; Becchi, Michel; Kremer, Laurent

2010-11-01

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.

Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop*

PubMed Central

Monteil, Arnaud; Chausson, Patrick; Boutourlinsky, Katia; Mezghrani, Alexandre; Huc-Brandt, Sylvaine; Blesneac, Iulia; Bidaud, Isabelle; Lemmers, Céline; Leresche, Nathalie; Lambert, Régis C.; Lory, Philippe

2015-01-01

Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (Δ1-Cav3.2; Ser423–Pro542) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, Δ1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of Δ1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels. PMID:25931121

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

PubMed

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

Evaluation of T-111 forced-convection loop tested with lithium at 1370 C. [free convection

NASA Technical Reports Server (NTRS)

Devan, J. H.; Long, E. L., Jr.

1975-01-01

A T-111 alloy (Ta-8% W-2% Hf) forced-convection loop containing molten lithium was operated 3000 hr at a maximum temperature of 1370 C. Flow velocities up to 6.3 m/sec were used, and the results of this forced-convection loop are very similar to those observed in lower velocity thermal-convection loops of T-111 containing lithium. Weight changes were determined at 93 positions around the loop. The maximum dissolution rate occurred at the maximum wall temperature of the loop and was less than 1.3 microns/year. Mass transfer of hafnium, nitrogen, and, to a lesser extent, carbon occurred from the hotter to cooler regions. Exposed surfaces in the highest temperature region were found to be depleted in hafnium to a depth of 60 microns with no detectable change in tungsten content. There was some loss in room-temperature tensile strength for specimens exposed to lithium at 1370 C, attributable to depletion of hafnium and nitrogen and to attendant grain growth.

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

PubMed Central

Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

2014-01-01

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

Probing the role of highly conserved residues in triosephosphate isomerase--analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme.

PubMed

Bandyopadhyay, Debarati; Murthy, Mathur R N; Balaram, Hemalatha; Balaram, Padmanabhan

2015-10-01

Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodium falciparum triosephosphate isomerase (PfTIM) enzyme (EC 5.3.1.1) as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of Kd values for dimer dissociation (Q64N = 73.7 ± 9.2 nm and Q64E = 44.6 ± 8.4 nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 °C and 45 °C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity. Structural data have been deposited in the Protein Data Bank under accession numbers 4ZZ9, 5BMW, 5BMX, 5BNK and 5BRB. © 2015 FEBS.

Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

PubMed Central

Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

PubMed

Li, Chang-Ling; Yang, Jin-Gang; Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125

Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis

PubMed Central

Zaccardi, Margot J; O'Rourke, Kathleen F; Yezdimer, Eric M; Loggia, Laura J; Woldt, Svenja; Boehr, David D

2014-01-01

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site β1α1 and β2α2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent

T-wave loop area from a pre-implant 12-lead ECG is associated with appropriate ICD shocks

PubMed Central

Hnatkova, Katerina; Friede, Tim; Malik, Marek; Zabel, Markus

2017-01-01

Aims In implantable cardioverter-defibrillator (ICD) patients, predictors of ICD shocks and mortality are needed to improve patient selection. Electrocardiographic (ECG) markers are simple to obtain and have been demonstrated to predict mortality. We aimed to assess the association of T-wave loop area and circularity with ICD shocks. Methods The study investigated patients with ICDs implanted between 1998 and 2010 for whom digital 12-lead ECGs (Schiller CS200 ECG-Network) of sufficient quality were obtained within 1 month prior to the implantation. T-wave loop area and circularity were calculated. Follow-up data of appropriate shocks were obtained during ICD clinic visits that included reviews of device stored electrograms. Results A total of 605 patients (82% males) were included; 68% had ischemic cardiomyopathy and 72% were treated for primary prevention. Over 3.8±1.4 years of follow-up, 114 patients (19%) experienced appropriate shock(s). Those with smaller T-wave loop area received fewer shocks (TLA, hazard ratio, HR, per increase of 1 technical unit, 0.71; [95% confidence interval, 0.53–0.94]; P = 0.02) and those with larger T-wave loop circularity (TLC) representing rounder T wave loop received more shocks (HR per 1% TLC increase 2.96; [0.85–10.36]; P = 0.09). When the quartile containing the largest TLA and TLC values, respectively, were compared to the remaining cases, TLA remained significantly associated with fewer and TLC with more frequent shocks also after multivariate adjustment for clinical variables (HR, 0.59 [0.35–0.99], P = 0.044; and 1.64 [1.08–2.49], P = 0.021, respectively). Conclusions The size and shape of the T-wave loop calculated from pre-implantation 12-lead ECGs are associated with appropriate ICD shocks. PMID:28291831

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

PubMed Central

Paslawski, Wojciech; Lillelund, Ove K.; Kristensen, Julie Veje; Schafer, Nicholas P.; Baker, Rosanna P.; Urban, Sinisa; Otzen, Daniel E.

2015-01-01

Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated. PMID:26056273

Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast.

PubMed

Bartish, Galyna; Moradi, Hossein; Nygård, Odd

2007-10-01

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

Modulation of HIV Protease Flexibility by the T80N Mutation

PubMed Central

Zhou, Hao; Li, Shangyang; Badger, John; Nalivaika, Ellen; Cai, Yufeng; Foulkes-Murzycki, Jennifer; Schiffer, Celia; Makowski, Lee

2015-01-01

The flexibility of HIV protease plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80 which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the potential influence of the T80N mutation on HIVp flexibility, wide-angle scattering (WAXS) data was measured for a series of HIV protease variants. Starting with a calculated WAXS pattern from a rigid atomic model, the modulations in the intensity distribution caused by structural fluctuations in the protein were predicted by simple analytic methods and compared to the experimental data. An analysis of T80N WAXS data shows that this variant is significantly more rigid than the WT across all length scales. The effects of this single point mutation extend throughout the protein, so as to alter the mobility of amino acids in the enzymatic core. These results support the contentions that significant protein flexibility extends throughout HIV protease and is critical to catalytic function. PMID:25488402

The Thr-His Connection on the Distal Heme of Catalase-Related Hemoproteins: A Hallmark of Reaction with Fatty Acid Hydroperoxides.

PubMed

Mashhadi, Zahra; Newcomer, Marcia E; Brash, Alan R

2016-11-03

This review focuses on a group of heme peroxidases that retain the catalase fold in structure, yet show little or no reaction with hydrogen peroxide. Instead of having a role in oxidative defense, these enzymes are involved in secondary metabolite biosynthesis. The prototypical enzyme is catalase-related allene oxide synthase, an enzyme that converts a specific fatty acid hydroperoxide to the corresponding allene oxide (epoxide). Other catalase-related enzymes form allylic epoxides, aldehydes, or a bicyclobutane fatty acid. In all catalases (including these relatives), a His residue on the distal face of the heme is absolutely required for activity. Its immediate neighbor in sequence as well as in 3 D space is conserved as Val in true catalases and Thr in the fatty acid hydroperoxide-metabolizing enzymes. Thr-His on the distal face of the heme is critical in switching the substrate specificity from H 2 O 2 to fatty acid hydroperoxide. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis

PubMed Central

Wang, Qian; Wei, Haojie; Du, Juan; Cao, Yan; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Chen, Dandan; Ma, Wei

2016-01-01

Abstract Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division. PMID:26636626

Immunogenicity of T7 bacteriophage nanoparticles displaying G-H loop of foot-and-mouth disease virus (FMDV).

PubMed

Xu, Hai; Bao, Xi; Lu, Yu; Liu, Yamei; Deng, Bihua; Wang, Yiwei; Xu, Yue; Hou, Jibo

2017-06-01

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses worldwide. The G-H loop of the FMDV VP1 structural protein is the major neutralizing antigenic site. However, a fully protective G-H loop peptide vaccine requires the addition of promiscuous Th sites from a source outside VP1. Thus, we demonstrated the potential of T7 bacteriophage based nanoparticles displaying a genetically fused G-H loop peptide (T7-GH) as a FMDV vaccine candidate. Recombinant T7-GH phage was constructed by inserting the G-H loop coding region into the T7 Select 415-1b vector. Purified T7-GH phage nanoparticles were analyzed by SDS-PAGE, Western blot and Dot-ELISA. Pigs seronegative for FMDV exposure were immunized with T7-GH nanoparticles along with the adjuvant Montanide ISA206, and two commercially available FMDV vaccines (InactVac and PepVac). Humoral and cellular immune responses, as well as protection against virulent homologous virus challenge were assessed following single dose immunization. Pigs immunized T7-GH developed comparable anti-VP1 antibody titers to PepVac, although lower LPBE titers than was induced by InactVac. Antigen specific lymphocyte proliferation was detected in T7-GH group similar to that of PepVac group, however, weaker than InactVac group. Pigs immunized with T7-GH developed a neutralizing antibody response stronger than PepVac, but weaker than InactVac. Furthermore, 80% (4/5) of T7-GH immunized pigs were protected from challenge with virulent homologous virus. These findings demonstrate that the T7-GH phage nanoparticles were effective in eliciting antigen specific immune responses in pigs, highlighting the value of such an approach in the research and development of FMDV vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Intracellular segment between transmembrane helices S0 and S1 of BK channel α subunit contains two amphipathic helices connected by a flexible loop

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

Shi, Pan; High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, 230031; Li, Dong

2013-08-02

Highlights: •The loop between S0 and S1 of BK channel was overexpressed and purified in DPC. •NMR studies indicated BK-IS1 contained two helices connected by a flexible loop. •Mg{sup 2+} titration of BK-IS1 indicated two possible binding sites of divalent ions. -- Abstract: The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca{sup 2+} and Mg{sup 2+}, as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0–S6) including an extra helix S0. Themore » intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg{sup 2+} coordination. In this study, BK-IS1 (44–113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide {sup 1}H–{sup 15}N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg{sup 2+}. Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.« less

RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.

PubMed

Vannier, Jean-Baptiste; Pavicic-Kaltenbrunner, Visnja; Petalcorin, Mark I R; Ding, Hao; Boulton, Simon J

2012-05-11

T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere. Copyright © 2012 Elsevier Inc. All rights reserved.

Discovery of Critical Residues for Viral Entry and Inhibition through Structural Insight of HIV-1 Fusion Inhibitor CP621–652*

PubMed Central

Chong, Huihui; Yao, Xue; Qiu, Zonglin; Qin, Bo; Han, Ruiyun; Waltersperger, Sandro; Wang, Meitian; Cui, Sheng; He, Yuxian

2012-01-01

The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the 621QIWNNMT627 motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621–652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621–652 complexed by T21. We find that the 621QIWNNMT627 motif does not maintain the α-helical conformation. Instead, residues Met626 and Thr627 form a unique hook-like structure (denoted as M-T hook), in which Thr627 redirects the peptide chain to position Met626 above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met626 caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621–652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition. PMID:22511760

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

PubMed Central

del Val, Coral; White, Stephen H.

2014-01-01

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

PubMed

Korkosh, V S; Zhorov, B S; Tikhonov, D B

2016-05-01

The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

PubMed Central

Wang, Guirong; Christensen, Neil D; Wigdahl, Brian; Guttentag, Susan H; Floros, Joanna

2003-01-01

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions. PMID:12356334

Sex-specific effect of CPB2 Ala147Thr but not Thr325Ile variants on the risk of venous thrombosis: A comprehensive meta-analysis

PubMed Central

Zwingerman, Nora; Medina-Rivera, Alejandra; Kassam, Irfahan; Wilson, Michael D.; Morange, Pierre-Emmanuel; Trégouët, David-Alexandre; Gagnon, France

2017-01-01

Background Thrombin activatable fibrinolysis inhibitor (TAFI), encoded by the Carboxypeptidase B2 gene (CPB2), is an inhibitor of fibrinolysis and plays a role in the pathogenesis of venous thrombosis. Experimental findings support a functional role of genetic variants in CPB2, while epidemiological studies have been unable to confirm associations with risk of venous thrombosis. Sex-specific effects could underlie the observed inconsistent associations between CPB2 genetic variants and venous thrombosis. Methods A comprehensive literature search was conducted for associations between Ala147Thr and Thr325Ile variants with venous thrombosis. Authors were contacted to provide sex-specific genotype counts from their studies. Combined and sex-specific random effects meta-analyses were used to estimate a pooled effect estimate for primary and secondary genetic models. Results A total of 17 studies met the inclusion criteria. A sex-specific meta-analysis applying a dominant model supported a protective effect of Ala147Thr on venous thrombosis in females (OR = 0.81, 95%CI: 0.68,0.97; p = 0.018), but not in males (OR = 1.06, 95%CI:0.96–1.16; p = 0.263). The Thr325Ile did not show a sex-specific effect but showed variation in allele frequencies by geographic region. A subgroup analysis of studies in European countries showed decreased risk, with a recessive model (OR = 0.83, 95%CI:0.71–0.97, p = 0.021) for venous thrombosis. Conclusions A comprehensive literature review, including unpublished data, provided greater statistical power for the analyses and decreased the likelihood of publication bias influencing the results. Sex-specific analyses explained apparent discrepancies across genetic studies of Ala147Thr and venous thrombosis. While, careful selection of genetic models based on population genetics, evolutionary and biological knowledge can increase power by decreasing the need to adjust for testing multiple models. PMID:28552956

Sex-specific effect of CPB2 Ala147Thr but not Thr325Ile variants on the risk of venous thrombosis: A comprehensive meta-analysis.

PubMed

Zwingerman, Nora; Medina-Rivera, Alejandra; Kassam, Irfahan; Wilson, Michael D; Morange, Pierre-Emmanuel; Trégouët, David-Alexandre; Gagnon, France

2017-01-01

Thrombin activatable fibrinolysis inhibitor (TAFI), encoded by the Carboxypeptidase B2 gene (CPB2), is an inhibitor of fibrinolysis and plays a role in the pathogenesis of venous thrombosis. Experimental findings support a functional role of genetic variants in CPB2, while epidemiological studies have been unable to confirm associations with risk of venous thrombosis. Sex-specific effects could underlie the observed inconsistent associations between CPB2 genetic variants and venous thrombosis. A comprehensive literature search was conducted for associations between Ala147Thr and Thr325Ile variants with venous thrombosis. Authors were contacted to provide sex-specific genotype counts from their studies. Combined and sex-specific random effects meta-analyses were used to estimate a pooled effect estimate for primary and secondary genetic models. A total of 17 studies met the inclusion criteria. A sex-specific meta-analysis applying a dominant model supported a protective effect of Ala147Thr on venous thrombosis in females (OR = 0.81, 95%CI: 0.68,0.97; p = 0.018), but not in males (OR = 1.06, 95%CI:0.96-1.16; p = 0.263). The Thr325Ile did not show a sex-specific effect but showed variation in allele frequencies by geographic region. A subgroup analysis of studies in European countries showed decreased risk, with a recessive model (OR = 0.83, 95%CI:0.71-0.97, p = 0.021) for venous thrombosis. A comprehensive literature review, including unpublished data, provided greater statistical power for the analyses and decreased the likelihood of publication bias influencing the results. Sex-specific analyses explained apparent discrepancies across genetic studies of Ala147Thr and venous thrombosis. While, careful selection of genetic models based on population genetics, evolutionary and biological knowledge can increase power by decreasing the need to adjust for testing multiple models.

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors*

PubMed Central

Chong, Huihui; Yao, Xue; Sun, Jianping; Qiu, Zonglin; Zhang, Meng; Waltersperger, Sandro; Wang, Meitian; Cui, Sheng; He, Yuxian

2012-01-01

CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors. PMID:22879603

Switch loop flexibility affects substrate transport of the AcrB efflux pump

DOE PAGES

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea; ...

2017-10-05

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutionsmore » on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.« less

Switch loop flexibility affects substrate transport of the AcrB efflux pump

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

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutionsmore » on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.« less

Mutation of a single residue in the S2-S3 loop of CNG channels alters the gating properties and sensitivity to inhibitors.

PubMed

Crary, J I; Dean, D M; Maroof, F; Zimmerman, A L

2000-12-01

We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.

Decoupling of a tight-fit transceiver phased array for human brain imaging at 9.4T: Loop overlapping rediscovered.

PubMed

Avdievich, Nikolai I; Giapitzakis, Ioannis-Angelos; Pfrommer, Andreas; Henning, Anke

2018-02-01

To improve the decoupling of a transceiver human head phased array at ultra-high fields (UHF, ≥ 7T) and to optimize its transmit (Tx) and receive (Rx) performance, a single-row eight-element (1 × 8) tight-fit transceiver overlapped loop array was developed and constructed. Overlapping the loops increases the RF field penetration depth but can compromise decoupling by generating substantial mutual resistance. Based on analytical modeling, we optimized the loop geometry and relative positioning to simultaneously minimize the resistive and inductive coupling and constructed a 9.4T eight-loop transceiver head phased array decoupled entirely by overlapping loops. We demonstrated that both the magnetic and electric coupling between adjacent loops is compensated at the same time by overlapping and nearly perfect decoupling (below -30 dB) can be obtained without additional decoupling strategies. Tx-efficiency and SNR of the overlapped array outperformed that of a common UHF gapped array of similar dimensions. Parallel Rx-performance was also not compromised due to overlapping the loops. As a proof of concept we developed and constructed a 9.4T (400 MHz) overlapped transceiver head array based on results of the analytical modeling. We demonstrated that at UHF overlapping loops not only provides excellent decoupling but also improves both Tx- and Rx-performance. Magn Reson Med 79:1200-1211, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

A Structural Biology and Protein Engineering Approach to the Development of Antidotes Against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents

DTIC Science & Technology

2015-05-01

structures that we reported earlier (Kryger et al [2000] Acta Crystallogr D Biol Crystallogr 56:1385-1394) were of complexes with the snake venom...interactions between conserved residues in the loop connecting α13 to α14 and residues from helices α18’-α19’, and, conversely, between residues in the...residues in the 4-helix bundle, including Glu376, Thr383, Asp384, Trp385, Gln508, Gln527, Phe535 and Lys538 (hAChE numbering), are strictly conserved in

Homology modeling and in silico site directed mutagenesis of pyruvate ferredoxin oxidoreductase from Clostridium thermocellum.

PubMed

Saranyah, Kannuchamy; Kalva, Sukesh; Mukund, Nisha; Singh, Sanjeev Kumar; Saleena, Lilly M

2015-01-01

Pyruvate ferredoxin oxidoreductase is the crucial enzyme that involves in bioethanol synthesis pathway of Clostridium thermocellum. It is an ethanologenic organism but has been investigated less on its enzyme structure. The amino acid sequence of Pyruvate ferredoxin oxidoreductase was derived from UNIPROT and the screened crystal structure was taken as the template for homology modeling using MODELLER 9V11. The model was loop refined and was validated using RMSD, ProSA and PROCHECK. The docking and per residue interaction studies were carried out to elucidate the interaction energies of amino acid residues with pyruvate. To enhance the binding of pyruvate with the enzyme, mutation studies were carried out by replacing Thr31 as it had a less interaction energy. Out of 10 mutants, T31N, T31Q and T31G were selected using potential energy and the residual energy calculations. Five nanoseconds explicit MD simulations were run for apo, wild type and mutants T31N, T31Q and T31G using Desmond. RMSD, RMSF, distance plots and H-bonds analysis proved T31G to be a favorable mutant for binding of pyruvate. Thus, modeling PFOR would help in profound understanding of its structural clefts and mutation studies would aid in improving the enzyme efficiency.

Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation.

PubMed

Lecchi, Silvia; Nelson, Clark J; Allen, Kenneth E; Swaney, Danielle L; Thompson, Katie L; Coon, Joshua J; Sussman, Michael R; Slayman, Carolyn W

2007-12-07

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase.

46 CFR 178.510 - Ballast.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 7 2012-10-01 2012-10-01 false Ballast. 178.510 Section 178.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Special Installations § 178.510 Ballast. (a) Any solid fixed ballast used to comply...

46 CFR 178.510 - Ballast.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false Ballast. 178.510 Section 178.510 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Special Installations § 178.510 Ballast. (a) Any solid fixed ballast used to comply...

49 CFR 178.345 - General design and construction requirements applicable to Specification DOT 406 (§ 178.346), DOT...

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false General design and construction requirements applicable to Specification DOT 406 (§ 178.346), DOT 407 (§ 178.347), and DOT 412 (§ 178.348) cargo tank motor vehicles. 178.345 Section 178.345 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS...

49 CFR 178.345 - General design and construction requirements applicable to Specification DOT 406 (§ 178.346), DOT...

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false General design and construction requirements applicable to Specification DOT 406 (§ 178.346), DOT 407 (§ 178.347), and DOT 412 (§ 178.348) cargo tank motor vehicles. 178.345 Section 178.345 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS...

49 CFR 178.345 - General design and construction requirements applicable to Specification DOT 406 (§ 178.346), DOT...

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false General design and construction requirements applicable to Specification DOT 406 (§ 178.346), DOT 407 (§ 178.347), and DOT 412 (§ 178.348) cargo tank motor vehicles. 178.345 Section 178.345 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS...

49 CFR 178.965 - Drop test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Drop test. 178.965 Section 178.965 Transportation... Packagings § 178.965 Drop test. (a) General. The drop test must be conducted for the qualification of all...) Special preparation for the drop test. Large Packagings must be filled in accordance with § 178.960. (c...

Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

PubMed Central

Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

2011-01-01

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

43 CFR 17.8 - Hearings.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 43 Public Lands: Interior 1 2012-10-01 2011-10-01 true Hearings. 17.8 Section 17.8 Public Lands: Interior Office of the Secretary of the Interior NONDISCRIMINATION IN FEDERALLY ASSISTED PROGRAMS OF THE DEPARTMENT OF THE INTERIOR Nondiscrimination on the Basis of Race, Color, or National Origin § 17.8 Hearings...

43 CFR 17.8 - Hearings.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Hearings. 17.8 Section 17.8 Public Lands: Interior Office of the Secretary of the Interior NONDISCRIMINATION IN FEDERALLY ASSISTED PROGRAMS OF THE DEPARTMENT OF THE INTERIOR Nondiscrimination on the Basis of Race, Color, or National Origin § 17.8 Hearings...

43 CFR 17.8 - Hearings.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 43 Public Lands: Interior 1 2014-10-01 2014-10-01 false Hearings. 17.8 Section 17.8 Public Lands: Interior Office of the Secretary of the Interior NONDISCRIMINATION IN FEDERALLY ASSISTED PROGRAMS OF THE DEPARTMENT OF THE INTERIOR Nondiscrimination on the Basis of Race, Color, or National Origin § 17.8 Hearings...

43 CFR 17.8 - Hearings.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Hearings. 17.8 Section 17.8 Public Lands: Interior Office of the Secretary of the Interior NONDISCRIMINATION IN FEDERALLY ASSISTED PROGRAMS OF THE DEPARTMENT OF THE INTERIOR Nondiscrimination on the Basis of Race, Color, or National Origin § 17.8 Hearings...

40 CFR 178.65 - Judicial review.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Judicial review. 178.65 Section 178.65... REQUESTS FOR HEARINGS Judicial Review § 178.65 Judicial review. An order issued under § 178.37 is final... of the order in the Federal Register. The failure to file a petition for judicial review within the...

21 CFR 178.3860 - Release agents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Release agents. 178.3860 Section 178.3860 Food and... and Production Aids § 178.3860 Release agents. Substances listed in paragraph (b) of this section may be safely used as release agents in petroleum wax complying with § 178.3710 and in polymeric resins...

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

PubMed

Klassen, Roland; Ciftci, Akif; Funk, Johanna; Bruch, Alexander; Butter, Falk; Schaffrath, Raffael

2016-12-15

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm 5 s 2 U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct 6 A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct 6 A37 or Ψ38/39 and mcm 5 U34 or s 2 U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNA Lys UUU or tRNA Gln UUG , respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm 5 U34 or s 2 U34, and this defect can be rescued by overexpression of tRNA Gln UUG Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

43 CFR 17.8 - Hearings.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Hearings. 17.8 Section 17.8 Public Lands... DEPARTMENT OF THE INTERIOR Nondiscrimination on the Basis of Race, Color, or National Origin § 17.8 Hearings. (a) Opportunity for hearing. Whenever an opportunity for a hearing is required by § 17.7(c...

NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana.

PubMed

Ye, Weihua; Spånning, Erika; Unnerståle, Sofia; Gotthold, David; Glaser, Elzbieta; Mäler, Lena

2012-10-01

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. © 2012 The Authors Journal compilation © 2012 FEBS.

Residual Highway Convolutional Neural Networks for in-loop Filtering in HEVC.

PubMed

Zhang, Yongbing; Shen, Tao; Ji, Xiangyang; Zhang, Yun; Xiong, Ruiqin; Dai, Qionghai

2018-08-01

High efficiency video coding (HEVC) standard achieves half bit-rate reduction while keeping the same quality compared with AVC. However, it still cannot satisfy the demand of higher quality in real applications, especially at low bit rates. To further improve the quality of reconstructed frame while reducing the bitrates, a residual highway convolutional neural network (RHCNN) is proposed in this paper for in-loop filtering in HEVC. The RHCNN is composed of several residual highway units and convolutional layers. In the highway units, there are some paths that could allow unimpeded information across several layers. Moreover, there also exists one identity skip connection (shortcut) from the beginning to the end, which is followed by one small convolutional layer. Without conflicting with deblocking filter (DF) and sample adaptive offset (SAO) filter in HEVC, RHCNN is employed as a high-dimension filter following DF and SAO to enhance the quality of reconstructed frames. To facilitate the real application, we apply the proposed method to I frame, P frame, and B frame, respectively. For obtaining better performance, the entire quantization parameter (QP) range is divided into several QP bands, where a dedicated RHCNN is trained for each QP band. Furthermore, we adopt a progressive training scheme for the RHCNN where the QP band with lower value is used for early training and their weights are used as initial weights for QP band of higher values in a progressive manner. Experimental results demonstrate that the proposed method is able to not only raise the PSNR of reconstructed frame but also prominently reduce the bit-rate compared with HEVC reference software.

49 CFR 178.345-1 - General requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Containers for Motor Vehicle Transportation § 178.345-1 General requirements. (a) Specification DOT 406, DOT 407 and DOT 412 cargo tank motor vehicles must conform to the requirements of this section in addition... definitions apply to §§ 178.345, 178.346, 178.347, and 178.348: Appurtenance means any cargo tank accessory...

49 CFR 178.345-1 - General requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Containers for Motor Vehicle Transportation § 178.345-1 General requirements. (a) Specification DOT 406, DOT 407 and DOT 412 cargo tank motor vehicles must conform to the requirements of this section in addition... definitions apply to §§ 178.345, 178.346, 178.347, and 178.348: Appurtenance means any cargo tank accessory...

49 CFR 178.345-1 - General requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Containers for Motor Vehicle Transportation § 178.345-1 General requirements. (a) Specification DOT 406, DOT 407 and DOT 412 cargo tank motor vehicles must conform to the requirements of this section in addition... definitions apply to §§ 178.345, 178.346, 178.347, and 178.348: Appurtenance means any cargo tank accessory...

Unbiased, scalable sampling of protein loop conformations from probabilistic priors.

PubMed

Zhang, Yajia; Hauser, Kris

2013-01-01

Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors

PubMed Central

2013-01-01

Background Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Results Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Conclusion Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion. PMID:24565175

Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.

PubMed

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-09-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. Copyright © 2014. Published by Elsevier B.V.

Buried polar residues in coiled-coil interfaces.

PubMed

Akey, D L; Malashkevich, V N; Kim, P S

2001-05-29

Coiled coils, estimated to constitute 3-5% of the encoded residues in most genomes, are characterized by a heptad repeat, (abcdefg)(n), where the buried a and d positions form the interface between multiple alpha-helices. Although generally hydrophobic, a substantial fraction ( approximately 20%) of these a- and d-position residues are polar or charged. We constructed variants of the well-characterized coiled coil GCN4-p1 with a single polar residue (Asn, Gln, Ser, or Thr) at either an a or a d position. The stability and oligomeric specificity of each variant were measured, and crystal structures of coiled-coil trimers with threonine or serine at either an a or a d position were determined. The structures show how single polar residues in the interface affect not only local packing, but also overall coiled-coil geometry as seen by changes in the Crick supercoil parameters and core cavity volumes.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

PubMed

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-11-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25

PubMed Central

Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

2013-01-01

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg−1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25. PMID:24804053

Multilevel D-loop PCR identification of hunting game.

PubMed

Parkanyi, V; Ondruska, L; Vasicek, D; Slamecka, J

2014-03-01

The control region of mtDNA (D-loop) was used for hair samples of the five hunting game species identification: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries musimon), and wild boar (Sus scrofa). For D-loop multilevel PCR detection scheme was applied in six primers (CE CVZV 1 = 5'-GATCACGAGCTTGATCACCA-3'; CE CVZV 2 = 5'-AGGAGTGGGCGATTTTAGGT-3'; DD CVZV 3 = 5'-CGCGTGAAACCAACAACCCGC-3'; DD CVZV 4 = 5'-CCGGGTCGGGGCCTTAGACG-3'; SSW CVZV 5 = 5'-ACACGTGCGTACACGCGCATA-3'; SSW CVZV 6 = 5'-GGTGCCTGCT T TCGTAGCACG-3') designed to identify unknown biological samples of the hunting game animals. The PCR reaction volume was 25 μl at conditions 95 °C for 2 min, 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 35 cycles, with last extension at 72 °C for 10 min. D-loop mtDNA amplicons of the game animals are characterized with specific PCR product sizes depending on species: red deer = 163 bp and 140 bp, fallow deer = 280 bp and 138 bp, roe deer = 303 bp, 280 bp, 160 bp and 138 bp, mouflon = 299 bp and 178 bp, wild boar = 137 bp and 229 bp.

49 CFR 178.345-4 - Joints.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Containers for Motor Vehicle Transportation § 178.345-4 Joints. (a) All joints between the cargo tank shell... accessible for inspection. [Amdt. 178-89, 54 FR 25022, June 12, 1989, as amended by Amdt. 178-118, 61 FR...

49 CFR 178.345-4 - Joints.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Containers for Motor Vehicle Transportation § 178.345-4 Joints. (a) All joints between the cargo tank shell... accessible for inspection. [Amdt. 178-89, 54 FR 25022, June 12, 1989, as amended by Amdt. 178-118, 61 FR...

49 CFR 178.345-4 - Joints.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Containers for Motor Vehicle Transportation § 178.345-4 Joints. (a) All joints between the cargo tank shell... accessible for inspection. [Amdt. 178-89, 54 FR 25022, June 12, 1989, as amended by Amdt. 178-118, 61 FR...

49 CFR 178.345-4 - Joints.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Containers for Motor Vehicle Transportation § 178.345-4 Joints. (a) All joints between the cargo tank shell... accessible for inspection. [Amdt. 178-89, 54 FR 25022, June 12, 1989, as amended by Amdt. 178-118, 61 FR...

49 CFR 178.345-4 - Joints.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Containers for Motor Vehicle Transportation § 178.345-4 Joints. (a) All joints between the cargo tank shell... accessible for inspection. [Amdt. 178-89, 54 FR 25022, June 12, 1989, as amended by Amdt. 178-118, 61 FR...

eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

PubMed

Thakur, Anil; Hinnebusch, Alan G

2018-05-01

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNA i ) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "P OUT " state to a closed conformation with TC more tightly bound in a "P IN " state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P IN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNA i D loop in the P IN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNA i We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNA i accommodation in the P IN state without influencing the P OUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNA i clash to an electrostatic attraction that stabilizes P IN and enhances selection of poor start codons in vivo.

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

PubMed Central

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W.; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-01-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. PMID:24792385

Mining protein loops using a structural alphabet and statistical exceptionality

PubMed Central

2010-01-01

Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a

Mining protein loops using a structural alphabet and statistical exceptionality.

PubMed

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 A). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of

Conformational states of mutant M13 coat proteins are regulated by transmembrane residues.

PubMed

Li, Z; Glibowicka, M; Joensson, C; Deber, C M

1993-03-05

Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core. SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus. Mutations to relatively polar residues (e.g. G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g. V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species. Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein. The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.

Cysteine Scanning Mutagenesis of TM4b-4c Loop of Glutamate Transporter EAAT1 Reveals Three Conformationally Sensitive Residues.

PubMed

Zhang, Wenlong; Zhang, Xiuping; Qu, Shaogang

2018-07-01

Glutamatergic synaptic transmitters are cleared from the synaptic cleft through excitatory amino acid transporters (EAATs) that are responsible for recycling glutamate and transporting it into neurons and glial cells. To probe the structural role of the TM4b-4c loop of EAAT1 ( Rattus norvegicus ), each of the 57 amino acid residues was mutated to cysteine. Thirteen of the single mutants have very low transport activity. Aqueous accessibility of the introduced cysteines from the remaining mutants was then explored by membrane-permeant and membrane-impermeant sulfhydryl reagents in different conditions. F190C, V238C, and A243C were affected by MTSET, whereas Q189C, F190C, V238C, A243C, and L244C were sensitive to MTSEA. Q189C and L244C transport activity was diminished in the presence of potassium, which is expected to favor the inward-facing conformation of the transporter. Inversely, L244C was protected by glutamate. The modification of A243C by MTSEA was enhanced by either potassium and glutamate or dl- threo -β-benzyloxyaspartate. From these results, we suggest that residues F190C, V238C, and A243C may be located near the extracellular surface, and the TM4b-4c loop forms multiple reentrant membrane loops on the cell surface. Alternatively, F190C, V238C, and A243C may function in the transport pathway, which is exposed to MTSET. In addition, Q189C, A243C, and L244C are conformationally sensitive and may play a role in the transport cycle. Copyright © 2018 by The Author(s).

21 CFR 178.3900 - Sodium pentachlorophenate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium pentachlorophenate. 178.3900 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3900 Sodium pentachlorophenate. Sodium... that contact food at temperatures not to exceed room temperature. The quantity of sodium...

21 CFR 178.3900 - Sodium pentachlorophenate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium pentachlorophenate. 178.3900 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3900 Sodium pentachlorophenate. Sodium... that contact food at temperatures not to exceed room temperature. The quantity of sodium...

21 CFR 178.3900 - Sodium pentachlorophenate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium pentachlorophenate. 178.3900 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3900 Sodium pentachlorophenate. Sodium... that contact food at temperatures not to exceed room temperature. The quantity of sodium...

21 CFR 178.3900 - Sodium pentachlorophenate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium pentachlorophenate. 178.3900 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3900 Sodium pentachlorophenate. Sodium... that contact food at temperatures not to exceed room temperature. The quantity of sodium...

49 CFR 178.33-1 - Compliance.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Compliance. 178.33-1 Section 178.33-1 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33-1 Compliance. (a) Required in all details. (b) [Reserved] ...

Albumin Redhill (-1 Arg, 320 Ala yields Thr): A glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site

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

Brennan, S.O.; Myles, T.; Peach, R.J.

1990-01-01

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind {sup 63}Ni{sup 2+} and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni{sup 2+} was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala {yields} Thr. Thismore » introduces as Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg {yields} Cys. The authors propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.« less

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

PubMed Central

Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth

2014-01-01

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. PMID:24562907

49 CFR 178.360-2 - Manufacture.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.360-2 Section 178.360-2 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.360-2 Manufacture. The ends of the vessel...

49 CFR 178.33-6 - Manufacture.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.33-6 Section 178.33-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33-6 Manufacture. (a) By appliances and methods that will...

49 CFR 178.360-3 - Dimensions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Dimensions. 178.360-3 Section 178.360-3 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.360-3 Dimensions. (a) The inside diameter...

49 CFR 178.338-2 - Material.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Material. 178.338-2 Section 178.338-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers for Motor Vehicle Transportation § 178.338-2 Material. (a) All material used in the construction...

49 CFR 178.338-2 - Material.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Material. 178.338-2 Section 178.338-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers for Motor Vehicle Transportation § 178.338-2 Material. (a) All material used in the construction...

49 CFR 178.338-2 - Material.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Material. 178.338-2 Section 178.338-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers for Motor Vehicle Transportation § 178.338-2 Material. (a) All material used in the construction...

49 CFR 178.338-2 - Material.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Material. 178.338-2 Section 178.338-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers for Motor Vehicle Transportation § 178.338-2 Material. (a) All material used in the construction...

49 CFR 178.33-1 - Compliance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Compliance. 178.33-1 Section 178.33-1 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33-1 Compliance. (a) Required in all details. (b...

Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

PubMed Central

Shimamura, A; Nakano, Y J; Mukasa, H; Kuramitsu, H K

1994-01-01

The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs. PMID:8050997

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor*

PubMed Central

Clayton, Cecilea C.; Donthamsetti, Prashant; Lambert, Nevin A.; Javitch, Jonathan A.; Neve, Kim A.

2014-01-01

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified. PMID:25336643

49 CFR 178.347-3 - Manhole assemblies.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Manhole assemblies. 178.347-3 Section 178.347-3... Containers for Motor Vehicle Transportation § 178.347-3 Manhole assemblies. Each manhole assembly must conform to § 178.345-5, except that each manhole assembly must be capable of withstanding internal fluid...

49 CFR 178.347-3 - Manhole assemblies.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Manhole assemblies. 178.347-3 Section 178.347-3... Containers for Motor Vehicle Transportation § 178.347-3 Manhole assemblies. Each manhole assembly must conform to § 178.345-5, except that each manhole assembly must be capable of withstanding internal fluid...

49 CFR 178.347-3 - Manhole assemblies.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Manhole assemblies. 178.347-3 Section 178.347-3... Containers for Motor Vehicle Transportation § 178.347-3 Manhole assemblies. Each manhole assembly must conform to § 178.345-5, except that each manhole assembly must be capable of withstanding internal fluid...

49 CFR 178.347-3 - Manhole assemblies.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Manhole assemblies. 178.347-3 Section 178.347-3... Containers for Motor Vehicle Transportation § 178.347-3 Manhole assemblies. Each manhole assembly must conform to § 178.345-5, except that each manhole assembly must be capable of withstanding internal fluid...

49 CFR 178.347-3 - Manhole assemblies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Manhole assemblies. 178.347-3 Section 178.347-3... Specifications for Containers for Motor Vehicle Transportation § 178.347-3 Manhole assemblies. Each manhole assembly must conform to § 178.345-5, except that each manhole assembly must be capable of withstanding...

49 CFR 178.605 - Hydrostatic pressure test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Hydrostatic pressure test. 178.605 Section 178.605... Packagings and Packages § 178.605 Hydrostatic pressure test. (a) General. The hydrostatic pressure test must... intended to contain liquids and be performed periodically as specified in § 178.601(e). This test is not...

49 CFR 178.605 - Hydrostatic pressure test.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Hydrostatic pressure test. 178.605 Section 178.605... Packagings and Packages § 178.605 Hydrostatic pressure test. (a) General. The hydrostatic pressure test must... intended to contain liquids and be performed periodically as specified in § 178.601(e). This test is not...

28 CFR 35.178 - State immunity.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 1 2013-07-01 2013-07-01 false State immunity. 35.178 Section 35.178 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION ON THE BASIS OF DISABILITY IN STATE AND LOCAL GOVERNMENT SERVICES Compliance Procedures § 35.178 State immunity. A State shall not be immune...

28 CFR 35.178 - State immunity.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 1 2014-07-01 2014-07-01 false State immunity. 35.178 Section 35.178 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION ON THE BASIS OF DISABILITY IN STATE AND LOCAL GOVERNMENT SERVICES Compliance Procedures § 35.178 State immunity. A State shall not be immune...

28 CFR 35.178 - State immunity.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 1 2012-07-01 2012-07-01 false State immunity. 35.178 Section 35.178 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION ON THE BASIS OF DISABILITY IN STATE AND LOCAL GOVERNMENT SERVICES Compliance Procedures § 35.178 State immunity. A State shall not be immune...

49 CFR 178.360-3 - Dimensions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Dimensions. 178.360-3 Section 178.360-3 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Packagings for Class 7 (Radioactive) Materials § 178.360-3 Dimensions. (a) The inside diameter of the vessel...

49 CFR 178.360-3 - Dimensions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Dimensions. 178.360-3 Section 178.360-3 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Packagings for Class 7 (Radioactive) Materials § 178.360-3 Dimensions. (a) The inside diameter of the vessel...

49 CFR 178.360-3 - Dimensions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Dimensions. 178.360-3 Section 178.360-3 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Packagings for Class 7 (Radioactive) Materials § 178.360-3 Dimensions. (a) The inside diameter of the vessel...

49 CFR 178.608 - Vibration standard.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Vibration standard. 178.608 Section 178.608... Packagings and Packages § 178.608 Vibration standard. (a) Each packaging must be capable of withstanding, without rupture or leakage, the vibration test procedure outlined in this section. (b) Test method. (1...

49 CFR 178.608 - Vibration standard.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Vibration standard. 178.608 Section 178.608... Testing of Non-bulk Packagings and Packages § 178.608 Vibration standard. (a) Each packaging must be capable of withstanding, without rupture or leakage, the vibration test procedure outlined in this section...

49 CFR 178.608 - Vibration standard.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Vibration standard. 178.608 Section 178.608... Packagings and Packages § 178.608 Vibration standard. (a) Each packaging must be capable of withstanding, without rupture or leakage, the vibration test procedure outlined in this section. (b) Test method. (1...

49 CFR 178.608 - Vibration standard.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Vibration standard. 178.608 Section 178.608... Packagings and Packages § 178.608 Vibration standard. (a) Each packaging must be capable of withstanding, without rupture or leakage, the vibration test procedure outlined in this section. (b) Test method. (1...

49 CFR 178.608 - Vibration standard.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Vibration standard. 178.608 Section 178.608... Packagings and Packages § 178.608 Vibration standard. (a) Each packaging must be capable of withstanding, without rupture or leakage, the vibration test procedure outlined in this section. (b) Test method. (1...

28 CFR 35.178 - State immunity.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 1 2010-07-01 2010-07-01 false State immunity. 35.178 Section 35.178 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION ON THE BASIS OF DISABILITY IN STATE AND LOCAL GOVERNMENT SERVICES Compliance Procedures § 35.178 State immunity. A State shall not be immune...

28 CFR 35.178 - State immunity.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 1 2011-07-01 2011-07-01 false State immunity. 35.178 Section 35.178 Judicial Administration DEPARTMENT OF JUSTICE NONDISCRIMINATION ON THE BASIS OF DISABILITY IN STATE AND LOCAL GOVERNMENT SERVICES Compliance Procedures § 35.178 State immunity. A State shall not be immune...

49 CFR 178.255-15 - Report.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Report. 178.255-15 Section 178.255-15 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-15 Report. (a) A copy of the manufacturer's data report required by Section VIII...

49 CFR 178.255-15 - Report.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Report. 178.255-15 Section 178.255-15 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-15 Report. (a) A copy of the manufacturer's data report required by Section VIII...

Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor.

PubMed

Wang, Jinti; Yarov-Yarovoy, Vladimir; Kahn, Roy; Gordon, Dalia; Gurevitz, Michael; Scheuer, Todd; Catterall, William A

2011-09-13

The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.

7 CFR 17.8 - Ocean transportation.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 1 2012-01-01 2012-01-01 false Ocean transportation. 17.8 Section 17.8 Agriculture... THE AGRICULTURAL TRADE DEVELOPMENT AND ASSISTANCE ACT OF 1954, AS AMENDED § 17.8 Ocean transportation. (a) General. (1) This section applies to the financing of ocean freight or ocean freight differential...

7 CFR 17.8 - Ocean transportation.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 1 2014-01-01 2014-01-01 false Ocean transportation. 17.8 Section 17.8 Agriculture... THE AGRICULTURAL TRADE DEVELOPMENT AND ASSISTANCE ACT OF 1954, AS AMENDED § 17.8 Ocean transportation. (a) General. (1) This section applies to the financing of ocean freight or ocean freight differential...

7 CFR 17.8 - Ocean transportation.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 1 2013-01-01 2013-01-01 false Ocean transportation. 17.8 Section 17.8 Agriculture... THE AGRICULTURAL TRADE DEVELOPMENT AND ASSISTANCE ACT OF 1954, AS AMENDED § 17.8 Ocean transportation. (a) General. (1) This section applies to the financing of ocean freight or ocean freight differential...

7 CFR 17.8 - Ocean transportation.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 1 2011-01-01 2011-01-01 false Ocean transportation. 17.8 Section 17.8 Agriculture... THE AGRICULTURAL TRADE DEVELOPMENT AND ASSISTANCE ACT OF 1954, AS AMENDED § 17.8 Ocean transportation. (a) General. (1) This section applies to the financing of ocean freight or ocean freight differential...

49 CFR 178.255-2 - Material.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Material. 178.255-2 Section 178.255-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-2 Material. (a) Material used in the tank must be steel of good weldable quality...

49 CFR 178.604 - Leakproofness test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Leakproofness test. 178.604 Section 178.604... Packagings and Packages § 178.604 Leakproofness test. (a) General. The leakproofness test must be performed... the test results are not affected; and (2) This test is not required for inner packagings of...

49 CFR 178.985 - Vibration test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Vibration test. 178.985 Section 178.985... Packagings § 178.985 Vibration test. (a) General. All rigid Large Packaging and flexible Large Packaging design types must be capable of withstanding the vibration test. (b) Test method. (1) A sample Large...

42 CFR 415.178 - Anesthesia services.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 3 2010-10-01 2010-10-01 false Anesthesia services. 415.178 Section 415.178 Public..., AND RESIDENTS IN CERTAIN SETTINGS Physician Services in Teaching Settings § 415.178 Anesthesia... schedule payment may be made if a physician is involved in a single anesthesia procedure involving an...

42 CFR 415.178 - Anesthesia services.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 3 2011-10-01 2011-10-01 false Anesthesia services. 415.178 Section 415.178 Public..., AND RESIDENTS IN CERTAIN SETTINGS Physician Services in Teaching Settings § 415.178 Anesthesia... schedule payment may be made if a physician is involved in a single anesthesia procedure involving an...

49 CFR 178.255-2 - Material.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Material. 178.255-2 Section 178.255-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-2 Material. (a) Material used in the tank must be steel of good weldable quality...

49 CFR 178.255-2 - Material.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Material. 178.255-2 Section 178.255-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-2 Material. (a) Material used in the tank must be steel of good weldable quality...

49 CFR 178.255-2 - Material.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Material. 178.255-2 Section 178.255-2 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-2 Material. (a) Material used in the tank must be steel of good weldable quality...

49 CFR 178.985 - Vibration test.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Vibration test. 178.985 Section 178.985... Packagings § 178.985 Vibration test. (a) General. All rigid Large Packaging and flexible Large Packaging design types must be capable of withstanding the vibration test. (b) Test method. (1) A sample Large...

49 CFR 178.985 - Vibration test.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Vibration test. 178.985 Section 178.985... Packagings § 178.985 Vibration test. (a) General. All rigid Large Packaging and flexible Large Packaging design types must be capable of withstanding the vibration test. (b) Test method. (1) A sample Large...

49 CFR 178.985 - Vibration test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Vibration test. 178.985 Section 178.985... Testing of Large Packagings § 178.985 Vibration test. (a) General. All rigid Large Packaging and flexible Large Packaging design types must be capable of withstanding the vibration test. (b) Test method. (1) A...

49 CFR 178.985 - Vibration test.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Vibration test. 178.985 Section 178.985... Packagings § 178.985 Vibration test. (a) General. All rigid Large Packaging and flexible Large Packaging design types must be capable of withstanding the vibration test. (b) Test method. (1) A sample Large...

21 CFR 178.3900 - Sodium pentachlorophenate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium pentachlorophenate. 178.3900 Section 178... § 178.3900 Sodium pentachlorophenate. Sodium pentachlorophenate may be safely used as a preservative for... temperature. The quantity of sodium pentachlorophenate used shall not exceed 0.5 percent by weight of ammonium...

Radiopharmaceutical composition containing tantalum-178 and process therefor

DOEpatents

Neirinckx, Rudi D.; Holman, B. Leonard; Davis, Michael A.; Harris, Gale I.

1989-05-16

A physiologically acceptable solution of tantalum-178 having an activity of 0.1 to 200 millicuries per milliliter of tantalum-178 solution is provided. The solution is obtained from tungsten-178 bound to a column of an anion exchange resin which forms tantalum-178 in situ by eluting the column with a hydrochloric acid solution containing hydrogen peroxide to form an acidic solution of tantalum-178. The acidic solution of tantalum-178 then is neutralized.

49 CFR 178.347-4 - Pressure relief.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Pressure relief. 178.347-4 Section 178.347-4... Containers for Motor Vehicle Transportation § 178.347-4 Pressure relief. (a) Each cargo tank must be equipped with a pressure and vacuum relief system in accordance with § 178.345-10 and this section. (b) Type and...

49 CFR 178.347-4 - Pressure relief.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Pressure relief. 178.347-4 Section 178.347-4... Containers for Motor Vehicle Transportation § 178.347-4 Pressure relief. (a) Each cargo tank must be equipped with a pressure and vacuum relief system in accordance with § 178.345-10 and this section. (b) Type and...

49 CFR 178.347-4 - Pressure relief.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Pressure relief. 178.347-4 Section 178.347-4... Containers for Motor Vehicle Transportation § 178.347-4 Pressure relief. (a) Each cargo tank must be equipped with a pressure and vacuum relief system in accordance with § 178.345-10 and this section. (b) Type and...

49 CFR 178.347-4 - Pressure relief.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Pressure relief. 178.347-4 Section 178.347-4... Containers for Motor Vehicle Transportation § 178.347-4 Pressure relief. (a) Each cargo tank must be equipped with a pressure and vacuum relief system in accordance with § 178.345-10 and this section. (b) Type and...

49 CFR 178.318-2 - Container.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Container. 178.318-2 Section 178.318-2... Specifications for Containers for Motor Vehicle Transportation § 178.318-2 Container. (a) Every container for... individual cell in the body of the container, into which each such cap shall snugly fit. There shall be...

49 CFR 178.606 - Stacking test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Stacking test. 178.606 Section 178.606... Packagings and Packages § 178.606 Stacking test. (a) General. All packaging design types other than bags must be subjected to a stacking test. (b) Number of test samples. Three test samples are required for each...

49 CFR 178.1065 - Righting test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Righting test. 178.1065 Section 178.1065... Containers § 178.1065 Righting test. (a) General. The righting test must be conducted for the qualification... preparation for the righting test. Flexible Bulk Container design types must be filled to not less than 95% of...

49 CFR 178.1070 - Tear test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tear test. 178.1070 Section 178.1070... Containers § 178.1070 Tear test. (a) General. The tear test must be conducted for the qualification of all of Flexible Bulk Containers design types. (b) Special preparation for the tear test. Flexible Bulk Container...

49 CFR 178.1045 - Drop test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Drop test. 178.1045 Section 178.1045... Containers § 178.1045 Drop test. (a) General. The drop test must be conducted for the qualification of all... subpart. (b) Special preparation for the drop test. Flexible Bulk Containers must be filled to their...

Substrate specificity of human protein arginine methyltransferase 7 (PRMT7): the importance of acidic residues in the double E loop.

PubMed

Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G

2014-11-21

Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

49 CFR 178.603 - Drop test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Drop test. 178.603 Section 178.603 Transportation... Packagings and Packages § 178.603 Drop test. (a) General. The drop test must be conducted for the... than flat drops, the center of gravity of the test packaging must be vertically over the point of...

49 CFR 178.818 - Tear test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Tear test. 178.818 Section 178.818 Transportation... § 178.818 Tear test. (a) General. The tear test must be conducted for the qualification of all flexible IBC design types. (b) Special preparation for the tear test. The flexible IBC must be filled to not...

49 CFR 178.819 - Vibration test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Vibration test. 178.819 Section 178.819... Testing of IBCs § 178.819 Vibration test. (a) General. The vibration test must be conducted for the... vibration test. (b) Test method. (1) A sample IBC, selected at random, must be filled and closed as for...

49 CFR 178.801 - General requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... which differs in surface treatment; (ii) A rigid plastic IBC or composite IBC which differs with regard to additives used to comply with §§ 178.706(c), 178.707(c) or 178.710(c); (iii) A packaging which... representative of the type of service equipment that will be fitted to any finished IBC body under the design...

49 CFR 178.801 - General requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... which differs in surface treatment; (ii) A rigid plastic IBC or composite IBC which differs with regard to additives used to comply with §§ 178.706(c), 178.707(c) or 178.710(c); (iii) A packaging which... representative of the type of service equipment that will be fitted to any finished IBC body under the design...

42 CFR 413.178 - Bad debts.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Bad debts. 413.178 Section 413.178 Public Health...) Services and Organ Procurement Costs § 413.178 Bad debts. Link to an amendment published at 75 FR 49199, Aug. 12, 2010. (a) CMS will reimburse each facility its allowable Medicare bad debts, as defined in...

49 CFR 178.33-8 - Tests.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Tests. 178.33-8 Section 178.33-8 Transportation... Containers, and Linings § 178.33-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.33-8 - Tests.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Tests. 178.33-8 Section 178.33-8 Transportation... Containers, and Linings § 178.33-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.356-3 - Tests.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Tests. 178.356-3 Section 178.356-3 Transportation... Packagings for Class 7 (Radioactive) Materials § 178.356-3 Tests. (a) Leakage test—Each inner liner assembly... must be held for at least 30 seconds. Liners failing to pass this test may not be used until repairs...

49 CFR 178.356-3 - Tests.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tests. 178.356-3 Section 178.356-3 Transportation... Packagings for Class 7 (Radioactive) Materials § 178.356-3 Tests. (a) Leakage test—Each inner liner assembly... must be held for at least 30 seconds. Liners failing to pass this test may not be used until repairs...

49 CFR 178.356-3 - Tests.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Tests. 178.356-3 Section 178.356-3 Transportation... Packagings for Class 7 (Radioactive) Materials § 178.356-3 Tests. (a) Leakage test—Each inner liner assembly... must be held for at least 30 seconds. Liners failing to pass this test may not be used until repairs...

49 CFR 178.33-8 - Tests.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tests. 178.33-8 Section 178.33-8 Transportation... Containers, and Linings § 178.33-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.33-8 - Tests.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Tests. 178.33-8 Section 178.33-8 Transportation... Containers, and Linings § 178.33-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.33-8 - Tests.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Tests. 178.33-8 Section 178.33-8 Transportation... Inside Containers, and Linings § 178.33-8 Tests. (a) One out of each lot of 25,000 containers or less... less, successively produced per day, shall constitute a lot and if the test container shall fail, the...

49 CFR 178.356-3 - Tests.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Tests. 178.356-3 Section 178.356-3 Transportation... Packagings for Class 7 (Radioactive) Materials § 178.356-3 Tests. (a) Leakage test—Each inner liner assembly... must be held for at least 30 seconds. Liners failing to pass this test may not be used until repairs...

21 CFR 178.3120 - Animal glue.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Animal glue. 178.3120 Section 178.3120 Food and... and Production Aids § 178.3120 Animal glue. Animal glue may be safely used as a component of articles..., transporting, or holding food, subject to the provisions of this section. (a) Animal glue consists of the...

21 CFR 178.3120 - Animal glue.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Animal glue. 178.3120 Section 178.3120 Food and... and Production Aids § 178.3120 Animal glue. Animal glue may be safely used as a component of articles..., transporting, or holding food, subject to the provisions of this section. (a) Animal glue consists of the...

21 CFR 178.3120 - Animal glue.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Animal glue. 178.3120 Section 178.3120 Food and... and Production Aids § 178.3120 Animal glue. Animal glue may be safely used as a component of articles..., transporting, or holding food, subject to the provisions of this section. (a) Animal glue consists of the...

21 CFR 178.3120 - Animal glue.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Animal glue. 178.3120 Section 178.3120 Food and... and Production Aids § 178.3120 Animal glue. Animal glue may be safely used as a component of articles..., transporting, or holding food, subject to the provisions of this section. (a) Animal glue consists of the...

Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

PubMed Central

Perkins, Daya I.; Trudell, James R.; Asatryan, Liana; Alkana, Ronald L.

2012-01-01

Recent studies highlighted the importance of loop 2 of α1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the β hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC50 but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC50 while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC50 and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the β hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs. PMID:22357974

Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling.

PubMed

Shacham, Sharon; Cheifetz, Maya N; Fridkin, Mati; Pawson, Adam J; Millar, Robert P; Naor, Zvi

2005-08-12

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.

The controversial p.Met34Thr variant in GJB2 gene: Two siblings, one genotype, two phenotypes.

PubMed

Lameiras, Ana Rita; Gonçalves, Ana Cláudia; Santos, Ricardo; O'Neill, Assunção; Reis, Luís Roque Dos; Matos, Tiago Daniel; Fialho, Graça; Caria, Helena; Escada, Pedro

2015-08-01

Recent advances in molecular genetics have increased the identification of genes and mutations responsible for inherited forms of hearing loss (HL), enabling early detection of these cases. Approximately, 60% of early-onset HL cases are due to genetic causes, of which 70% are non-syndromic. Of these, 75-80% are inherited in an autosomal recessive pattern (DFNB). Mutations in GJB2 gene, coding for connexin 26 (Cx26), are the major cause of autosomal recessive hereditary HL, but some GJB2 mutations are yet of unclear or controversial significance. The aim of the present study was to identify the etiology of hearing loss, and correlate genotype-phenotype, in two Portuguese siblings with profound and moderate non-syndromic sensorineural bilateral HL. The affected subjects and their parents underwent audiological and genetic study. Molecular analysis of GJB2 gene was performed, searching for mutations in the coding region and receptor splicing site by automated sequencing. The onset and the degree of HL were different in the two affected subjects. However, the same GJB2 genotype [p.Met34Thr]+[p.Arg184Pro] was identified in both siblings. The c.551G>C (p.Arg184Pro) and c.101T>C (p.Met34Thr) missense variants were inherited from the father and mother, respectively, both heterozygous carriers of these variants. The clinical and genetic data here presented suggest that the non-syndromic sensorineural HL of these two Portuguese siblings might be due to the presence of p.Met34Thr and p.Arg184Pro variants in compound heterozygosity. If so, p.Met34Thr variant could have function as a hypomorphic allele that may cause HL depending on the opposing GJB2 allele. The observed phenotypic variability may not, however, be solely explained by variable expression of this genotype. A putative modifier gene or mutations in another HL-associated gene could probably be contributing to the severe HL in one of the siblings. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Effect of T- and C-loop mutations on the Herbaspirillum seropedicae GlnB protein in nitrogen signalling.

PubMed

Bonatto, Ana C; Souza, Emanuel M; Pedrosa, Fábio O; Yates, M Geoffrey; Benelli, Elaine M

2005-01-01

Proteins of the PII family are found in species of all kingdoms. Although these proteins usually share high identity, their functions are specific to the different organisms. Comparison of structural data from Escherichia coli GlnB and GlnK and Herbaspirillum seropedicae GlnB showed that the T-loop and C-terminus were variable regions. To evaluate the role of these regions in signal transduction by the H. seropedicae GlnB protein, four mutants were constructed: Y51F, G108A/P109a, G108W and Q3R/T5A. The activities of the native and mutated proteins were assayed in an E. coli background constitutively expressing the Klebsiella pneumoniae nifLA operon. The results suggested that the T-loop and C-terminus regions of H. seropedicae GlnB are involved in nitrogen signal transduction.

Lung cancer risk associated with Thr495Pro polymorphism of GHR in Chinese population.

PubMed

Cao, Guochun; Lu, Hongna; Feng, Jifeng; Shu, Jian; Zheng, Datong; Hou, Yayi

2008-04-01

The incidence of lung cancer has been increasing over recent decades. Previous studies showed that polymorphisms of the genes involved in carcinogen-detoxication, DNA repair and cell cycle control comprise risk factors for lung cancer. Recent observations revealed that the growth hormone receptor (GHR) might play important roles in carcinogenesis and Rudd et al. found that the Thr495Pro polymorphism of GHR was strongly associated with lung cancer risk in Caucasians living in the UK (OR = 12.98, P = 0.0019, 95% CI: 1.77-infinity). To test whether this variant of GHR would modify the risk of lung cancer in Chinese population, we compared the polymorphism between 778 lung cancer patients and 781 healthy control subjects. Our results indicate that the frequency of 495Thr (2.8%) allele in cases was significantly higher than in controls (OR = 2.04, P = 0.006, 95% CI: 1.21-3.42) which indicated this allele might be a risk factor for lung cancer. Further analyses revealed Thr495Pro variant was associated with lung cancer in the subpopulation with higher risk for lung cancer: male subpopulation, still-smokers subpopulation and the subpopulation with familial history of cancer. In different histological types of lung cancer, Thr495Pro SNP was significantly associated with small cell and squamous cell lung cancer, but not with adenocarcinoma, which suggested a potential interaction between this polymorphism and metabolic pathways related to smoking. The potential gene-environment interaction on lung cancer risk was evaluated using MDR software. A significant redundant interaction between Thr495Pro polymorphism and smoking dose and familial history of cancer was identified and the combination of genetic factors and smoking status or familial history of cancer barely increased the cancer risk prediction accuracy. In conclusion, our results suggested that the Thr495Pro polymorphism of GHR was associated with the risk of lung cancer in a redundant interaction with smoking and

40 CFR 763.178 - Recordkeeping.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Recordkeeping. 763.178 Section 763.178 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT ASBESTOS Prohibition of the Manufacture, Importation, Processing, and Distribution in Commerce of Certain Asbestos...

40 CFR 264.178 - Closure.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Closure. 264.178 Section 264.178 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR... from the containment system. Remaining containers, liners, bases, and soil containing or contaminated...

Method of Implementing Digital Phase-Locked Loops

NASA Technical Reports Server (NTRS)

Stephens, Scott A. (Inventor); Thomas, J. Brooks (Inventor)

1997-01-01

In a new formulation for digital phase-locked loops, loop-filter constants are determined from loop roots that can each be selectively placed in the s-plane on the basis of a new set of parameters, each with simple and direct physical meaning in terms of loop noise bandwidth, root-specific decay rate, and root-specific damping. Loops of first to fourth order are treated in the continuous-update approximation (B(sub L)T approaches 0) and in a discrete-update formulation with arbitrary B(sub L)T. Deficiencies of the continuous-update approximation in large-B(sub L)T applications are avoided in the new discrete-update formulation.

Molecular Dynamics Simulations and Structural Analysis to Decipher Functional Impact of a Twenty Residue Insert in the Ternary Complex of Mus musculus TdT Isoform

PubMed Central

Mutt, Eshita; Sowdhamini, Ramanathan

2016-01-01

Insertions/deletions are common evolutionary tools employed to alter the structural and functional repertoire of protein domains. An insert situated proximal to the active site or ligand binding site frequently impacts protein function; however, the effect of distal indels on protein activity and/or stability are often not studied. In this paper, we have investigated a distal insert, which influences the function and stability of a unique DNA polymerase, called terminal deoxynucleotidyl transferase (TdT). TdT (EC:2.7.7.31) is a monomeric 58 kDa protein belonging to family X of eukaryotic DNA polymerases and known for its role in V(D)J recombination as well as in non-homologous end-joining (NHEJ) pathways. Two murine isoforms of TdT, with a length difference of twenty residues and having different biochemical properties, have been studied. All-atom molecular dynamics simulations at different temperatures and interaction network analyses were performed on the short and long-length isoforms. We observed conformational changes in the regions distal to the insert position (thumb subdomain) in the longer isoform, which indirectly affects the activity and stability of the enzyme through a mediating loop (Loop1). A structural rationale could be provided to explain the reduced polymerization rate as well as increased thermosensitivity of the longer isoform caused by peripherally located length variations within a DNA polymerase. These observations increase our understanding of the roles of length variants in introducing functional diversity in protein families in general. PMID:27311013

Curvature versus v-bends in a group B titanium T-loop spring.

PubMed

Martins, Renato Parsekian; Buschang, Peter H; Viecilli, Rodrigo; dos Santos-Pinto, Ary

2008-05-01

To compare the system of forces acting on curvature and preactivated V-bends in titanium T-loop springs (TTLSs) made of 0.017- x 0.025-inch TMA (titanium molibdenium alloy) wire. Pictures of TTLSs preactivated by curvature and V-bends were inserted in the LOOP software program to design both TTLSs. Symmetry was assured using the program. Both TTLSs used the same amount (length) of wire and had the same angulation between their anterior and posterior extremities when passive. The loops were activated 7 mm, and forces and moments were registered after each 0.5 mm of deactivation. The brackets were at the same height, separated by 23 mm and angulated 0 degrees . The preactivated curvature TTLS delivered horizontal forces ranging from 34 gF to 456 gF, while the TTLS preactivated by V-bends delivered forces ranging from 54 gF to 517 gF. The forces decreased more (30 gF vs 33 gF) with every 0.5 mm of activation on the preactivated V-bend TTLS than on the preactivated curvature TTLS. Vertical forces were low and clinically insignificant for both TTLSs. The moment to force (MF) ratios were systematically higher on the preactivated curvature than on the preactivated V-bend TTLS (from 5.8 mm to 38.8 mm vs 4.7 mm to 28.3 mm). Although both loops show symmetrical moments in their anterior and posterior extremities and can be used for group B anchorage, the curvature preactivated TTLS delivers lower horizontal forces and higher MF ratios than the acute preactivated V-bend TTLS.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases.

PubMed

Viviani, Vadim R; Silva Neto, Antonio J; Arnoldi, Frederico G C; Barbosa, João A R G; Ohmiya, Yoshihiro

2008-01-01

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.

40 CFR 265.178 - Air emission standards.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 27 2012-07-01 2012-07-01 false Air emission standards. 265.178 Section 265.178 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... DISPOSAL FACILITIES Use and Management of Containers § 265.178 Air emission standards. The owner or...

40 CFR 265.178 - Air emission standards.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 26 2014-07-01 2014-07-01 false Air emission standards. 265.178 Section 265.178 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... DISPOSAL FACILITIES Use and Management of Containers § 265.178 Air emission standards. The owner or...

40 CFR 265.178 - Air emission standards.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Air emission standards. 265.178 Section 265.178 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... DISPOSAL FACILITIES Use and Management of Containers § 265.178 Air emission standards. The owner or...

40 CFR 265.178 - Air emission standards.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 26 2011-07-01 2011-07-01 false Air emission standards. 265.178 Section 265.178 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... DISPOSAL FACILITIES Use and Management of Containers § 265.178 Air emission standards. The owner or...

Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

PubMed

Tanaka, Jun; Fukamizo, Tamo; Ohnuma, Takayuki

2017-05-01

The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Violations of K-Conservation in 178Hf

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

Hayes, A. B.; Cline, D.; Wu, C. Y.

2006-03-13

Coulomb excitation of K{pi}=6+(t1/2=77 ns), 8-(t1/2=4.0 s) and 16+(t1/2=31 y) 178Hf isomers has led to the measurement of a set of E{lambda} matrix elements, coupling the isomer bands to the {gamma}- and ground state bands. The resulting matrix elements, derived using a coupled-channel semiclassical Coulomb excitation search code, have been used to probe the K-components in the wave functions and revealed the onset and saturation of K-mixing in low-K bands, whereas K-mixing is negligible in the high-K bands. The implications can be applied to other quadrupole-deformed nuclei. An upper limit on the Coulomb depopulation yield of the 16+ isomer wasmore » calculated based on the present set of matrix elements.« less

Phosphorylation of a conserved Thr357 in yeast Nedd4-like ubiquitin ligase Rsp5 is involved in down-regulation of the general amino acid permease Gap1.

PubMed

Sasaki, Toshiya; Takagi, Hiroshi

2013-06-01

Rsp5, an essential HECT-type ubiquitin ligase, is the only yeast Saccharomyces cerevisiae member of the Nedd4 family. Rsp5 triggers the ubiquitination-dependent endocytosis of the general amino acid permease Gap1 in response to a good nitrogen source. Previously, we showed that the Thr357Ala/Lys764Glu variant Rsp5 induces the constitutive inactivation of Gap1, which is mainly involved in uptake of the toxic proline analogue, l-azetidine-2-carboxylate (AZC). Here, our experimental results indicated that the Thr357Ala substitution in the substrate-recognizing WW2 domain of Rsp5 constitutively causes the down-regulation of four proline permeases (Gap1, Put4, Agp1 and Gnp1), leading to AZC tolerance to yeast cells. In RSP5(T357A) cells, Gap1 was highly ubiquitinated and constantly delivered to the vacuole from the Golgi without sorting to the plasma membrane. Analyses of RSP5 mutants using antiphosphopeptide antibody suggest that Thr phosphorylation occurred in all three WW domains and, interestingly, that Thr357 in the WW2 domain was phosphorylated, in agreement with the in vitro result for the mouse Rsp5 orthologue. Furthermore, the phosphorylation-mimic mutant (Thr357Asp) showed strong sensitivity to AZC. From these results, we propose a possible mechanism involved in the regulation of Rsp5 activity for Gap1 down-regulation via the phosphorylation of a conserved Thr357 in the Nedd4 family. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Role of Thr399Ile and Asp299Gly polymorphisms of toll-like receptor-4 gene in acute dental abscess.

PubMed

Miri-Moghaddam, Ebrahim; Farhad-Mollashahi, Narges; Baghaee, Elnaz; Bazi, Ali; Garme, Yasaman

2017-02-01

Apical Periodontitis (AP) is an inflammatory disease that affects the tissues surrounding the root end of a tooth. The disease which is caused by endodontic infections presents in different clinical ways including development of an acute abscess. Recent studies have provided information suggesting role of a multitude of factors in pathogenesis of acute apical abscess (AAA). In this case-control study, our goal was to evaluate the frequency and potential role of two common polymorphisms of toll like receptor-4 (TLR-4) gene; Thr399Ile (1196 C>T) and Asp299Gly (+896 A>G), in 50 patients with AAA as cases and 50 patients with asymptomatic apical periodontitis (AAP) as controls. Saliva sample containing mucosal epithelial cells was used for DNA extraction. Polymorphisms were detected by Tetra-ARMS (Amplification Refractory Mutation System) PCR method. Statistical analyses were carried out in SPSS 21 software. Homozygous wild type (CC) and heterozygous (CT) genotypes of Thr399Ile polymorphism were detected in 84% and 16% of AAA patients respectively. In controls, respective ratios were 94% (CC) and 6% (CT). Observed difference was not statistically significant ( P >0.05) for distribution of these genotypes. The mutant homozygous (TT) genotype of this polymorphism was identified in neither of the participants. Overall, T allele frequency was obtained 8% in AAA and 3% in AAP (OR=2.6, 95% CI; 0. 6-10.6, p >0.05). For Asp299Gly polymorphism, no individual was detected with the mutant allele in case or control groups. Our results indicated a possible role for Thr399Ile polymorphism in acute presentations of abscess in AAA. However, the impact of this polymorphism needs to be more assessed in future studies. Key words: Genetic polymorphism, periapical abscess, periapical periodontitis, toll-like receptor 4.

Role of Thr399Ile and Asp299Gly polymorphisms of toll-like receptor-4 gene in acute dental abscess

PubMed Central

Miri-Moghaddam, Ebrahim; Baghaee, Elnaz; Bazi, Ali; Garme, Yasaman

2017-01-01

Background Apical Periodontitis (AP) is an inflammatory disease that affects the tissues surrounding the root end of a tooth. The disease which is caused by endodontic infections presents in different clinical ways including development of an acute abscess. Recent studies have provided information suggesting role of a multitude of factors in pathogenesis of acute apical abscess (AAA). In this case-control study, our goal was to evaluate the frequency and potential role of two common polymorphisms of toll like receptor-4 (TLR-4) gene; Thr399Ile (1196 C>T) and Asp299Gly (+896 A>G), in 50 patients with AAA as cases and 50 patients with asymptomatic apical periodontitis (AAP) as controls. Material and Methods Saliva sample containing mucosal epithelial cells was used for DNA extraction. Polymorphisms were detected by Tetra-ARMS (Amplification Refractory Mutation System) PCR method. Statistical analyses were carried out in SPSS 21 software. Results Homozygous wild type (CC) and heterozygous (CT) genotypes of Thr399Ile polymorphism were detected in 84% and 16% of AAA patients respectively. In controls, respective ratios were 94% (CC) and 6% (CT). Observed difference was not statistically significant (P>0.05) for distribution of these genotypes. The mutant homozygous (TT) genotype of this polymorphism was identified in neither of the participants. Overall, T allele frequency was obtained 8% in AAA and 3% in AAP (OR=2.6, 95% CI; 0. 6-10.6, p>0.05). For Asp299Gly polymorphism, no individual was detected with the mutant allele in case or control groups. Conclusions Our results indicated a possible role for Thr399Ile polymorphism in acute presentations of abscess in AAA. However, the impact of this polymorphism needs to be more assessed in future studies. Key words:Genetic polymorphism, periapical abscess, periapical periodontitis, toll-like receptor 4. PMID:28210435

49 CFR 178.33a-6 - Manufacture.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.33a-6 Section 178.33a-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33a-6 Manufacture. (a) By appliances and methods that...

49 CFR 178.33b-6 - Manufacture.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Manufacture. 178.33b-6 Section 178.33b-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33b-6 Manufacture. (a) Each container must be...

49 CFR 178.605 - Hydrostatic pressure test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Hydrostatic pressure test. 178.605 Section 178.605... Testing of Non-bulk Packagings and Packages § 178.605 Hydrostatic pressure test. (a) General. The hydrostatic pressure test must be conducted for the qualification of all metal, plastic, and composite...

21 CFR 178.1005 - Hydrogen peroxide solution.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Hydrogen peroxide solution. 178.1005 Section 178... SANITIZERS Substances Utilized To Control the Growth of Microorganisms § 178.1005 Hydrogen peroxide solution. Hydrogen peroxide solution identified in this section may be safely used to sterilize polymeric food...

21 CFR 178.1005 - Hydrogen peroxide solution.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Hydrogen peroxide solution. 178.1005 Section 178... SANITIZERS Substances Utilized To Control the Growth of Microorganisms § 178.1005 Hydrogen peroxide solution. Hydrogen peroxide solution identified in this section may be safely used to sterilize polymeric food...

21 CFR 178.1005 - Hydrogen peroxide solution.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Hydrogen peroxide solution. 178.1005 Section 178... SANITIZERS Substances Utilized To Control the Growth of Microorganisms § 178.1005 Hydrogen peroxide solution. Hydrogen peroxide solution identified in this section may be safely used to sterilize polymeric food...

21 CFR 178.1005 - Hydrogen peroxide solution.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Hydrogen peroxide solution. 178.1005 Section 178... SANITIZERS Substances Utilized To Control the Growth of Microorganisms § 178.1005 Hydrogen peroxide solution. Hydrogen peroxide solution identified in this section may be safely used to sterilize polymeric food...

49 CFR 178.33b-5 - Material.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of polyethylene...

49 CFR 178.33b-5 - Material.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of polyethylene...

49 CFR 178.33b-5 - Material.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of polyethylene...

49 CFR 178.33b-5 - Material.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of polyethylene...

21 CFR 178.3520 - Industrial starch-modified.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Industrial starch-modified. 178.3520 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3520 Industrial starch-modified. Industrial starch... provisions of this section. (a) Industrial starch-modified is identified as follows: (1) A food starch...

21 CFR 178.3520 - Industrial starch-modified.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Industrial starch-modified. 178.3520 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3520 Industrial starch-modified. Industrial starch... provisions of this section. (a) Industrial starch-modified is identified as follows: (1) A food starch...

49 CFR 178.33a-1 - Compliance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Compliance. 178.33a-1 Section 178.33a-1 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33a-1 Compliance. (a) Required in all details. (b...

49 CFR 178.33b-1 - Compliance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Compliance. 178.33b-1 Section 178.33b-1 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33b-1 Compliance. (a) Required in all details. (b...

Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding.

PubMed

Jacob, Maik H; D'Souza, Roy N; Schwarzlose, Thomas; Wang, Xiaojuan; Huang, Fang; Haas, Elisha; Nau, Werner M

2018-04-26

Protein folding can be described as a probabilistic succession of events in which the peptide chain forms loops closed by specific amino acid residue contacts, herein referred to as loop nodes. To measure loop rates, several photophysical methods have been introduced where a pair of optically active probes is incorporated at selected chain positions and the excited probe undergoes contact quenching (CQ) upon collision with the second probe. The quenching mechanisms involved triplet-triplet energy transfer, photoinduced electron transfer, and collision-induced fluorescence quenching, where the fluorescence of Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, is quenched by tryptophan. The discrepancy between the loop rates afforded from these three CQ techniques has, however, remained unresolved. In analyzing this discrepancy, we now report two short-distance FRET methods where Dbo acts as an energy acceptor in combination with tryptophan and naphtylalanine, two donors with largely different fluorescence lifetimes of 1.3 and 33 ns, respectively. Despite the different quenching mechanisms, the rates from FRET and CQ methods were, surprisingly, of comparable magnitude. This combination of FRET and CQ data led to a unifying physical model and to the conclusion that the rate of loop formation in folding reactions varies not only with the kind and number of residues that constitute the chain but also in particular with the size and properties of the residues that constitute the loop node.

The Staphylococcus aureus autoinducer-2 synthase LuxS is regulated by Ser/Thr phosphorylation.

PubMed

Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

2010-12-01

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.

21 CFR 178.3800 - Preservatives for wood.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Preservatives for wood. 178.3800 Section 178.3800... Certain Adjuvants and Production Aids § 178.3800 Preservatives for wood. Preservatives may be safely used... to accomplish the technical effect of protecting the wood from decay, mildew, and water absorption...

21 CFR 178.3800 - Preservatives for wood.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Preservatives for wood. 178.3800 Section 178.3800... Certain Adjuvants and Production Aids § 178.3800 Preservatives for wood. Preservatives may be safely used... to accomplish the technical effect of protecting the wood from decay, mildew, and water absorption...

49 CFR 178.255-8 - Safety devices.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Safety devices. 178.255-8 Section 178.255-8 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-8 Safety devices. (a) See § 173.315(i) of this subchapter. (b) [Reserved] [Amdt...

49 CFR 178.255-8 - Safety devices.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Safety devices. 178.255-8 Section 178.255-8 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Portable Tanks § 178.255-8 Safety devices. (a) See § 173.315(i) of this subchapter. (b...

21 CFR 178.1005 - Hydrogen peroxide solution.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Hydrogen peroxide solution. 178.1005 Section 178... Growth of Microorganisms § 178.1005 Hydrogen peroxide solution. Hydrogen peroxide solution identified in...)(1) of this section. (a) Identity. For the purpose of this section, hydrogen peroxide solution is an...

49 CFR 178.33a-5 - Material.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Material. 178.33a-5 Section 178.33a-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33a-5 Material. (a) Uniform quality steel plate such as black plate, electrotin...

49 CFR 178.345-5 - Manhole assemblies.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Manhole assemblies. 178.345-5 Section 178.345-5... Containers for Motor Vehicle Transportation § 178.345-5 Manhole assemblies. (a) Each cargo tank with capacity... manhole, fill opening and washout assembly must be structurally capable of withstanding, without leakage...

49 CFR 178.345-5 - Manhole assemblies.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Manhole assemblies. 178.345-5 Section 178.345-5... Containers for Motor Vehicle Transportation § 178.345-5 Manhole assemblies. (a) Each cargo tank with capacity... manhole, fill opening and washout assembly must be structurally capable of withstanding, without leakage...

49 CFR 178.345-5 - Manhole assemblies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Manhole assemblies. 178.345-5 Section 178.345-5... Specifications for Containers for Motor Vehicle Transportation § 178.345-5 Manhole assemblies. (a) Each cargo... diameter. (b) Each manhole, fill opening and washout assembly must be structurally capable of withstanding...

49 CFR 178.345-5 - Manhole assemblies.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Manhole assemblies. 178.345-5 Section 178.345-5... Containers for Motor Vehicle Transportation § 178.345-5 Manhole assemblies. (a) Each cargo tank with capacity... manhole, fill opening and washout assembly must be structurally capable of withstanding, without leakage...

49 CFR 178.345-5 - Manhole assemblies.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Manhole assemblies. 178.345-5 Section 178.345-5... Containers for Motor Vehicle Transportation § 178.345-5 Manhole assemblies. (a) Each cargo tank with capacity... manhole, fill opening and washout assembly must be structurally capable of withstanding, without leakage...

49 CFR 178.33a-5 - Material.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Material. 178.33a-5 Section 178.33a-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33a-5 Material. (a) Uniform quality steel plate such as black plate, electrotin...

49 CFR 178.33a-5 - Material.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Material. 178.33a-5 Section 178.33a-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33a-5 Material. (a) Uniform quality steel plate such as black plate, electrotin...

49 CFR 178.33a-5 - Material.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Material. 178.33a-5 Section 178.33a-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33a-5 Material. (a) Uniform quality steel plate such as black plate, electrotin...

49 CFR 178.337-14 - Gauging devices.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Gauging devices. 178.337-14 Section 178.337-14... Specifications for Containers for Motor Vehicle Transportation § 178.337-14 Gauging devices. (a) Liquid level... subchapter. (2) Each cargo tank used in carbon dioxide, refrigerated liquid or nitrous oxide, refrigerated...

21 CFR 178.3720 - Petroleum wax, synthetic.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Petroleum wax, synthetic. 178.3720 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3720 Petroleum wax, synthetic. Synthetic petroleum wax may be safely used in applications and under the same conditions where naturally derived petroleum wax...

21 CFR 178.3720 - Petroleum wax, synthetic.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Petroleum wax, synthetic. 178.3720 Section 178.3720... Certain Adjuvants and Production Aids § 178.3720 Petroleum wax, synthetic. Synthetic petroleum wax may be safely used in applications and under the same conditions where naturally derived petroleum wax is...

21 CFR 178.3800 - Preservatives for wood.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Preservatives for wood. 178.3800 Section 178.3800... Certain Adjuvants and Production Aids § 178.3800 Preservatives for wood. Preservatives may be safely used... to accomplish the technical effect of protecting the wood from decay, mildew, and water absorption...

21 CFR 178.3800 - Preservatives for wood.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Preservatives for wood. 178.3800 Section 178.3800... Certain Adjuvants and Production Aids § 178.3800 Preservatives for wood. Preservatives may be safely used... to accomplish the technical effect of protecting the wood from decay, mildew, and water absorption...

21 CFR 178.3290 - Chromic chloride complexes.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Chromic chloride complexes. 178.3290 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3290 Chromic chloride complexes. Myristo chromic chloride complex and stearato chromic chloride complex may be safely used as release agents in the closure...

Pro-oncogenic function of HIP-55/Drebrin-like (DBNL) through Ser269/Thr291-phospho-sensor motifs

PubMed Central

Park, Hae Ryon; Shi, Zhi; Li, Zenggang; Pham, Cau Dinh; Du, Yuhong; Khuri, Fadlo R.; Zhang, Youyi; Han, Qide

2014-01-01

HIP-55 (HPK1-interacting protein of 55 kDa, also named DBNL, SH3P7, and mAbp1) is a multidomain adaptor protein that is critical for organ development and the immune response. Here, we report the coupling of HIP-55 to cell growth control through its 14-3-3-binding phospho-Ser/Thr-sensor sites. Using affinity chromatography, we found HIP-55 formed a complex with 14-3-3 proteins, revealing a new node in phospho-Ser/Thr-mediated signaling networks. In addition, we demonstrated that HIP-55 is required for proper cell growth control. Enforced HIP-55 expression promoted proliferation, colony formation, migration, and invasion of lung cancer cells while silencing of HIP-55 reversed these effects. Importantly, HIP-55 was found to be upregulated in lung cancer cell lines and in tumor tissues of lung cancer patients. Upregulated HIP-55 was required to promote the growth of tumors in a xenograft animal model. However, tumors with S269A/T291A-mutated HIP-55, which ablates 14-3-3 binding, exhibited significantly reduced sizes, supporting a vital role of the HIP-55/14-3-3 protein interaction node in transmitting oncogenic signals. Mechanistically, HIP-55-mediated tumorigenesis activity appears to be in part mediated by antagonizing the tumor suppressor function of HPK1. Thus, the HIP-55–mediated oncogenic pathway, through S269/T291, may be exploited for the development of new therapeutic strategies. PMID:24912570

Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations.

PubMed

Chillemi, Giovanni; D'Annessa, Ilda; Fiorani, Paola; Losasso, Carmen; Benedetti, Piero; Desideri, Alessandro

2008-10-01

The role of Thr729 in modulating the enzymatic function of human topoisomerase I has been characterized by molecular dynamics (MD) simulation. In detail, the structural-dynamical behaviour of the Thr729Lys and the Thr729Pro mutants have been characterized because of their in vivo and in vitro functional properties evidenced in the accompanying paper. Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding. MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket. On the other hand, the Thr729Pro mutant maintains the wild-type structural scaffold, only increasing its rigidity. The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.

Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations

PubMed Central

Chillemi, Giovanni; D’Annessa, Ilda; Fiorani, Paola; Losasso, Carmen; Benedetti, Piero; Desideri, Alessandro

2008-01-01

The role of Thr729 in modulating the enzymatic function of human topoisomerase I has been characterized by molecular dynamics (MD) simulation. In detail, the structural–dynamical behaviour of the Thr729Lys and the Thr729Pro mutants have been characterized because of their in vivo and in vitro functional properties evidenced in the accompanying paper. Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding. MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket. On the other hand, the Thr729Pro mutant maintains the wild-type structural scaffold, only increasing its rigidity. The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant. PMID:18765473

Effect of Ala54Thr polymorphism of FABP2 on anthropometric and biochemical variables in response to a moderate-fat diet.

PubMed

Martinez-Lopez, Erika; Garcia-Garcia, Maritza R; Gonzalez-Avalos, Jorge M; Maldonado-Gonzalez, Montserrat; Ruiz-Madrigal, Bertha; Vizmanos, Barbara; Hernandez-Nazara, Zamira; Roman, Sonia; Panduro, Arturo

2013-01-01

To analyze the effect of the fatty acid-binding protein (FABP2) gene Ala54Thr polymorphism on anthropometric and biochemical variables in response to a moderate-fat diet in overweight or obese subjects. One hundred nine subjects with a body mass index ≥ 25 kg/m(2) were studied. Participants underwent a dietary intervention that consisted of 30% fat (saturated fat <7% of total calories), 15% protein, and 55% carbohydrates. The FABP2 genotypes were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Anthropometric and biochemical data were measured at baseline, 1 mo, and 2 mo of nutritional intervention. The mean age was 38.6 ± 11.3 y and the mean body mass index 32.7 ± 6.1 kg/m(2), with 20 men (18%) and 89 women (82%). Fifty-three patients (48.6%) had genotype Ala54Ala (wild-type group) and 56 patients had genotype Ala54Thr/Thr54Thr (51.4%, mutant group). At baseline, no significant difference was found between the FABP2 genotypes groups, except for the carbohydrate intake and resting metabolic rate, which were higher in the Ala54Thr/Thr54Thr group (P < 0.05). At 2 mo, participants had lost 6.8% of their initial weight. The Ala54Thr/Thr54Thr group compared with the Ala54Ala group showed significant decreases in the parameters of weight (-7.5 versus -4.2 kg), body mass index (-2.1 versus -1.2 kg/m(2)), waist circumference (-7.6 versus -5.2 cm), waist-to-hip ratio (-0.04 versus -0.02), and C-reactive protein (-1.4 versus -0.76 mg/L), respectively (P < 0.05). After the resting metabolic rate was adjusted, the decreases in waist circumference, waist-to-hip ratio, and C-reactive protein remained significant between the two groups. This study showed that the Thr54 allele carriers responded better to a moderate-fat diet. Copyright © 2013 Elsevier Inc. All rights reserved.

49 CFR 178.255-11 - Tank mountings.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Tank mountings. 178.255-11 Section 178.255-11... Portable Tanks § 178.255-11 Tank mountings. (a) Tanks shall be designed and fabricated with mountings to... requirement. (b) All tank mountings such as skids, fastenings, brackets, cradles, lifting lugs, etc., intended...

49 CFR 178.255-11 - Tank mountings.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Tank mountings. 178.255-11 Section 178.255-11... Portable Tanks § 178.255-11 Tank mountings. (a) Tanks shall be designed and fabricated with mountings to... requirement. (b) All tank mountings such as skids, fastenings, brackets, cradles, lifting lugs, etc., intended...

49 CFR 178.255-11 - Tank mountings.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Tank mountings. 178.255-11 Section 178.255-11... Portable Tanks § 178.255-11 Tank mountings. (a) Tanks shall be designed and fabricated with mountings to... requirement. (b) All tank mountings such as skids, fastenings, brackets, cradles, lifting lugs, etc., intended...

49 CFR 178.255-11 - Tank mountings.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tank mountings. 178.255-11 Section 178.255-11... Portable Tanks § 178.255-11 Tank mountings. (a) Tanks shall be designed and fabricated with mountings to... requirement. (b) All tank mountings such as skids, fastenings, brackets, cradles, lifting lugs, etc., intended...

49 CFR 178.814 - Hydrostatic pressure test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Hydrostatic pressure test. 178.814 Section 178.814... Testing of IBCs § 178.814 Hydrostatic pressure test. (a) General. The hydrostatic pressure test must be... preparation for the hydrostatic pressure test. For metal IBCs, the test must be carried out before the fitting...

49 CFR 178.605 - Hydrostatic pressure test.

Code of Federal Regulations, 2012 CFR

2012-10-01

... hydraulic pressure (gauge) applied, taken at the top of the receptacle, and determined by any one of the... 49 Transportation 3 2012-10-01 2012-10-01 false Hydrostatic pressure test. 178.605 Section 178.605... Packagings and Packages § 178.605 Hydrostatic pressure test. (a) General. The hydrostatic pressure test must...

49 CFR 178.605 - Hydrostatic pressure test.

Code of Federal Regulations, 2014 CFR

2014-10-01

... hydraulic pressure (gauge) applied, taken at the top of the receptacle, and determined by any one of the... 49 Transportation 3 2014-10-01 2014-10-01 false Hydrostatic pressure test. 178.605 Section 178.605... Packagings and Packages § 178.605 Hydrostatic pressure test. (a) General. The hydrostatic pressure test must...

21 CFR 178.3520 - Industrial starch-modified.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Industrial starch-modified. 178.3520 Section 178... § 178.3520 Industrial starch-modified. Industrial starch-modified may be safely used as a component of..., transporting, or holding food, subject to the provisions of this section. (a) Industrial starch-modified is...

49 CFR 178.33a-1 - Compliance.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Compliance. 178.33a-1 Section 178.33a-1 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33a-1 Compliance. (a) Required in all details. (b) [Reserved] [Order 71, 31 FR...

49 CFR 178.33b-1 - Compliance.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Compliance. 178.33b-1 Section 178.33b-1 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-1 Compliance. (a) Required in all details. (b) [Reserved] [74 FR 2268, Jan...

Residual Gas and Dust around Transition Objects and Weak T Tauri Stars

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

Doppmann, Greg W.; Najita, Joan R.; Carr, John S., E-mail: gdoppmann@keck.hawaii.edu, E-mail: najita@noao.edu, E-mail: carr@nrl.navy.mil

Residual gas in disks around young stars can spin down stars, circularize the orbits of terrestrial planets, and whisk away the dusty debris that is expected to serve as a signpost of terrestrial planet formation. We have carried out a sensitive search for residual gas and dust in the terrestrial planet region surrounding young stars ranging in age from a few to ∼10 Myr. Using high-resolution 4.7 μ m spectra of transition objects (TOs) and weak T Tauri stars, we searched for weak continuum excesses and CO fundamental emission, after making a careful correction for the stellar contribution to themore » observed spectrum. We find that the CO emission from TOs is weaker and located farther from the star than CO emission from nontransition T Tauri stars with similar stellar accretion rates. The difference is possibly the result of chemical and/or dynamical effects (i.e., a low CO abundance or close-in low-mass planets). The weak T Tauri stars show no CO fundamental emission down to low flux levels (5 × 10{sup −20} to 10{sup −18} W m{sup −2}). We illustrate how our results can be used to constrain the residual disk gas content in these systems and discuss their potential implications for star and planet formation.« less

21 CFR 178.3800 - Preservatives for wood.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Preservatives for wood. 178.3800 Section 178.3800... § 178.3800 Preservatives for wood. Preservatives may be safely used on wooden articles that are used or... protecting the wood from decay, mildew, and water absorption. (b) The substances permitted are as follows...

49 CFR 178.338-9 - Holding time.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Holding time. 178.338-9 Section 178.338-9... Specifications for Containers for Motor Vehicle Transportation § 178.338-9 Holding time. (a) “Holding time” is the time, as determined by testing, that will elapse from loading until the pressure of the contents...

49 CFR 178.255-12 - Pressure test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Pressure test. 178.255-12 Section 178.255-12... Portable Tanks § 178.255-12 Pressure test. (a) Each completed portable tank prior to application of lining... the test, and applying a pressure of 60 psig. The tank shall be capable of holding the prescribed...

49 CFR 178.347-4 - Pressure relief.

Code of Federal Regulations, 2010 CFR

2010-10-01

... adequate vapor and liquid capacity to limit the tank pressure to the cargo tank test pressure at maximum... 49 Transportation 2 2010-10-01 2010-10-01 false Pressure relief. 178.347-4 Section 178.347-4... Specifications for Containers for Motor Vehicle Transportation § 178.347-4 Pressure relief. (a) Each cargo tank...

The Staphylococcus aureus Autoinducer-2 Synthase LuxS Is Regulated by Ser/Thr Phosphorylation▿

PubMed Central

Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J.; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

2010-01-01

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis. PMID:20870760

T-REX Version 1.5 User's Guide for Calculating Pesticide Residues on Avian and Mammalian Food Items - Appendix B - Initial Pesticide Residues on Arthropods

EPA Pesticide Factsheets

Obtain empirical data from the scientific literature and registrant-submitted studies to refine the initial residue assumptions of pesticides on arthropods that are simulated with TIM, T-REX, and T-HERPS

49 CFR 178.345-11 - Tank outlets.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Tank outlets. 178.345-11 Section 178.345-11... Containers for Motor Vehicle Transportation § 178.345-11 Tank outlets. (a) General. As used in this section, “loading/unloading outlet” means any opening in the cargo tank wall used for loading or unloading of lading...

49 CFR 178.345-11 - Tank outlets.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tank outlets. 178.345-11 Section 178.345-11... Containers for Motor Vehicle Transportation § 178.345-11 Tank outlets. (a) General. As used in this section, “loading/unloading outlet” means any opening in the cargo tank wall used for loading or unloading of lading...

49 CFR 178.345-11 - Tank outlets.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Tank outlets. 178.345-11 Section 178.345-11... Containers for Motor Vehicle Transportation § 178.345-11 Tank outlets. (a) General. As used in this section, “loading/unloading outlet” means any opening in the cargo tank wall used for loading or unloading of lading...

49 CFR 178.345-11 - Tank outlets.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Tank outlets. 178.345-11 Section 178.345-11... Containers for Motor Vehicle Transportation § 178.345-11 Tank outlets. (a) General. As used in this section, “loading/unloading outlet” means any opening in the cargo tank wall used for loading or unloading of lading...

49 CFR 178.255-11 - Tank mountings.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Tank mountings. 178.255-11 Section 178.255-11... Specifications for Portable Tanks § 178.255-11 Tank mountings. (a) Tanks shall be designed and fabricated with mountings to provide a secure base in transit. “Skids” or similar devices shall be deemed to comply with...

49 CFR 178.338-9 - Holding time.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Holding time. 178.338-9 Section 178.338-9... Containers for Motor Vehicle Transportation § 178.338-9 Holding time. (a) “Holding time” is the time, as...) Holding time test. (1) The test to determine holding time must be performed by charging the tank with a...

49 CFR 178.338-9 - Holding time.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Holding time. 178.338-9 Section 178.338-9... Containers for Motor Vehicle Transportation § 178.338-9 Holding time. (a) “Holding time” is the time, as...) Holding time test. (1) The test to determine holding time must be performed by charging the tank with a...

49 CFR 178.338-9 - Holding time.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Holding time. 178.338-9 Section 178.338-9... Containers for Motor Vehicle Transportation § 178.338-9 Holding time. (a) “Holding time” is the time, as...) Holding time test. (1) The test to determine holding time must be performed by charging the tank with a...

49 CFR 178.346-4 - Outlets.

Code of Federal Regulations, 2010 CFR

2010-10-01

... must conform to § 178.345-11 and this section. (b) External self-closing stop-valves are not authorized as an alternative to internal self-closing stop-valves on loading/unloading outlets. [Amdt. 178-89...

Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia

PubMed Central

Atak, Sinem; Langlhofer, Georg; Schaefer, Natascha; Kessler, Denise; Meiselbach, Heike; Delto, Carolyn; Schindelin, Hermann; Villmann, Carmen

2015-01-01

Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. PMID:26733802

Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

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

McCormick, D.J.; Griesmann, G.E.; Huang, Z.

1986-03-05

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodiesmore » to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.« less

49 CFR 178.33a-8 - Tests.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Tests. 178.33a-8 Section 178.33a-8 Transportation... Inside Containers, and Linings § 178.33a-8 Tests. (a) One out of each lot of 25,000 containers or less... less, successively produced per day, shall constitute a lot and if the test container shall fail, the...

49 CFR 178.33a-8 - Tests.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Tests. 178.33a-8 Section 178.33a-8 Transportation... Containers, and Linings § 178.33a-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.33a-8 - Tests.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Tests. 178.33a-8 Section 178.33a-8 Transportation... Containers, and Linings § 178.33a-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.33a-8 - Tests.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Tests. 178.33a-8 Section 178.33a-8 Transportation... Containers, and Linings § 178.33a-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.33a-8 - Tests.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tests. 178.33a-8 Section 178.33a-8 Transportation... Containers, and Linings § 178.33a-8 Tests. (a) One out of each lot of 25,000 containers or less, successively..., successively produced per day, shall constitute a lot and if the test container shall fail, the lot shall be...

49 CFR 178.338-2 - Material.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Material. 178.338-2 Section 178.338-2 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS FOR PACKAGINGS...

49 CFR 178.255-2 - Material.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Material. 178.255-2 Section 178.255-2 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS FOR PACKAGINGS...

49 CFR 178.33-5 - Material.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Material. 178.33-5 Section 178.33-5 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS FOR PACKAGINGS Specifications for...

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase

PubMed Central

Webb, Michael E.; Yorke, Briony A.; Kershaw, Tom; Lovelock, Sarah; Lobley, Carina M. C.; Kilkenny, Mairi L.; Smith, Alison G.; Blundell, Tom L.; Pearson, Arwen R.; Abell, Chris

2014-01-01

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation. PMID:24699660

46 CFR 178.210 - Stability information.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false Stability information. 178.210 Section 178.210 Shipping... information. (a) Stability information (stability details indicated on the Certificate of Inspection, a... this section. Enough stability information, including stability calculations and assumptions made to...

46 CFR 178.210 - Stability information.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Stability information. 178.210 Section 178.210 Shipping... information. (a) Stability information (stability details indicated on the Certificate of Inspection, a... this section. Enough stability information, including stability calculations and assumptions made to...

All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

PubMed Central

Xu, Wu; Amire-Brahimi, Benjamin; Xie, Xiao-Jun; Huang, Liying; Ji, Jun-Yuan

2014-01-01

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate

Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities

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

Tommaso, Anne di; Hagen, Jussara; Tompkins, Van

2009-04-15

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala{sup 14} and Thr{sup 31}) were found to destabilize the protein while two others (Val{sup 24} and Ala{sup 41}) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significantmore » biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val{sup 24} was required for p53-independent growth suppression whereas multiple residues (Val{sup 24}, Thr{sup 31}, Ala{sup 41} and His{sup 60}) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.« less

49 CFR 178.358-6 - Typical assembly detail.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Typical assembly detail. 178.358-6 Section 178.358-6 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS... PACKAGINGS Specifications for Packagings for Class 7 (Radioactive) Materials § 178.358-6 Typical assembly...

49 CFR 178.356-5 - Typical assembly detail.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Typical assembly detail. 178.356-5 Section 178.356-5 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS... PACKAGINGS Specifications for Packagings for Class 7 (Radioactive) Materials § 178.356-5 Typical assembly...

49 CFR 178.510 - Standards for wooden barrels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Standards for wooden barrels. 178.510 Section 178.510 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS...-bulk Performance-Oriented Packaging Standards § 178.510 Standards for wooden barrels. (a) The following...

49 CFR 178.510 - Standards for wooden barrels.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for wooden barrels. 178.510 Section 178.510 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.510 Standards for wooden barrels. (a) The...

Mechanism of autophosphorylation of mycobacterial PknB explored by molecular dynamics simulations.

PubMed

Damle, Nikhil P; Mohanty, Debasisa

2014-07-22

Mycobacterial Ser/Thr kinase, PknB, is essential for the growth of the pathogen. Unphosphorylated PknB is catalytically inactive, and its activation requires autophosphorylation of Thr residues on the activation loop. Autophosphorylation can in principle take place via two distinct mechanisms. Intermolecular trans autophosphorylation involves dimerization and phosphorylation of the activation loop of one chain in the catalytic pocket of the other chain. On the other hand, intramolecular cis autophosphorylation involves phosphorylation of the activation loop of the kinases in its own catalytic pocket within a monomer. On the basis of the crystal structure of PknB in the front-to-front dimeric form, it is currently believed that activation of PknB involves trans autophosphorylation. However, because of the lack of coordinates of the activation loop in the crystal structures, atomic details of the conformational changes associated with activation are yet to be deciphered. Therefore, to understand the conformational transitions associated with activation via autophosphorylation, a series of explicit solvent molecular dynamics simulations with a duration of 1 μs have been performed on each of the phosphorylated and nonphosphorylated forms of the PknB catalytic domain in monomeric and dimeric states. Simulations on phosphorylated PknB revealed a differential network of crucial electrostatic and hydrophobic residues that stabilize the phosphorylated form in the active conformation. Interestingly, in our simulations on nonphosphorylated monomers, the activation loop was observed to fold into its own active site, thereby opening the novel possibility of activation through intramolecular cis autophosphorylation. Thus, our simulations suggest that autophosphorylation of PknB might also involve cis initiation followed by trans amplification as reported for other eukaryotic kinases based on recent reaction kinetics studies.

49 CFR 178.356-5 - Typical assembly detail.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Typical assembly detail. 178.356-5 Section 178.356-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.356-5 Typical assembly detail. (a...

49 CFR 178.358-6 - Typical assembly detail.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Typical assembly detail. 178.358-6 Section 178.358-6 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.358-6 Typical assembly detail. (a...

49 CFR 178.356-5 - Typical assembly detail.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Typical assembly detail. 178.356-5 Section 178.356-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.356-5 Typical assembly detail. (a...

49 CFR 178.356-5 - Typical assembly detail.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Typical assembly detail. 178.356-5 Section 178.356-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.356-5 Typical assembly detail. (a...

49 CFR 178.358-6 - Typical assembly detail.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Typical assembly detail. 178.358-6 Section 178.358-6 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.358-6 Typical assembly detail. (a...

49 CFR 178.358-6 - Typical assembly detail.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Typical assembly detail. 178.358-6 Section 178.358-6 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS... Specifications for Packagings for Class 7 (Radioactive) Materials § 178.358-6 Typical assembly detail. (a...

7 CFR 800.178 - Summary revocation of licenses.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Summary revocation of licenses. 800.178 Section 800.178 Agriculture Regulations of the Department of Agriculture (Continued) GRAIN INSPECTION, PACKERS AND... Licenses and Authorizations (for Individuals Only) § 800.178 Summary revocation of licenses. Licenses may...

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

PubMed Central

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

2006-01-01

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors. PMID:16477027

The m1A(58) modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI.

PubMed

Dégut, Clément; Ponchon, Luc; Folly-Klan, Marcia; Barraud, Pierre; Tisné, Carine

2016-03-01

The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of Iron-Binding Ability Between Thr70-NapA and Ser70-NapA of Helicobacter pylori.

PubMed

Shan, Weiran; Kung, Hsiang-Fu; Ge, Ruiguang

2016-06-01

The neutrophil-activating protein (NapA) of Helicobacter pylori (H. pylori), with DNA-binding and iron seizing properties, is a fundamental virulence factor involved in H. pylori-related diseases. Compared with Ser70-NapA strain, Thr70-NapA strain is more intimately correlated with iron-deficiency anemia. To investigate whether two types of proteins differ in iron-binding ability, mutated Thr70-NapA and Ser70-NapA strains were established. Isothermal titration calorimetry (ITC) method was conducted to measure the binding between the NapA protein and Fe(2+) . The structural changes of NapA protein were also tested during iron interaction by fast protein liquid chromatography (FPLC) and circular dichroism (CD) methods. DNA-binding assay was performed for evaluate the affinity of both mutated and wild types of NapA with DNA. Mutated Thr70-NapA had higher iron-binding ability than wild Ser70-NapA. The structural stability of Thr70-NapA was disrupted and became more active along with the rising concentration of Fe(2+) , whereas no similar association was observed between Ser70-NapA and Fe(2+) level. When the iron/protein molar ratio ranged from 10 to 20, both Ser70-NapA and Thr70-NapA displayed weaker DNA-binding ability. Thr70-NapA has much stronger ability to sequester ferrous ion compared with Ser70-NapA in H. pylori. In addition, the DNA-binding property of NapA is dependent upon the Fe(2+) concentration. © 2015 John Wiley & Sons Ltd.

7 CFR 28.178 - Submission of cotton samples.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Submission of cotton samples. 28.178 Section 28.178... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Classification for Foreign Growth Cotton § 28.178 Submission of cotton samples. Samples of cotton submitted to a Classing Office for classification and/or...

7 CFR 28.178 - Submission of cotton samples.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Submission of cotton samples. 28.178 Section 28.178... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Classification for Foreign Growth Cotton § 28.178 Submission of cotton samples. Samples of cotton submitted to a Classing Office for classification and/or...

7 CFR 28.178 - Submission of cotton samples.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Submission of cotton samples. 28.178 Section 28.178... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Classification for Foreign Growth Cotton § 28.178 Submission of cotton samples. Samples of cotton submitted to a Classing Office for classification and/or...

7 CFR 28.178 - Submission of cotton samples.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Submission of cotton samples. 28.178 Section 28.178... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Classification for Foreign Growth Cotton § 28.178 Submission of cotton samples. Samples of cotton submitted to a Classing Office for classification and/or...

7 CFR 28.178 - Submission of cotton samples.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Submission of cotton samples. 28.178 Section 28.178... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Classification for Foreign Growth Cotton § 28.178 Submission of cotton samples. Samples of cotton submitted to a Classing Office for classification and/or...

46 CFR 178.420 - Drainage of cockpit vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Drainage of cockpit vessels. 178.420 Section 178.420 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.420 Drainage of cockpit...

46 CFR 178.420 - Drainage of cockpit vessels.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 7 2012-10-01 2012-10-01 false Drainage of cockpit vessels. 178.420 Section 178.420 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.420 Drainage of cockpit...

46 CFR 178.420 - Drainage of cockpit vessels.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Drainage of cockpit vessels. 178.420 Section 178.420 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.420 Drainage of cockpit...

46 CFR 178.420 - Drainage of cockpit vessels.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Drainage of cockpit vessels. 178.420 Section 178.420 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.420 Drainage of cockpit...

46 CFR 178.420 - Drainage of cockpit vessels.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false Drainage of cockpit vessels. 178.420 Section 178.420 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.420 Drainage of cockpit...

49 CFR 178.813 - Leakproofness test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Leakproofness test. 178.813 Section 178.813... Leakproofness test. (a) General. The leakproofness test must be conducted for the qualification of all IBC... pressure or intended to contain liquids. (b) Special preparation for the leakproofness test. Vented...

50 CFR 622.178 - At-sea observer coverage.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false At-sea observer coverage. 622.178 Section 622.178 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC...-Grouper Fishery of the South Atlantic Region § 622.178 At-sea observer coverage. (a) Required coverage. (1...

50 CFR 622.178 - At-sea observer coverage.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false At-sea observer coverage. 622.178 Section 622.178 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC...-Grouper Fishery of the South Atlantic Region § 622.178 At-sea observer coverage. (a) Required coverage. (1...

46 CFR 178.440 - Drainage of open boats.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 7 2012-10-01 2012-10-01 false Drainage of open boats. 178.440 Section 178.440 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.440 Drainage of open boats. The deck...

46 CFR 178.440 - Drainage of open boats.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Drainage of open boats. 178.440 Section 178.440 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.440 Drainage of open boats. The deck...

46 CFR 178.440 - Drainage of open boats.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Drainage of open boats. 178.440 Section 178.440 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.440 Drainage of open boats. The deck...

46 CFR 178.440 - Drainage of open boats.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Drainage of open boats. 178.440 Section 178.440 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.440 Drainage of open boats. The deck...

46 CFR 178.440 - Drainage of open boats.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false Drainage of open boats. 178.440 Section 178.440 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.440 Drainage of open boats. The deck...

49 CFR 178.707 - Standards for composite IBCs.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for composite IBCs. 178.707 Section 178... PACKAGINGS IBC Performance-Oriented Standards § 178.707 Standards for composite IBCs. (a) The provisions in this section apply to composite IBCs intended to contain solids and liquids. To complete the marking...

21 CFR 178.3930 - Terpene resins.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Terpene resins. 178.3930 Section 178.3930 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND SANITIZERS Certain Adjuvants...

21 CFR 178.3930 - Terpene resins.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Terpene resins. 178.3930 Section 178.3930 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND SANITIZERS Certain Adjuvants...

21 CFR 178.3710 - Petroleum wax.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Petroleum wax. 178.3710 Section 178.3710 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND SANITIZERS Certain Adjuvants...

21 CFR 178.3710 - Petroleum wax.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Petroleum wax. 178.3710 Section 178.3710 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND SANITIZERS Certain Adjuvants...

RCD+: Fast loop modeling server.

PubMed

López-Blanco, José Ramón; Canosa-Valls, Alejandro Jesús; Li, Yaohang; Chacón, Pablo

2016-07-08

Modeling loops is a critical and challenging step in protein modeling and prediction. We have developed a quick online service (http://rcd.chaconlab.org) for ab initio loop modeling combining a coarse-grained conformational search with a full-atom refinement. Our original Random Coordinate Descent (RCD) loop closure algorithm has been greatly improved to enrich the sampling distribution towards near-native conformations. These improvements include a new workflow optimization, MPI-parallelization and fast backbone angle sampling based on neighbor-dependent Ramachandran probability distributions. The server starts by efficiently searching the vast conformational space from only the loop sequence information and the environment atomic coordinates. The generated closed loop models are subsequently ranked using a fast distance-orientation dependent energy filter. Top ranked loops are refined with the Rosetta energy function to obtain accurate all-atom predictions that can be interactively inspected in an user-friendly web interface. Using standard benchmarks, the average root mean squared deviation (RMSD) is 0.8 and 1.4 Å for 8 and 12 residues loops, respectively, in the challenging modeling scenario in where the side chains of the loop environment are fully remodeled. These results are not only very competitive compared to those obtained with public state of the art methods, but also they are obtained ∼10-fold faster. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

49 CFR 178.255-13 - Repair of tanks.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Repair of tanks. 178.255-13 Section 178.255-13 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Portable Tanks § 178.255-13 Repair of tanks. (a) Tanks failing to meet the test may be repaired and...

49 CFR 178.505 - Standards for aluminum drums.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for aluminum drums. 178.505 Section 178... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.505 Standards for aluminum drums. (a) The following are the identification codes for aluminum drums: (1) 1B1 for a non-removable head aluminum drum...

49 CFR 178.338-16 - Inspection and testing.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Inspection and testing. 178.338-16 Section 178.338... PACKAGINGS Specifications for Containers for Motor Vehicle Transportation § 178.338-16 Inspection and testing... Section VIII of the ASME Code. The welder and the welding procedure must be qualified in accordance with...

49 CFR 178.33b-8 - Production tests.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Production tests. 178.33b-8 Section 178.33b-8 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... Specifications for Inside Containers, and Linings § 178.33b-8 Production tests. (a) Burst Testing. (1) One out of...

49 CFR 178.337-10 - Accident damage protection.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Accident damage protection. 178.337-10 Section 178... Specifications for Containers for Motor Vehicle Transportation § 178.337-10 Accident damage protection. (a) All... sacrificial device must be located in the piping system outboard of the stop valve and within the accident...

Structure of the MazF-mt9 toxin, a tRNA-specific endonuclease from Mycobacterium tuberculosis.

PubMed

Chen, Ran; Tu, Jie; Liu, Zhihui; Meng, Fanrong; Ma, Pinyun; Ding, Zhishan; Yang, Chengwen; Chen, Lei; Deng, Xiangyu; Xie, Wei

2017-05-06

Tuberculosis (TB) is a severe disease caused by Mycobacterium tuberculosis (M. tb) and the well-characterized M. tb MazE/F proteins play important roles in stress adaptation. Recently, the MazF-mt9 toxin has been found to display endonuclease activities towards tRNAs but the mechanism is unknown. We hereby present the crystal structure of apo-MazF-mt9. The enzyme recognizes tRNA Lys with a central UUU motif within the anticodon loop, but is insensitive to the sequence context outside of the loop. Based on our crystallographic and biochemical studies, we identified key residues for catalysis and proposed the potential tRNA-binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

LoopX: A Graphical User Interface-Based Database for Comprehensive Analysis and Comparative Evaluation of Loops from Protein Structures.

PubMed

Kadumuri, Rajashekar Varma; Vadrevu, Ramakrishna

2017-10-01

Due to their crucial role in function, folding, and stability, protein loops are being targeted for grafting/designing to create novel or alter existing functionality and improve stability and foldability. With a view to facilitate a thorough analysis and effectual search options for extracting and comparing loops for sequence and structural compatibility, we developed, LoopX a comprehensively compiled library of sequence and conformational features of ∼700,000 loops from protein structures. The database equipped with a graphical user interface is empowered with diverse query tools and search algorithms, with various rendering options to visualize the sequence- and structural-level information along with hydrogen bonding patterns, backbone φ, ψ dihedral angles of both the target and candidate loops. Two new features (i) conservation of the polar/nonpolar environment and (ii) conservation of sequence and conformation of specific residues within the loops have also been incorporated in the search and retrieval of compatible loops for a chosen target loop. Thus, the LoopX server not only serves as a database and visualization tool for sequence and structural analysis of protein loops but also aids in extracting and comparing candidate loops for a given target loop based on user-defined search options.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic investigations of a unique editing domain from archaebacteria.

PubMed

Dwivedi, Shweta; Kruparani, Shobha P; Sankaranarayanan, Rajan

2004-09-01

Threonyl-tRNA synthetase (ThrRS) faces a crucial double-discrimination problem during the translation of genetic code. Most ThrRSs from the archaeal kingdom possess a unique editing domain that differs from those of eubacteria and eukaryotes. In order to understand the structural basis of the editing mechanism in archaea, the editing module of ThrRS from Pyrococcus abyssi comprising of the first 183 amino-acid residues was cloned, expressed, purified and crystallized. The crystals belong to the trigonal space group P3(1(2))21, with one molecule in the asymmetric unit.

49 CFR 178.819 - Vibration test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Vibration test. 178.819 Section 178.819... Vibration test. (a) General. The vibration test must be conducted for the qualification of all rigid IBC design types. Flexible IBC design types must be capable of withstanding the vibration test. (b) Test...

49 CFR 178.819 - Vibration test.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Vibration test. 178.819 Section 178.819... Vibration test. (a) General. The vibration test must be conducted for the qualification of all rigid IBC design types. Flexible IBC design types must be capable of withstanding the vibration test. (b) Test...

49 CFR 178.819 - Vibration test.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Vibration test. 178.819 Section 178.819... Vibration test. (a) General. The vibration test must be conducted for the qualification of all rigid IBC design types. Flexible IBC design types must be capable of withstanding the vibration test. (b) Test...

49 CFR 178.819 - Vibration test.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Vibration test. 178.819 Section 178.819... Vibration test. (a) General. The vibration test must be conducted for the qualification of all rigid IBC design types. Flexible IBC design types must be capable of withstanding the vibration test. (b) Test...

Communication between the N and C Termini Is Required for Copper-stimulated Ser/Thr Phosphorylation of Cu(I)-ATPase (ATP7B)*

PubMed Central

Braiterman, Lelita T.; Gupta, Arnab; Chaerkady, Raghothama; Cole, Robert N.; Hubbard, Ann L.

2015-01-01

The Wilson disease protein ATP7B exhibits copper-dependent trafficking. In high copper, ATP7B exits the trans-Golgi network and moves to the apical domain of hepatocytes where it facilitates elimination of excess copper through the bile. Copper levels also affect ATP7B phosphorylation. ATP7B is basally phosphorylated in low copper and becomes more phosphorylated (“hyperphosphorylated”) in elevated copper. The functional significance of hyperphosphorylation remains unclear. We showed that hyperphosphorylation occurs even when ATP7B is restricted to the trans-Golgi network. We performed comprehensive phosphoproteomics of ATP7B in low versus high copper, which revealed that 24 Ser/Thr residues in ATP7B could be phosphorylated, and only four of these were copper-responsive. Most of the phosphorylated sites were found in the N- and C-terminal cytoplasmic domains. Using truncation and mutagenesis, we showed that inactivation or elimination of all six N-terminal metal binding domains did not block copper-dependent, reversible, apical trafficking but did block hyperphosphorylation in hepatic cells. We showed that nine of 15 Ser/Thr residues in the C-terminal domain were phosphorylated. Inactivation of 13 C-terminal phosphorylation sites reduced basal phosphorylation and eliminated hyperphosphorylation, suggesting that copper binding at the N terminus propagates to the ATP7B C-terminal region. C-terminal mutants with either inactivating or phosphomimetic substitutions showed little effect upon copper-stimulated trafficking, indicating that trafficking does not depend on phosphorylation at these sites. Thus, our studies revealed that copper-dependent conformational changes in the N-terminal region lead to hyperphosphorylation at C-terminal sites, which seem not to affect trafficking and may instead fine-tune copper sequestration. PMID:25666620

49 CFR 178.517 - Standards for plastic boxes.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Standards for plastic boxes. 178.517 Section 178...-bulk Performance-Oriented Packaging Standards § 178.517 Standards for plastic boxes. (a) The following are identification codes for plastic boxes: (1) 4H1 for an expanded plastic box; and (2) 4H2 for a...

49 CFR 178.338-16 - Inspection and testing.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Specifications for Containers for Motor Vehicle Transportation § 178.338-16 Inspection and testing. (a) General... of the test pressure and held for a sufficient time to permit inspection of the cargo tank for leaks... 49 Transportation 3 2012-10-01 2012-10-01 false Inspection and testing. 178.338-16 Section 178.338...

49 CFR 178.338-16 - Inspection and testing.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Specifications for Containers for Motor Vehicle Transportation § 178.338-16 Inspection and testing. (a) General... of the test pressure and held for a sufficient time to permit inspection of the cargo tank for leaks... 49 Transportation 3 2011-10-01 2011-10-01 false Inspection and testing. 178.338-16 Section 178.338...

49 CFR 178.338-16 - Inspection and testing.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Specifications for Containers for Motor Vehicle Transportation § 178.338-16 Inspection and testing. (a) General... of the test pressure and held for a sufficient time to permit inspection of the cargo tank for leaks... 49 Transportation 3 2013-10-01 2013-10-01 false Inspection and testing. 178.338-16 Section 178.338...

49 CFR 178.517 - Standards for plastic boxes.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Standards for plastic boxes. 178.517 Section 178...-bulk Performance-Oriented Packaging Standards § 178.517 Standards for plastic boxes. (a) The following are identification codes for plastic boxes: (1) 4H1 for an expanded plastic box; and (2) 4H2 for a...

49 CFR 178.517 - Standards for plastic boxes.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Standards for plastic boxes. 178.517 Section 178...-bulk Performance-Oriented Packaging Standards § 178.517 Standards for plastic boxes. (a) The following are identification codes for plastic boxes: (1) 4H1 for an expanded plastic box; and (2) 4H2 for a...

49 CFR 178.517 - Standards for plastic boxes.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Standards for plastic boxes. 178.517 Section 178...-bulk Performance-Oriented Packaging Standards § 178.517 Standards for plastic boxes. (a) The following are identification codes for plastic boxes: (1) 4H1 for an expanded plastic box; and (2) 4H2 for a...

XRCC3 Thr241Met Polymorphism is not Associated with Lung Cancer Risk in a Romanian Population.

PubMed

Catana, Andreea; Pop, Monica; Marginean, Dragos Horea; Blaga, Ioana Cristina; Porojan, Mihai Dumitru; Popp, Radu Anghel; Pop, Ioan Victor

2016-01-01

Deoxyribonucleic Acid (DNA) repair mechanisms play a critical role in protecting the cellular genome against carcinogens. X-ray cross-complementing gene 3 (XRCC3) is involved in DNA repair and therefore certain genetic polymorphisms that occur in DNA repair genes may affect the ability to repair DNA defects and may represent a risk factor in carcinogenesis. The purpose of our study was to investigate the association between XRCC3 gene substitution of Threonine with Methionine in codon 241 of XRCC3 gene (Thr241Met) polymorphism and the risk of lung cancer, in a Romanian population. We recruited 93 healthy controls and 85 patients with lung cancer, all smokers. Thr241Met, XRCC3 gene genotyping was determined by multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis (OR, recessive model), did not revealed an increased risk for lung cancer, for the variant 241Met allele and Thr241Met genotypes (p=0.138, OR=0.634, CI=0.348-1.157; p=0.023, OR=0.257, CI=0.085-6.824). Also, there were no positive statistical associations between Thr241Met polymorphism of XRCC3 gene, gender, tobacco and various histopathological tumor type of lung cancer. In conclusion, the results of the study suggest that the XRCC3 gene Thr241Met polymorphism is not associated with an increased risk for the development of lung cancer in this Romanian group.

The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel.

PubMed

Minor, D L; Lin, Y F; Mobley, B C; Avelar, A; Jan, Y N; Jan, L Y; Berger, J M

2000-09-01

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.

Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

PubMed Central

Ben-Shimon, Avraham; Niv, Masha Y.

2011-01-01

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions. PMID:22125489

49 CFR 178.33b-8 - Production tests.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Production tests. 178.33b-8 Section 178.33b-8... Containers, and Linings § 178.33b-8 Production tests. (a) Burst Testing. (1) One out of each lot of 5,000...,000 containers or less, successively produced per day, shall constitute a lot and if the test...

49 CFR 178.517 - Standards for plastic boxes.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for plastic boxes. 178.517 Section 178... PACKAGINGS Non-bulk Performance-Oriented Packaging Standards § 178.517 Standards for plastic boxes. (a) The following are identification codes for plastic boxes: (1) 4H1 for an expanded plastic box; and (2) 4H2 for a...

49 CFR 178.504 - Standards for steel drums.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for steel drums. 178.504 Section 178.504...-bulk Performance-Oriented Packaging Standards § 178.504 Standards for steel drums. (a) The following are identification codes for steel drums: (1) 1A1 for a non-removable head steel drum; and (2) 1A2 for...

49 CFR 178.504 - Standards for steel drums.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Standards for steel drums. 178.504 Section 178.504...-Oriented Packaging Standards § 178.504 Standards for steel drums. (a) The following are identification codes for steel drums: (1) 1A1 for a non-removable head steel drum; and (2) 1A2 for a removable head...

49 CFR 178.504 - Standards for steel drums.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Standards for steel drums. 178.504 Section 178.504...-Oriented Packaging Standards § 178.504 Standards for steel drums. (a) The following are identification codes for steel drums: (1) 1A1 for a non-removable head steel drum; and (2) 1A2 for a removable head...

49 CFR 178.504 - Standards for steel drums.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Standards for steel drums. 178.504 Section 178.504...-Oriented Packaging Standards § 178.504 Standards for steel drums. (a) The following are identification codes for steel drums: (1) 1A1 for a non-removable head steel drum; and (2) 1A2 for a removable head...

49 CFR 178.504 - Standards for steel drums.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Standards for steel drums. 178.504 Section 178.504...-Oriented Packaging Standards § 178.504 Standards for steel drums. (a) The following are identification codes for steel drums: (1) 1A1 for a non-removable head steel drum; and (2) 1A2 for a removable head...

49 CFR 178.817 - Righting test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Righting test. 178.817 Section 178.817... Righting test. (a) General. The righting test must be conducted for the qualification of all flexible IBCs designed to be lifted from the top or side. (b) Special preparation for the righting test. The flexible IBC...

49 CFR 178.810 - Drop test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Drop test. 178.810 Section 178.810 Transportation... Drop test. (a) General. The drop test must be conducted for the qualification of all IBC design types... the drop test. (1) Metal, rigid plastic, and composite IBCs intended to contain solids must be filled...

21 CFR 178.1010 - Sanitizing solutions.

Code of Federal Regulations, 2014 CFR

2014-04-01

...-terpineol and alpha[p-(1,1,3,3-tetramethylbutyl)phenyl] -omega-hydroxy-poly (oxyethylene) produced with one... parts per million of alpha[p-(1,1,3,3-tetramethylbutyl) phenyl]-omega-hydroxypoly (oxyethylene). (14... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sanitizing solutions. 178.1010 Section 178.1010...

21 CFR 178.3120 - Animal glue.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Animal glue. 178.3120 Section 178.3120 Food and... Animal glue. Animal glue may be safely used as a component of articles intended for use in producing... the provisions of this section. (a) Animal glue consists of the proteinaceous extractives obtained...

42 CFR 415.178 - Anesthesia services.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 3 2014-10-01 2014-10-01 false Anesthesia services. 415.178 Section 415.178 Public... Anesthesia services. (a) General rule. (1) For services furnished prior to January 1, 2010, an unreduced physician fee schedule payment may be made if a physician is involved in a single anesthesia procedure...

42 CFR 415.178 - Anesthesia services.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 3 2013-10-01 2013-10-01 false Anesthesia services. 415.178 Section 415.178 Public... Anesthesia services. (a) General rule. (1) For services furnished prior to January 1, 2010, an unreduced physician fee schedule payment may be made if a physician is involved in a single anesthesia procedure...

42 CFR 415.178 - Anesthesia services.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 3 2012-10-01 2012-10-01 false Anesthesia services. 415.178 Section 415.178 Public... Anesthesia services. (a) General rule. (1) For services furnished prior to January 1, 2010, an unreduced physician fee schedule payment may be made if a physician is involved in a single anesthesia procedure...

Protein Phosphatase 1-α Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle.

PubMed

Sharma, Pragya; Arias, Edward B; Cartee, Gregory D

2016-09-01

Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) and Ser(588) by Akt is essential for insulin's full effect on glucose transport. However, protein phosphorylation is determined by the balance of actions by kinases and phosphatases, and the specific phosphatase(s) controlling AS160 dephosphorylation is (are) unknown. Accordingly, we assessed roles of highly expressed skeletal muscle serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used an AS160 dephosphorylation assay. Lysates from insulin-stimulated skeletal muscle were treated with pharmacological phosphatase inhibitors and assessed for AS160 Ser(588) and Thr(642) dephosphorylation. AS160 dephosphorylation on both phosphorylation sites was unaltered by PP2B or PP2C inhibitors. Okadaic acid (low dose inhibits PP2A; high dose inhibits PP1) delayed AS160 Ser(588) (both doses) and Thr(642) (high dose only) dephosphorylation concomitant with greater Akt phosphorylation (both doses). AS160 was coimmunoprecipitated with PP1-α but not with PP1-β, PP1-γ1, or PP2A. Recombinant inhibitor-2 protein (a selective PP1 inhibitor) delayed AS160 dephosphorylation on both phosphorylation sites without altering Akt phosphorylation. Furthermore, knockdown of PP1-α but not PP1-β or PP1-γ1 by small interfering RNA caused greater AS160 Ser(588) and Thr(642) phosphorylation concomitant with unaltered Akt phosphorylation. Together, these results identified PP1-α as a regulator of AS160 Thr(642) and Ser(588) dephosphorylation in skeletal muscle. © 2016 by the American Diabetes Association.

49 CFR 178.818 - Tear test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Tear test. 178.818 Section 178.818 Transportation... Tear test. (a) General. The tear test must be conducted for the qualification of all flexible IBC design types. (b) Special preparation for the tear test. The flexible IBC must be filled to not less than...

21 CFR 178.3860 - Release agents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... bran wax For use only in plastics intended for contact with dry foods identified as Type VIII in table... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Release agents. 178.3860 Section 178.3860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN...

21 CFR 178.3860 - Release agents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... bran wax For use only in plastics intended for contact with dry foods identified as Type VIII in table... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Release agents. 178.3860 Section 178.3860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN...

21 CFR 178.3860 - Release agents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... centimeter (1.29 milligrams per square inch) of backing. Rice bran wax For use only in plastics intended for... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Release agents. 178.3860 Section 178.3860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) INDIRECT FOOD...

49 CFR 178.345-6 - Supports and anchoring.

Code of Federal Regulations, 2010 CFR

2010-10-01

... support elements must include the stresses indicated in § 178.345-3(b) and as generated by the loads... in lieu of a frame must be supported in such a manner that the resulting stress levels in the cargo... must include the stresses indicated in § 178.345-3(b) and as generated by the loads described in § 178...

49 CFR 178.521 - Standards for paper bags.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Standards for paper bags. 178.521 Section 178.521...-Oriented Packaging Standards § 178.521 Standards for paper bags. (a) The following are identification codes for paper bags: (1) 5M1 for a multi-wall paper bag; and (2) 5M2 for a multi-wall water-resistant paper...

49 CFR 178.521 - Standards for paper bags.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for paper bags. 178.521 Section 178.521...-bulk Performance-Oriented Packaging Standards § 178.521 Standards for paper bags. (a) The following are identification codes for paper bags: (1) 5M1 for a multi-wall paper bag; and (2) 5M2 for a multi-wall water...

Plasma urotensin-2 level and Thr21Met but not Ser89Asn polymorphisms of the urotensin-2 gene are associated with migraines.

PubMed

Geyik, Sırma; Ergun, Sercan; Kuzudişli, Samiye; Şensoy, Figen; Temiz, Ebru; Altunışık, Erman; Korkmaz, Murat; Dağlı, Hasan; Kul, Seval; Akçalı, Aylin; Neyal, Ayşe Münife

2016-01-01

Urotensin-II (U-II) is a peptide recognized by its potent vasoconstrictor activity in many vascular events, however the role of urotensin-II in migraine has not been considered yet. The molecular mechanisms and genetics of migraine have not been fully clarified yet, but it is well-known that vascular changes considerably contribute in pathophysiology of migraine and also its complications. The aim of this study was to analyze the plasma U-II levels along with genotype distributions and allele frequencies for UTS2 Thr21Met and Ser89Asn polymorphisms among the patients with migraine without aura (MWoA). One hundred eighty-six patients with MWoA and 171 healthy individuals were included in this study. Plasma U-II levels were measured in attack free period. The genotype and allele frequencies for the Thr21Met (T21M) and Ser89Asn (S89N) polymorphisms in the UTS2 gene were analyzed. Plasma U-II levels were significantly higher in MWoA patients (p = 0.002). We detected a significant association between the T21M polymorphism in the UTS2 gene and migraine (53.8 % in patients, 40.4 % in controls, p = 0.035), but not with S89N polymorphism (p = 0.620). A significant relationship was found between U-II levels and MIDAS score (β = 0.508, p = 0.001). Our study suggests that U-II may play a role in migraine pathogenesis; also Thr21Met polymorphism was associated with the risk of migraine disease. Further studies are needed for considering the role of U-II in migraine pathophysiology and for deciding if UTS2 gene may be a novel candidate gene in migraine cases.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed

Sakoda, H; Imanaka, T

1992-02-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed Central

Sakoda, H; Imanaka, T

1992-01-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH. Images PMID:1735726

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

PubMed

Pliszka, Barbara; Martin, Brian M; Karczewska, Emilia

2008-02-01

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

49 CFR 178.255 - Specification 60; steel portable tanks.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Specification 60; steel portable tanks. 178.255 Section 178.255 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND... PACKAGINGS Specifications for Portable Tanks § 178.255 Specification 60; steel portable tanks. ...

49 CFR 178.255 - Specification 60; steel portable tanks.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Specification 60; steel portable tanks. 178.255 Section 178.255 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND... PACKAGINGS Specifications for Portable Tanks § 178.255 Specification 60; steel portable tanks. ...

49 CFR 178.255 - Specification 60; steel portable tanks.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Specification 60; steel portable tanks. 178.255 Section 178.255 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS... FOR PACKAGINGS Specifications for Portable Tanks § 178.255 Specification 60; steel portable tanks. ...

49 CFR 178.255 - Specification 60; steel portable tanks.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Specification 60; steel portable tanks. 178.255 Section 178.255 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND... PACKAGINGS Specifications for Portable Tanks § 178.255 Specification 60; steel portable tanks. ...

49 CFR 178.255 - Specification 60; steel portable tanks.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Specification 60; steel portable tanks. 178.255 Section 178.255 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND... PACKAGINGS Specifications for Portable Tanks § 178.255 Specification 60; steel portable tanks. ...

Residual Gas and Dust around Transition Objects and Weak T Tauri Stars

NASA Astrophysics Data System (ADS)

Doppmann, Greg W.; Najita, Joan R.; Carr, John S.

2017-02-01

Residual gas in disks around young stars can spin down stars, circularize the orbits of terrestrial planets, and whisk away the dusty debris that is expected to serve as a signpost of terrestrial planet formation. We have carried out a sensitive search for residual gas and dust in the terrestrial planet region surrounding young stars ranging in age from a few to ˜10 Myr. Using high-resolution 4.7 μm spectra of transition objects (TOs) and weak T Tauri stars, we searched for weak continuum excesses and CO fundamental emission, after making a careful correction for the stellar contribution to the observed spectrum. We find that the CO emission from TOs is weaker and located farther from the star than CO emission from nontransition T Tauri stars with similar stellar accretion rates. The difference is possibly the result of chemical and/or dynamical effects (I.e., a low CO abundance or close-in low-mass planets). The weak T Tauri stars show no CO fundamental emission down to low flux levels (5 × 10-20 to 10-18 W m-2). We illustrate how our results can be used to constrain the residual disk gas content in these systems and discuss their potential implications for star and planet formation. Data presented herein were obtained at the W. M. Keck Observatory from telescope time allocated to the National Aeronautics and Space Administration through the agency’s scientific partnership with the California Institute of Technology and the University of California. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation.

49 CFR 178.33a-5 - Material.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Material. 178.33a-5 Section 178.33a-5 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS FOR PACKAGINGS...

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

PubMed

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

7 CFR 1.78 - Development of special working relationships.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 1 2014-01-01 2014-01-01 false Development of special working relationships. 1.78 Section 1.78 Agriculture Office of the Secretary of Agriculture ADMINISTRATIVE REGULATIONS Cooperative Production of Television Films § 1.78 Development of special working relationships. (a) Preliminary. Prior to...

7 CFR 1.78 - Development of special working relationships.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 1 2011-01-01 2011-01-01 false Development of special working relationships. 1.78 Section 1.78 Agriculture Office of the Secretary of Agriculture ADMINISTRATIVE REGULATIONS Cooperative Production of Television Films § 1.78 Development of special working relationships. (a) Preliminary. Prior to...

7 CFR 1.78 - Development of special working relationships.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 1 2013-01-01 2013-01-01 false Development of special working relationships. 1.78 Section 1.78 Agriculture Office of the Secretary of Agriculture ADMINISTRATIVE REGULATIONS Cooperative Production of Television Films § 1.78 Development of special working relationships. (a) Preliminary. Prior to...

7 CFR 1.78 - Development of special working relationships.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 1 2012-01-01 2012-01-01 false Development of special working relationships. 1.78 Section 1.78 Agriculture Office of the Secretary of Agriculture ADMINISTRATIVE REGULATIONS Cooperative Production of Television Films § 1.78 Development of special working relationships. (a) Preliminary. Prior to...

7 CFR 1.78 - Development of special working relationships.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Development of special working relationships. 1.78 Section 1.78 Agriculture Office of the Secretary of Agriculture ADMINISTRATIVE REGULATIONS Cooperative Production of Television Films § 1.78 Development of special working relationships. (a) Preliminary. Prior to...

Loop versus divided colostomy for the management of anorectal malformations.

PubMed

Oda, Omar; Davies, Dafydd; Colapinto, Kimberly; Gerstle, J Ted

2014-01-01

The purpose of this study was to compare the clinical outcomes of loop and divided colostomies in patients with anorectal malformations (ARM). We performed a retrospective cohort study reviewing the medical records of all patients with ARM managed with diverting colostomies between 2000 and 2010 at our institution. Independent variables and outcomes of stoma complications were analyzed by parametric measures and logistic regression. One hundred forty-four patients managed with a colostomy for ARM were evaluated (37.5% females, 50.7% loop, 49.3% divided). The incidence of patients with loop and divided colostomies who developed stoma-related complications was 31.5 and 15.5%, respectively (p=0.031). The incidence of prolapse was 17.8 and 2.8%, respectively (p=0.005). Multivariable-logistic regression controlling for other significant independent variables found loop colostomies to be positively associated with the development of a stoma complication (OR 3.13, 95%CI (1.09, 8.96), p=0.033). When individual complications were evaluated, it was only stoma prolapse that was more likely in patients with loop colostomies (OR 8.75, 95%CI (1.74, 44.16), p=0.009). Because of the higher incidence of prolapse, loop colostomies were found to be associated with a higher total incidence of complications than divided stomas. The development of other complications, including urinary tract infections (UTIs) and megarectum, were independent of the type of colostomy performed. © 2014.

Influence of ALA54THR polymorphism of fatty acid binding protein 2 on lifestyle modification response in obese subjects.

PubMed

de Luis, D A; Aller, R; Izaola, O; Sagrado, M Gonzalez; Conde, R

2006-01-01

It has been found that the expression of fatty acid binding protein 2 (FABP2) mRNA is under dietary control. A G-to-A transition at codon 54 of FABP2 results in an amino acid substitution (from Ala 54 to Thr 54). This polymorphism was associated with high insulin resistance and high fasting insulin concentrations. The aim of our study was to investigate the influence of Thr54 polymorphism in the FABP2 protein on the response to a lifestyle modification (Mediterranean hypocaloric diet and exercise) in obese patients. A population of 69 obese (body mass index > 30) nondiabetic outpatients was analyzed in a prospective way. Before and after 3 months of the lifestyle modification program, indirect calorimetry, tetrapolar electrical bioimpedance measurement, blood pressure recording, serial assessment of the nutritional intake (3 days of written food records), and biochemical analysis were performed. The lifestyle modification program consisted of a hypocaloric diet (1,520 kcal; 52% carbohydrates, 25% lipids, and 23% proteins). The exercise program consisted of aerobic exercise for at least three times/week (60 min each). Statistical analysis was performed for combined Ala54/Thr54 and Thr54/Thr54 as a mutant group and wild-type Ala54/Ala54 as second group. The mean age was 45.5 +/- 16.7 years, the mean body mass index was 34.1 +/- 5.1, and there were 14 males (20.3%) and 55 females (79.7%) with a weight loss of 3.17 +/- 3.5 kg (3.5%). Thirty-seven patients (53.6%) had the genotype Ala54/Ala54 (wild-type group) and 32 (46.4%) patients either the genotype Ala54/Thr54 (26 patients, 30.2%) or the genotype Thr54/Thr54 (6 patients, 16.2%). The percentage of responders (weight loss) was similar in both groups (89.2 vs. 90.6%). In the wild-type group, body mass index, weight, fat mass, low-density lipoprotein cholesterol level, and waist circumference decreased, whereas the VO2 (oxygen consumption) increased. In the mutant group, glucose, body mass index, weight, waist

28 CFR 0.178 - Redelegation of authority.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Redelegation of authority. 0.178 Section 0.178 Judicial Administration DEPARTMENT OF JUSTICE ORGANIZATION OF THE DEPARTMENT OF JUSTICE Assigning Responsibility Concerning Applications for Orders Compelling Testimony or Production of Evidence...

Residual pathological stage at radical cystectomy significantly impacts outcomes for initial T2N0 bladder cancer.

PubMed

Isbarn, Hendrik; Karakiewicz, Pierre I; Shariat, Shahrokh F; Capitanio, Umberto; Palapattu, Ganesh S; Sagalowsky, Arthur I; Lotan, Yair; Schoenberg, Mark P; Amiel, Gilad E; Lerner, Seth P; Sonpavde, Guru

2009-08-01

We hypothesized that in patients with T2N0 stage disease at transurethral bladder tumor resection a lower residual cancer stage (P1N0 or less) at radical cystectomy may correlate with improved outcomes relative to those with residual P2N0 disease. We analyzed 208 patients with T2N0 stage disease at transurethral bladder tumor resection whose tumors were organ confined at radical cystectomy (P2 or lower, pN0). None received perioperative chemotherapy. Kaplan-Meier as well as univariable and multivariable Cox regression models addressed the effect of residual pT stage at radical cystectomy on recurrence and cancer specific mortality rates. Covariates consisted of age, gender, grade, lymphovascular invasion, carcinoma in situ, number of lymph nodes removed and year of surgery. Residual pT stage at radical cystectomy was P0 in 24 (11.5%) patients, Pa in 9 (4.3%), PCIS in 22 (10.6%), P1 in 35 (16.8%) and P2 in 118 (56.7%). Median followup of censored patients was 55.7 months for recurrence and 52.1 months for cancer specific mortality analyses. The 5-year recurrence-free survival rates of patients with P0/Pa/PCIS, P1 and P2 stage disease were 100%, 85% and 75%, respectively. The 5-year cancer specific survival rates for the same cohorts were 100%, 93% and 81%, respectively. On multivariable analysis the effect of residual stage P1 or lower at radical cystectomy achieved independent predictor status for recurrence (adjusted HR 0.20, p = 0.002) and cancer specific mortality (adjusted HR 0.24, p = 0.02). Down staging from initial T2N0 bladder cancer at transurethral bladder tumor resection to lower stage at radical cystectomy significantly reduces recurrence and cancer specific mortality. Further validation of this finding is warranted.

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Improved Speech Coding Based on Open-Loop Parameter Estimation

NASA Technical Reports Server (NTRS)

Juang, Jer-Nan; Chen, Ya-Chin; Longman, Richard W.

2000-01-01

A nonlinear optimization algorithm for linear predictive speech coding was developed early that not only optimizes the linear model coefficients for the open loop predictor, but does the optimization including the effects of quantization of the transmitted residual. It also simultaneously optimizes the quantization levels used for each speech segment. In this paper, we present an improved method for initialization of this nonlinear algorithm, and demonstrate substantial improvements in performance. In addition, the new procedure produces monotonically improving speech quality with increasing numbers of bits used in the transmitted error residual. Examples of speech encoding and decoding are given for 8 speech segments and signal to noise levels as high as 47 dB are produced. As in typical linear predictive coding, the optimization is done on the open loop speech analysis model. Here we demonstrate that minimizing the error of the closed loop speech reconstruction, instead of the simpler open loop optimization, is likely to produce negligible improvement in speech quality. The examples suggest that the algorithm here is close to giving the best performance obtainable from a linear model, for the chosen order with the chosen number of bits for the codebook.

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

PubMed

Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

2016-01-01

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

Eukaryotic-like Ser/Thr Protein Kinases SpkC/F/K Are Involved in Phosphorylation of GroES in the Cyanobacterium Synechocystis

PubMed Central

Zorina, Anna; Stepanchenko, Natalia; Novikova, Galina V.; Sinetova, Maria; Panichkin, Vladimir B.; Moshkov, Igor E.; Zinchenko, Vladislav V.; Shestakov, Sergey V.; Suzuki, Iwane; Murata, Norio; Los, Dmitry A.

2011-01-01

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES. PMID:21551175

49 CFR 178.345-13 - Pressure and leakage tests.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Pressure and leakage tests. 178.345-13 Section 178... PACKAGINGS Specifications for Containers for Motor Vehicle Transportation § 178.345-13 Pressure and leakage tests. (a) Each cargo tank must be pressure and leakage tested in accordance with this section and...

21 CFR 178.3600 - Methyl glucoside-coconut oil ester.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Methyl glucoside-coconut oil ester. 178.3600 Section 178.3600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Production Aids § 178.3600 Methyl glucoside-coconut oil ester. Methyl glucoside-coconut oil ester identified...

Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization.

PubMed

Peters, K; Hinz, H J; Cesareni, G

1997-10-01

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.

Early-onset progressive ataxia associated with the first CACNA1A mutation identified within the I-II loop.

PubMed

Cricchi, F; Di Lorenzo, C; Grieco, G S; Rengo, C; Cardinale, A; Racaniello, M; Santorelli, F M; Nappi, G; Pierelli, F; Casali, C

2007-03-15

Familial hemiplegic migraine type 1, spinocerebellar ataxia type 6 (SCA6) and episodic ataxia type 2 (EA2) are allelic disorders associated with mutations in the CACNA1A gene, which encodes the alpha1 subunit of the P/Q-type calcium channel (Ca(V)2.1). SCA6 and EA2 share a number of clinical features, such as prominent cerebellar involvement and good response to acetazolamide therapy. However, while SCA6 develops as a late-onset, progressive ataxia, EA2 has an earlier, and episodic, onset. We report on two sisters with a heterogeneous clinical phenotype. The first developed progressive cerebellar ataxia after age 30, without noticeable episodes of vertigo or headache. A 1 year trial with acetazolamide did not produce significant results. The other reported episodes of vertigo, headache and gait imbalance since late childhood, with good response to acetazolamide, before developing moderate chronic cerebellar ataxia. Brain MRI showed cerebellar atrophy, especially in the vermis, in both patients. Direct sequencing of CACNA1A identified a heterozygous 1360G>A mutation in exon 11 resulting in the substitution of alanine for threonine at residue 454 (p.Ala454Thr). This is the first description of a change residing in the cytoplasmic I-II loop associated with a clinical phenotype.

21 CFR 178.3280 - Castor oil, hydrogenated.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Castor oil, hydrogenated. 178.3280 Section 178.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND...

21 CFR 178.3280 - Castor oil, hydrogenated.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Castor oil, hydrogenated. 178.3280 Section 178.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND...

21 CFR 178.3280 - Castor oil, hydrogenated.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Castor oil, hydrogenated. 178.3280 Section 178.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND SANITIZERS...

21 CFR 178.3280 - Castor oil, hydrogenated.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Castor oil, hydrogenated. 178.3280 Section 178.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADJUVANTS, PRODUCTION AIDS, AND...

Loop Integrands for Scattering Amplitudes from the Riemann Sphere

NASA Astrophysics Data System (ADS)

Geyer, Yvonne; Mason, Lionel; Monteiro, Ricardo; Tourkine, Piotr

2015-09-01

The scattering equations on the Riemann sphere give rise to remarkable formulas for tree-level gauge theory and gravity amplitudes. Adamo, Casali, and Skinner conjectured a one-loop formula for supergravity amplitudes based on scattering equations on a torus. We use a residue theorem to transform this into a formula on the Riemann sphere. What emerges is a framework for loop integrands on the Riemann sphere that promises to have a wide application, based on off-shell scattering equations that depend on the loop momentum. We present new formulas, checked explicitly at low points, for supergravity and super-Yang-Mills amplitudes and for n -gon integrands at one loop. Finally, we show that the off-shell scattering equations naturally extend to arbitrary loop order, and we give a proposal for the all-loop integrands for supergravity and planar super-Yang-Mills theory.

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

PubMed

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.

Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

PubMed

Bondoc, Jasper Marc G; Wolf, Nina M; Ndichuck, Michael; Abad-Zapatero, Celerino; Movahedzadeh, Farahnaz

2017-09-01

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis ( Mt ), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in V max and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH - in the Thr84 residue of the wildtype of Mt FBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

Thumb-loops up for catalysis: a structure/function investigation of a functional loop movement in a GH11 xylanase

PubMed Central

Paës, Gabriel; Cortés, Juan; Siméon, Thierry; O'Donohue, Michael J.; Tran, Vinh

2012-01-01

Dynamics is a key feature of enzyme catalysis. Unfortunately, current experimental and computational techniques do not yet provide a comprehensive understanding and description of functional macromolecular motions. In this work, we have extended a novel computational technique, which combines molecular modeling methods and robotics algorithms, to investigate functional motions of protein loops. This new approach has been applied to study the functional importance of the so-called thumb-loop in the glycoside hydrolase family 11 xylanase from Thermobacillus xylanilyticus (Tx-xyl). The results obtained provide new insight into the role of the loop in the glycosylation/deglycosylation catalytic cycle, and underline the key importance of the nature of the residue located at the tip of the thumb-loop. The effect of mutations predicted in silico has been validated by in vitro site-directed mutagenesis experiments. Overall, we propose a comprehensive model of Tx-xyl catalysis in terms of substrate and product dynamics by identifying the action of the thumb-loop motion during catalysis. PMID:24688637

49 CFR 178.33b-7 - Design qualification test.

Code of Federal Regulations, 2010 CFR

2010-10-01

..., each new design must be drop tested as follows: Three groups of twenty-five filled containers must be... 49 Transportation 2 2010-10-01 2010-10-01 false Design qualification test. 178.33b-7 Section 178... PACKAGINGS Specifications for Inside Containers, and Linings § 178.33b-7 Design qualification test. (a) Drop...

49 CFR 178.345-13 - Pressure and leakage tests.

Code of Federal Regulations, 2014 CFR