scholarly journals Crystal structure of an alleged mannosylphosphate transferase Ktr6p at 3.06 Å

2014 ◽  
Vol 70 (a1) ◽  
pp. C1671-C1671
Author(s):  
Dmitry Rodionov ◽  
Daniella Marks ◽  
Pedro Romero ◽  
Albert Berghuis
Keyword(s):  
Room Temperature ◽  
Sequence Similarity ◽  
Transferase Activity ◽  
Molecular Replacement ◽  
Sequence Identity ◽  
Mannose Binding ◽  
The Difference ◽  
Resolution Dataset ◽  
Close Homolog

Ktr6p is an alleged mannosylphosphate transferase from yeast Golgi. It has been implicated in decorating both O-linked and N-lined glycans with mannosylphosphate in vivo. However, based on sequence similarity, Ktr6p belongs to GT15 family of α-1,2-mannosyltransferases. To address this disagreement, the soluble portion of Ktr6p was expressed in P. pastoris and purified by liquid chromatography. The purified protein, GDP-mannose and various acceptors were used in a number of direct and indirect activity assays, however, neither manosyltransferase nor mannosylphosphate transferase activity was detected. Ktr6p was crystallized in a number of PEG- containing conditions, but the crystals resisted all attempts at cryoprotection. Three crystals were used to collect a 3.06 Å resolution dataset on a home source at room temperature. The crystals belong to P 21 21 21 spacegroup with 2 molecules per asymmetric unit. The structure was solved by molecular replacement using a structure of Kre2p, a close homolog from GT15 family (40% sequence identity). The structure was refined to R/Rfree 16.1%/21.2% The overall structure of Ktr6p is very similar to the structure of Kre2p having less than 2 Å overall backbone RMSD. However even at 3 Å resolution the difference in the active site is striking. The guanine moiety binding pocked is occluded by a well-ordered loop making GDP-mannose binding impossible in this conformation. Several aminoacid substitutions in the Mn2+ coordinating environment suggest that Ktr6 does not depend on manganese for its postulated activity. These observations indicate that Ktr6p functions quite differently from Kre2p.

scholarly journals Interfacial polarization of in vivo rat sciatic nerve with crush injury studied via broadband dielectric spectroscopy

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252589
Author(s):  
Risa Otagiri ◽  
Hideki Kawai ◽  
Masanobu Takatsuka ◽  
Naoki Shinyashiki ◽  
Akira Ito ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Dielectric Spectroscopy ◽  
Peripheral Nerves ◽  
Room Temperature ◽  
Cell Structure ◽  
Interfacial Polarization ◽  
Broadband Dielectric Spectroscopy ◽  
Sciatic Nerves ◽  
The Difference

Electrical stimulation is one of the candidates for elongation-driven regeneration of damaged peripheral nerves. Different organs and tissues have an inherent cell structure and size. This leads to variation in the tissue-specific electrical properties of the frequency of interfacial polarization. Although nervous tissues have a membrane potential, the electrical reaction inside these tissues following electrical stimulation from outside remains unexplored. Furthermore, the pathophysiological reaction of an injured nerve is unclear. Here, we investigated the electrical reaction of injured and non-injured rat sciatic nerves via broadband dielectric spectroscopy. Crush injured and non-injured sciatic nerves of six 12-week-old male Lewis rats were used, 6 days after infliction of the injury. Both sides of the nerves (with and without injury) were exposed, and impedance measurements were performed at room temperature (approximately 25°C) at frequencies ranging from 100 mHz to 5.5 MHz and electric potential ranging from 0.100 to 1.00 V. The measured interfacial polarization potentially originated from the polarization by ion transport around nerve membranes at frequencies between 3.2 kHz and 1.6 MHz. The polarization strength of the injured nerves was smaller than that of non-injured nerves. However, the difference in polarization between injured and non-injured nerves might be caused by inflammation and edema. The suitable frequency range of the interfacial polarization can be expected to be critical for electrical stimulation of injured peripheral nerves.

scholarly journals Identification of a Novel Family of Nonclassic Yeast Phosphatidylinositol Transfer Proteins Whose Function Modulates Phospholipase D Activity and Sec14p-independent Cell Growth

2000 ◽  
Vol 11 (6) ◽  
pp. 1989-2005 ◽  
Author(s):  
Xinmin Li ◽  
Sheri M. Routt ◽  
Zhigang Xie ◽  
Xiaoxia Cui ◽  
Min Fang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Phospholipase D ◽  
Sequence Similarity ◽  
Primary Sequence ◽  
Transfer Protein ◽  
Sequence Identity ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24–65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet,SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate thatSFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with “bypass-Sec14p” mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

scholarly journals β1 Tubulin Rather Than β2 Tubulin Is the Preferred Binding Target for Carbendazim in Fusarium graminearum

2016 ◽  
Vol 106 (9) ◽  
pp. 978-985 ◽  
Author(s):  
Yujun Zhou ◽  
Yuanye Zhu ◽  
Yanjun Li ◽  
Yabing Duan ◽  
Rongsheng Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusarium Graminearum ◽  
Amino Acid Sequence Identity ◽  
Deletion Strain ◽  
Protein Accumulation ◽  
High Concentration ◽  
Sequence Identity ◽  
Increased Sensitivity ◽  
The Difference

Tubulins are the proposed target of anticancer drugs, anthelminthics, and fungicides. In Fusarium graminearum, β2 tubulin has been reported to be the binding target of methyl benzimidazole carbamate (MBC) fungicides. However, the function of F. graminearum β1 tubulin, which shares 76% amino acid sequence identity with β2 tubulin, in MBC sensitivity has been unclear. In this study, MBC sensitivity relative to that of a parental strain (2021) was significantly reduced in a β1 tubulin deletion strain but increased in a β2 tubulin deletion strain, suggesting that β1 tubulin was involved in the MBC sensitivity of F. graminearum. When strain 2021 was grown in a medium with a low or high concentration of the MBC fungicide carbendazim (0.5 or 1.4 μg/ml), the protein accumulation levels were reduced by 47 and 87%, respectively, for β1 tubulin but only by 6 and 24%, respectively, for β2 tubulin. This result was consistent with observations that MBC fungicides are more likely to disrupt β1 tubulin microtubules rather than β2 tubulin microtubules in GFP-β tubulin fusion mutants in vivo. Furthermore, sequence analysis indicated that a difference in tubulin amino acid 240 (240L in β1 versus 240F in β2) may explain the difference in MBC binding affinity; this result was consistent with the result that an F240L mutation in β2 tubulin greatly increased sensitivity to carbendazim in F. graminearum. We suggest that β1 tubulin rather than β2 tubulin is the preferred binding target for MBC fungicides in F. graminearum.

scholarly journals Gpi19, the Saccharomyces cerevisiae Homologue of Mammalian PIG-P, Is a Subunit of the Initial Enzyme for Glycosylphosphatidylinositol Anchor Biosynthesis

2005 ◽  
Vol 4 (11) ◽  
pp. 1801-1807 ◽  
Author(s):  
Heather A. Newman ◽  
Martin J. Romeo ◽  
Sarah E. Lewis ◽  
Benjamin C. Yan ◽  
Peter Orlean ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Transferase Activity ◽  
Its Gene ◽  
A Subunit ◽  
Glcnac Transferase

ABSTRACT Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast Saccharomyces cerevisiae. The uncharacterized yeast gene YDR437w encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene GPI19. Similar to other mutants in the yeast enzyme, temperature-sensitive gpi19 mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional gpi19 mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.

scholarly journals Factors influencing estimates of coordinate error for molecular replacement

2020 ◽  
Vol 76 (1) ◽  
pp. 19-27
Author(s):  
Kaushik S. Hatti ◽  
Airlie J. McCoy ◽  
Robert D. Oeffner ◽  
Massimo D. Sammito ◽  
Randy J. Read
Keyword(s):  
Error Estimates ◽  
Sequence Similarity ◽  
Protein Structures ◽  
Optimal Error Estimates ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity ◽  
Highly Correlated ◽  
Coordinate Error

Good prior estimates of the effective root-mean-square deviation (r.m.s.d.) between the atomic coordinates of the model and the target optimize the signal in molecular replacement, thereby increasing the success rate in difficult cases. Previous studies using protein structures solved by X-ray crystallography as models showed that optimal error estimates (refined after structure solution) were correlated with the sequence identity between the model and target, and with the number of residues in the model. Here, this work has been extended to find additional correlations between parameters of the model and the target and hence improved prior estimates of the coordinate error. Using a graph database, a curated set of 6030 molecular-replacement calculations using models that had been solved by X-ray crystallography was analysed to consider about 120 model and target parameters. Improved estimates were achieved by replacing the sequence identity with the Gonnet score for sequence similarity, as well as by considering the resolution of the target structure and the MolProbity score of the model. This approach was extended by analysing 12 610 additional molecular-replacement calculations where the model was determined by NMR. The median r.m.s.d. between pairs of models in an ensemble was found to be correlated with the estimated r.m.s.d. to the target. For models solved by NMR, the overall coordinate error estimates were larger than for structures determined by X-ray crystallography, and were more highly correlated with the number of residues.

scholarly journals Different Roles of DosS and DosT in the Hypoxic Adaptation of Mycobacteria

2010 ◽  
Vol 192 (19) ◽  
pp. 4868-4875 ◽  
Author(s):  
Min-Ju Kim ◽  
Kwang-Jin Park ◽  
In-Jeong Ko ◽  
Young Min Kim ◽  
Jeong-Il Oh
Keyword(s):  
Oxygen Tension ◽  
Response Regulator ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
High Sequence Similarity ◽  
Hypoxic Adaptation ◽  
The Difference ◽  
A Domains

ABSTRACT The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Reliability of a Single β-Thromboglobulin Measurement in a Diabetic Population : Importance of PGE1 in Anticoagulant Mixture

1987 ◽  
Vol 57 (02) ◽  
pp. 201-204 ◽  
Author(s):  
P Y Scarabin ◽  
L Strain ◽  
C A Ludlam ◽  
J Jones ◽  
E M Kohner
Keyword(s):  
In Vitro Release ◽  
Mean Value ◽  
Reliable Estimate ◽  
Diabetic Patients ◽  
Single Measurement ◽  
Diabetic Population ◽  
The Difference ◽  
Vitro Release

SummaryDuring the collection of samples for plasma β-thromboglobulin (β-TG) determination, it is well established that artificially high values can be observed due to in-vitro release. To estimate the reliability of a single β-TG measurement, blood samples were collected simultaneously from both arms on two separate occasions in 56 diabetic patients selected for a clinical trial. From each arm, blood was taken into two tubes containing an anticoagulant mixture with (tube A) and without (tube B) PGE!. The overall mean value of B-TG in tube B was 1.14 times higher than in tube A (p <0.01). The markedly large between-arms variation accounted for the most part of within-subject variation in both tubes and was significantly greater in tube B than in tube A. Based on the difference between B-TG values from both arms, the number of subjects with artifically high B-TG values was significantly higher in tube B than in tube A on each occasion (overall rate: 28% and 14% respectively). Estimate of between-occasions variation showed that B-TG levels were relatively stable for each subject between two occasions in each tube. It is concluded that the use of PGEi decreases falsely high B-TG levels, but a single measurement of B-TG does not provide a reliable estimate of the true B-TG value in vivo.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

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