40 CFR 721.10032 - Acrylic acid, polymer with substituted acrylamides (generic).

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.10032 Acrylic acid, polymer with substituted acrylamides (generic). (a... generically as acrylic acid, polymer with substituted acrylamides (PMN P-02-269) is subject to reporting under...

40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting under...

40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting under...

40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting under...

40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting under...

40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting under...

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

PubMed

Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

2017-04-01

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

40 CFR 721.530 - Substituted aliphatic acid halide (generic name).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted aliphatic acid halide... Specific Chemical Substances § 721.530 Substituted aliphatic acid halide (generic name). (a) Chemical... acid halide (PMN P-84-491) is subject to reporting under this section for the significant new uses...

40 CFR 721.530 - Substituted aliphatic acid halide (generic name).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Substituted aliphatic acid halide... Specific Chemical Substances § 721.530 Substituted aliphatic acid halide (generic name). (a) Chemical... acid halide (PMN P-84-491) is subject to reporting under this section for the significant new uses...

Functionalization of Carbon Nanotubes via Electrophilic Substitution Reaction in Polyphosphoric Acid

DTIC Science & Technology

2006-07-26

1 Title of proposed research: Functionalization of Carbon Nanotubes via Electrophilic Substitution Reaction in Polyphosphoric Acid Proposer: Jong...Choi, J.-Y.; Tan, L.-S.; Baek, J.-B. “Functionalization of carbon nanotubes via electrophilic substitution reaction in polyphosphoric acid” AFOSR...2006 4. TITLE AND SUBTITLE Functionalization of carbon nanotubes via electrophilic substitution reaction in polyphosphoric acid 5a. CONTRACT

Detecting Adaptation in Protein-Coding Genes Using a Bayesian Site-Heterogeneous Mutation-Selection Codon Substitution Model.

PubMed

Rodrigue, Nicolas; Lartillot, Nicolas

2017-01-01

Codon substitution models have traditionally attempted to uncover signatures of adaptation within protein-coding genes by contrasting the rates of synonymous and non-synonymous substitutions. Another modeling approach, known as the mutation-selection framework, attempts to explicitly account for selective patterns at the amino acid level, with some approaches allowing for heterogeneity in these patterns across codon sites. Under such a model, substitutions at a given position occur at the neutral or nearly neutral rate when they are synonymous, or when they correspond to replacements between amino acids of similar fitness; substitutions from high to low (low to high) fitness amino acids have comparatively low (high) rates. Here, we study the use of such a mutation-selection framework as a null model for the detection of adaptation. Following previous works in this direction, we include a deviation parameter that has the effect of capturing the surplus, or deficit, in non-synonymous rates, relative to what would be expected under a mutation-selection modeling framework that includes a Dirichlet process approach to account for across-codon-site variation in amino acid fitness profiles. We use simulations, along with a few real data sets, to study the behavior of the approach, and find it to have good power with a low false-positive rate. Altogether, we emphasize the potential of recent mutation-selection models in the detection of adaptation, calling for further model refinements as well as large-scale applications. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Coordination ability determined transition metal ions substitution of Tb in Tb-Asp fluorescent nanocrystals and a facile ions-detection approach.

PubMed

Duan, Jiazhi; Ma, Baojin; Liu, Feng; Zhang, Shan; Wang, Shicai; Kong, Ying; Du, Min; Han, Lin; Wang, Jianjun; Sang, Yuanhua; Liu, Hong

2018-04-26

Although the synthesis and fluorescent properties of lanthanide-amino acid complex nanostructures have been investigated extensively, limited studies have been reported on metal ions' substitution ability for the lanthanide ions in the complex and their effect on the fluorescent property. In this study, taking biocompatible Tb-aspartic acid (Tb-Asp) complex nanocrystals as a model, the substitution mechanism of metal ions, particularly transition metals, for Tb ions in Tb-Asp nanocrystals and the change in the fluorescent property of the Tb-Asp nanocrystals after substitution were systematically investigated. The experimental results illustrated that metal ions with higher electronegativity, higher valence, and smaller radius possess stronger ability for Tb ions' substitution in Tb-Asp nanocrystals. Based on the effect of substituting ions' concentration on the fluorescent property of Tb-Asp, a facile method for copper ions detection with high sensitivity was proposed by measuring the fluorescent intensity of Tb-Asp nanocrystals' suspensions containing different concentrations of copper ions. The good biocompatibility, great convenience of synthesis and sensitive detection ability make Tb-Asp nanocrystals a very low cost and effective material for metal ions detection, which also opens a new door for practical applications of metal-Asp coordinated nanocrystals.

40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to reporting...

40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to reporting...

40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to reporting...

40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to reporting...

40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to reporting...

Effect of substitution of low linolenic acid soybean oil for hydrogenated soybean oil on fatty acid intake.

PubMed

DiRienzo, Maureen A; Astwood, James D; Petersen, Barbara J; Smith, Kim M

2006-02-01

Low linolenic acid soybean oil (LLSO) has been developed as a substitute for hydrogenated soybean oil to reduce intake of trans FA while improving stability and functionality in processed foods. We assessed the dietary impact of substitution of LLSO for hydrogenated soybean oil (HSBO) used in several food categories. All substitutions were done using an assumption of 100% market penetration. The impact of this substitution on the intake of five FA and trans FA was assessed. Substitution of LLSO for current versions of HSBO resulted in a 45% decrease in intake of trans FA. Impacts on other FA intakes were within the realm of typical dietary intakes. No decrease in intake of alpha-linolenic acid was associated with the use of LLSO in place of HSBO because LLSO substitutes for HSBO that are already low in alpha-linolenic acid.

Elongated and substituted triazine-based tricarboxylic acid linkers for MOFs.

PubMed

Klinkebiel, Arne; Beyer, Ole; Malawko, Barbara; Lüning, Ulrich

2016-01-01

New triazine-based tricarboxylic acid linkers were prepared as elongated relatives of triazinetribenzoic acid (TATB). Additionally, functional groups (NO 2 , NH 2 , OMe, OH) were introduced for potential post-synthetic modification (PSM) of MOFs. Functionalized tris(4-bromoaryl)triazine "cores" ( 3a , 3b ) were obtained by unsymmetric trimerization mixing one equivalent of an acid chloride (OMe or NO 2 substituted) with two equivalents of an unsubstituted nitrile. Triple Suzuki coupling of the cores 3 with suitable phenyl- and biphenylboronic acid derivatives provided elongated tricarboxylic acid linkers as carboxylic acids 17 and 20 or their esters 16 and 19 . Reduction of the nitro group and cleavage of the methoxy group gave the respective amino and hydroxy-substituted triazine linkers.

Elongated and substituted triazine-based tricarboxylic acid linkers for MOFs

PubMed Central

Klinkebiel, Arne; Beyer, Ole; Malawko, Barbara

2016-01-01

New triazine-based tricarboxylic acid linkers were prepared as elongated relatives of triazinetribenzoic acid (TATB). Additionally, functional groups (NO2, NH2, OMe, OH) were introduced for potential post-synthetic modification (PSM) of MOFs. Functionalized tris(4-bromoaryl)triazine “cores” (3a,3b) were obtained by unsymmetric trimerization mixing one equivalent of an acid chloride (OMe or NO2 substituted) with two equivalents of an unsubstituted nitrile. Triple Suzuki coupling of the cores 3 with suitable phenyl- and biphenylboronic acid derivatives provided elongated tricarboxylic acid linkers as carboxylic acids 17 and 20 or their esters 16 and 19. Reduction of the nitro group and cleavage of the methoxy group gave the respective amino and hydroxy-substituted triazine linkers. PMID:28144293

Natural derivatives of diphenolic acid as substitutes for bisphenol-A

NASA Astrophysics Data System (ADS)

Ertl, Johanna; Cerri, Elisa; Rizzuto, Matteo; Caretti, Daniele

2014-05-01

Diphenolic acid had been originally used in the first epoxy resins and was later on forgotten as it was substituted by the cheaper bisphenol A. But in the recent years major health concerns have been raised as bisphenol A has a pseudo-hormonal effect on the body, playing the role of estrogen it can cause a severe impact on the organism, especially in males. Moreover it is produced from acetone and phenol, both from fossil, and thus limited resources. On the contrary, diphenolic acid is synthesized from levulinic acid and phenol. Levulinic acid being directly produced by hydrolysis of biomass. By substituting the fossil phenol with natural phenols from lignin or plant extraction we are able to synthesize a fully renewable substitute for bisphenol A. The reactions to yield an epoxy resin have been examined and the reactivity with epichlorohydrin is satisfying. Moreover, some of the derivatives of diphenolic acid have interesting curing properties and preliminary results show excellent properties of the cured resin, including thermal stability and pencil hardness.

40 CFR 721.10690 - Benzenedicarboxylic acid, polymer with substituted alkanediol, dodecanedioic acid, 1,2-ethanediol...

Code of Federal Regulations, 2014 CFR

2014-07-01

..., alkanediol,.alpha.-hydro-.omega.-hydroxypoly[oxyalkanediyl], 1,3-isobenzofurandione, methylene diphenyl...-ethanediol, alkanedioic acid, alkanediol,.alpha.-hydro-.omega.-hydroxypoly[oxyalkanediyl], 1,3... substituted alkanediol, dodecanedioic acid, 1,2-ethanediol, alkanedioic acid, alkanediol,.alpha.-hydro-.omega...

40 CFR 721.9798 - Benzenesulfonic acid, 2,2′-(1,2-ethenediyl)bis[5-[[4-substituted-6-substituted-1,3,5-triazin-2-yl...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Benzenesulfonic acid, 2,2â²-(1,2-ethenediyl)bis[5-[[4-substituted-6-substituted-1,3,5-triazin-2-yl]amino]-, sodium salt (generic). 721.9798... Substances § 721.9798 Benzenesulfonic acid, 2,2′-(1,2-ethenediyl)bis[5-[[4-substituted-6-substituted-1,3,5...

40 CFR 721.9798 - Benzenesulfonic acid, 2,2′-(1,2-ethenediyl)bis[5-[[4-substituted-6-substituted-1,3,5-triazin-2-yl...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Benzenesulfonic acid, 2,2â²-(1,2-ethenediyl)bis[5-[[4-substituted-6-substituted-1,3,5-triazin-2-yl]amino]-, sodium salt (generic). 721.9798... Substances § 721.9798 Benzenesulfonic acid, 2,2′-(1,2-ethenediyl)bis[5-[[4-substituted-6-substituted-1,3,5...

40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Naphthalenedisulfonic acid, [amino... Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo... naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy-[(methoxy-sulfophenyl)azo...

40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Naphthalenedisulfonic acid, [amino... Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo... naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy-[(methoxy-sulfophenyl)azo...

Detecting Single-Nucleotide Substitutions Induced by Genome Editing.

PubMed

Miyaoka, Yuichiro; Chan, Amanda H; Conklin, Bruce R

2016-08-01

The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells. © 2016 Cold Spring Harbor Laboratory Press.

Effects of tempering (annealing), acid hydrolysis, low-citric acid substitution on chemical and physicochemical properties of starches of four yam (Dioscorea spp.) cultivars.

PubMed

Falade, Kolawole O; Ayetigbo, Oluwatoyin E

2017-05-01

The effects of tempering (annealing), acid hydrolysis and low-citric acid substitution on chemical and physicochemical properties of starches of four Nigerian yam cultivars were investigated. Crude fat and protein contents of the native starches decreased significantly after the modifications, while nitrogen-free extract increased significantly with acid hydrolysis and citric acid substitution. Acid hydrolysis and low-citric acid substitution reduced the least concentration for gel formation of the starches from 4 to 2% w/v, but tempering had no effect. Swelling power of the starches reduced significantly, and water solubility increased significantly at 75 and 85 °C, especially with acid hydrolysis and low-citric acid substitution. However, tempering significantly reduced starch solubility in the four cultivars. Paste clarity of starches of white (29.17%), water (18.90%), yellow (30.90%) and bitter (10.57%) yams reduced significantly with tempering to 14.43, 11.83, 16.93 and 7.27%, but increased significantly with acid hydrolysis to 41.40, 35.37, 28.77 and 32.33%, and low-citric acid substitution to 36.60, 44.17, 50.67 and 14.33%, respectively. Pasting properties such as peak, trough, breakdown, final, and setback viscosities and peak time of native starches reduced significantly with acid hydrolysis and low-citric acid substitution, however, tempering significantly increased their pasting temperature, peak time, setback and final viscosities.

An injectable oxidated hyaluronic acid/adipic acid dihydrazide hydrogel as a vitreous substitute.

PubMed

Su, Wen-Yu; Chen, Ko-Hua; Chen, Yu-Chun; Lee, Yen-Hsien; Tseng, Ching-Li; Lin, Feng-Huei

2011-01-01

Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute. Koninklijke Brill NV, Leiden, 2011

40 CFR 721.1680 - Substituted benzoic acid (generic).

Code of Federal Regulations, 2010 CFR

2010-07-01

...). 721.1680 Section 721.1680 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.1680 Substituted benzoic acid (generic). (a) Chemical substance and significant new uses...

The effect of amino acid deletions and substitutions in the longest loop of GFP

PubMed Central

Flores-Ramírez, Gabriela; Rivera, Manuel; Morales-Pablos, Alfredo; Osuna, Joel; Soberón, Xavier; Gaytán, Paul

2007-01-01

Background The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region. Results In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein. Conclusion The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure. PMID:17594481

40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Fatty acid, reaction product with... Specific Chemical Substances § 721.6181 Fatty acid, reaction product with substituted oxirane, formaldehyde... as fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Fatty acid, reaction product with... Specific Chemical Substances § 721.6181 Fatty acid, reaction product with substituted oxirane, formaldehyde... as fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

Wide Tolerance to Amino Acids Substitutions In The OCTN1 Ergothioneine Transporter

PubMed Central

Frigeni, Marta; Iacobazzi, Francesco; Yin, Xue; Longo, Nicola

2016-01-01

Background Organic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease. Methods Here we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy. Results Substitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased Vmax, with modest changes in Km toward ergothioneine. Conclusions Our results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity. General significance The widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role. PMID:26994919

Synthesis, characterization, and subcellular localization studies of amino acid-substituted porphyrinic pigments

NASA Astrophysics Data System (ADS)

van Diggelen, Lisa; Khin, Hnin; Conner, Kip; Shao, Jenny; Sweezy, Margaretta; Jung, Anna H.; Isaac, Meden; Simonis, Ursula

2009-06-01

Stopping cancer in its path occurs when photosensitizers (PSs) induce apoptotic cell death after their exposure to light and the subsequent formation of reactive oxygen species. In pursuit of our hypothesis that mitochondrial localizing PSs will enhance the efficacy of the photosensitizing process in photodynamic therapy, since they provoke cell death by inducing apoptosis, we synthesized and characterized tetraphenylporphyrins (TPPs) that are substituted at the paraphenyl positions by two amino acids and two fluoro or hydroxyl groups, respectively. They were prepared according to the Lindsey-modified Adler-Longo methodology using trifluoromethanesulfonylchloride (CF3SO2Cl) as a catalyst instead of trifluoroacetic acid. The use of CF3SO2Cl yielded cleaner products in significantly higher yields. During the synthesis, not only the yields and work-up procedure of the TPPs were improved by using CF3SO2Cl as a catalyst, but also a better means of synthesizing the precursor dipyrromethanes was tested by using indium(III) chloride. Column chromatography, HPLC, and NMR spectroscopy were used to separate and characterize the di-amino acid-dihydroxy, or difluoro-substituted porphyrins and to ascertain their purity before subcellular localization studies were carried out. Studies using androgen-sensitive human prostate adenocarcinoma cells LNCaP revealed that certain amino acid substituted porphyrins that are positively charged in the slightly acidic medium of cancer cells are very useful in shedding light on the targets of TPPs in subcellular organelles of cancer cells. Although some of these compounds have properties of promising photosensitizers by revealing increased water solubility, acidic properties, and innate ability to provoke cell death by apoptosis, the cell killing efficacy of these TPPs is low. This correlates with their subcellular localization. The di-amino acid, di-hydroxy substituted TPPs localize mainly to the lysosomes, whereas the di-fluoro-substituted

40 CFR 721.1648 - Substituted benzenesulfonic acid salt (generic).

Code of Federal Regulations, 2010 CFR

2010-07-01

...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.1648 Substituted benzenesulfonic acid salt (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically...

Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids

USGS Publications Warehouse

Cortinas, I.; Field, J.A.; Kopplin, M.; Garbarino, J.R.; Gandolfi, A.J.; Sierra-Alvarez, R.

2006-01-01

Large quantities of arsenic are introduced into the environment through land application of poultry litter containing the organoarsenical feed additive roxarsone (3-nitro-4-hydroxyphenylarsonic acid). The objective of this study was to evaluate the bioconversion of roxarsone and related N-substituted phenylarsonic acid derivatives under anaerobic conditions. The results demonstrate that roxarsone is rapidly transformed in the absence of oxygen to the corresponding aromatic amine, 4-hydroxy-3-aminophenylarsonic acid (HAPA). The formation of HAPA is attributable to the facile reduction of the nitro group. Electron-donating substrates, such as hydrogen gas, glucose, and lactate, stimulated the rate of nitro group reduction, indicating a microbial role. During long-term incubations, HAPA and the closely related 4-aminophenylarsonic acid (4-APA) were slowly biologically eliminated by up to 99% under methanogenic and sulfate-reducing conditions, whereas little or no removal occurred in heat-killed inoculum controls. Arsenite and, to a lesser extent, arsenate were observed as products of the degradation. Freely soluble forms of the inorganic arsenical species accounted for 19-28% of the amino-substituted phenylarsonic acids removed. This constitutes the first report of a biologically catalyzed rupture of the phenylarsonic group under anaerobic conditions. ?? 2006 American Chemical Society.

Identification of single amino acid substitutions (SAAS) in neuraminidase from influenza a virus (H1N1) via mass spectrometry analysis coupled with de novo peptide sequencing.

PubMed

Peng, Qisheng; Wang, Zijian; Wu, Donglin; Li, Xiaoou; Liu, Xiaofeng; Sun, Wanchun; Liu, Ning

2016-08-01

Amino acid substitutions in the neuraminidase of the influenza virus are the main cause of the emergence of resistance to zanamivir or oseltamivir during seasonal influenza treatment; they are the result of non-synonymous mutations in the viral genome that can be successfully detected by polymer chain reaction (PCR)-based approaches. There is always an urgent need to detect variation in amino acid sequences directly at the protein level. Mass spectrometry coupled with de novo sequencing has been explored as an alternative and straightforward strategy for detecting amino acid substitutions, as well - this approach is the primary focus of the present study. Influenza virus (A/Puerto Rico/8/1934 H1N1) propagated in embryonated chicken eggs was purified by ultracentrifugation, followed by PNGase F treatment. The deglycosylated virion was lysed and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel band corresponding to neuraminidase was picked up and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three amino acid substitutions: R346K, S349 N, and S370I/L, in the neuraminidase from the influenza virus (A/Puerto Rico/8/1934 H1N1), which were located in three mutated peptides of the neuraminidase: YGNGVWIGK, TKNHSSR, and PNGWTETDI/LK, respectively. We found that the amino acid substitutions in the proteins of RNA viruses (including influenza A virus) resulting from non-synonymous gene mutations can indeed be directly analyzed via mass spectrometry, and that manual interpretation of the MS/MS data may be beneficial. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

40 CFR 721.5278 - Substituted naphthalenesulfonic acid, alkali salt.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted naphthalenesulfonic acid, alkali salt. 721.5278 Section 721.5278 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.5278...

Antimicrobial activity and stability of protonectin with D-amino acid substitutions.

PubMed

Qiu, Shuai; Zhu, Ranran; Zhao, Yanyan; An, Xiaoping; Jia, Fengjing; Peng, Jinxiu; Ma, Zelin; Zhu, Yuanyuan; Wang, Jiayi; Su, Jinhuan; Wang, Qingjun; Wang, Hailin; Li, Yuan; Wang, Kairong; Yan, Wenjin; Wang, Rui

2017-05-01

The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D-amino acids. Both the D-enantiomer of protonectin (D-prt) and D-Lys-protonectin (D-Lys-prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D-prt showed strong stability against trypsin, chymotrypsin and the human serum, while D-Lys-prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D-Lys-prt still kept typical α-helical structure in the membrane mimicking environment, while D-prt showed left hand α-helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D-amino acid substitution or partially D-amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D-prt and D-Lys-prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

From N-triisopropylsilylpyrrole to an optically active C-4 substituted pyroglutamic acid: total synthesis of penmacric acid.

PubMed

Berini, Christophe; Pelloux-Léon, Nadia; Minassian, Frédéric; Denis, Jean-Noël

2009-11-07

The stereoselective synthesis of penmacric acid, an optically active C-4 substituted pyroglutamic acid, has been efficiently achieved through an unusual 11-step sequence starting from simple N-triisopropylsilylpyrrole. The key-steps are the initial addition of the pyrrole nucleus onto a chiral nitrone and the obtention of the pyroglutamic acid moiety by reductive hydrogenation of the pyrrole followed by oxidation of the corresponding pyrrolidine into pyrrolidinone.

40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Reaction products of secondary alkyl... Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric acid... substances identified generically as reaction products of secondary alkyl amines with a substituted...

40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Reaction products of secondary alkyl... Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric acid... substances identified generically as reaction products of secondary alkyl amines with a substituted...

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

PubMed

Biswal, Jitendra K; Das, Biswajit; Sharma, Gaurav K; Khulape, Sagar A; Pattnaik, Bramhadev

2016-04-01

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142 → R or H142 → F or H142 → A substitutions resulted in non-infectious FMDV, H142 → D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

FTIRI Parameters describing Acid Phosphate Substitution in Biologic Hydroxyapatite

PubMed Central

Spevak, Lyudmila; Flach, Carol R.; Hunter, Tracey; Mendelsohn, Richard; Boskey, Adele

2013-01-01

Acid phosphate substitution into mineralized tissue is an important determinant of their mechanical properties and their response to treatment. This study identifies and validates Fourier Transform Infrared Spectroscopic Imaging (FTIRI) spectral parameters that provide information on the acid phosphate (HPO4) substitution into hydroxyapatite in developing mineralized tissues. Curve fitting and Fourier self-deconvolution were used to identify subband positions in model compounds (with and without HPO4). The intensity of subbands at 1127 cm−1 and 1110 cm−1 correlated with the acid phosphate content in these models. Peak height ratios of these subbands to the ν3 vibration at 1096 cm−1 found in stoichiometric apatite, were evaluated in the model compounds and mixtures thereof. FTIRI spectra of bones and teeth at different developmental ages were analyzed using these spectral parameters. Factor analysis (a chemometric technique) was also conducted on the tissue samples and resulted in factor loadings with spectral features corresponding to the HPO4 vibrations described above. Images of both factor correlation coefficients and the peak height ratios 1127cm−1/1096cm−1 and 1112cm−1/1096cm−1 demonstrated higher acid phosphate content in younger vs. more mature regions in the same specimen. Maps of the distribution of acid phosphate content will be useful for characterizing the extent of new bone formation, areas of potential decreased strength, and the effects of therapies such as those used in metabolic bone diseases (osteoporosis, chronic kidney disease) on mineral composition. Because of the wider range of values obtained with the 1127 cm−1/1096 cm−1 parameter compared to the 1110 cm−1/1096 cm−1 parameter, and the smaller scatter in the slope, it is suggested that this ratio should be the parameter of choice. PMID:23380987

40 CFR 721.321 - Substituted acrylamides and acrylic acid copolymer (generic).

Code of Federal Regulations, 2010 CFR

2010-07-01

... AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.321 Substituted acrylamides and acrylic acid copolymer (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance...

Amino acid-substituted gemini surfactant-based nanoparticles as safe and versatile gene delivery agents.

PubMed

Singh, Jagbir; Yang, Peng; Michel, Deborah; Verrall, Ronald E; Foldvari, Marianna; Badea, Ildiko

2011-05-01

Gene based therapy represents an important advance in the treatment of diseases that heretofore have had either no treatment or cure. To capitalize on the true potential of gene therapy, there is a need to develop better delivery systems that can protect these therapeutic biomolecules and deliver them safely to the target sites. Recently, we have designed and developed a series of novel amino acid-substituted gemini surfactants with the general chemical formula C(12)H(25) (CH(3))(2)N(+)-(CH(2))(3)-N(AA)-(CH(2))(3)-N(+) (CH(3))(2)-C(12)H(25) (AA= glycine, lysine, glycyl-lysine and, lysyl-lysine). These compounds were synthesized and tested in rabbit epithelial cells using a model plasmid and a helper lipid. Plasmid/gemini/lipid (P/G/L) nanoparticles formulated using these novel compounds achieved higher gene expression than the nanoparticles containing the parent unsubstituted compound. In this study, we evaluated the cytotoxicity of P/G/L nanoparticles and explored the relationship between transfection efficiency/toxicity and their physicochemical characteristics (such as size, binding properties, etc.). An overall low toxicity is observed for all complexes with no significant difference among substituted and unsubstituted compounds. An interesting result revealed by the dye exclusion assay suggests a more balanced protection of the DNA by the glycine and glycyl-lysine substituted compounds. Thus, the higher transfection efficiency is attributed to the greater biocompatibility and flexibility of the amino acid/peptide-substituted gemini surfactants and demonstrates the feasibility of using amino acid-substituted gemini surfactants as gene carriers for the treatment of diseases affecting epithelial tissue.

N-Substituted carbazolyloxyacetic acids modulate Alzheimer associated gamma-secretase.

PubMed

Narlawar, Rajeshwar; Pérez Revuelta, Blanca I; Baumann, Karlheinz; Schubenel, Robert; Haass, Christian; Steiner, Harald; Schmidt, Boris

2007-01-01

N-Sulfonylated and N-alkylated carbazolyloxyacetic acids were investigated for the inhibition and modulation of the Alzheimer's disease associated gamma-secretase. The introduction of a lipophilic substituent, which may vary from arylsulfone to alkyl, turned 2-carbazolyloxyacetic acids into potent gamma-secretase modulators. This resulted in the selective reduction of Abeta(42) and an increase of the less aggregatory Abeta(38) fragment by several compounds (e.g., 7d and 8c). Introduction of an electron donating group at position 6 and 8 of N-substituted carbazolyloxyacetic acids either decreased the activity or inversed modulation. The most active compounds displayed activity on amyloid precursor protein (APP) overexpressing cell lines in the low micromolar range and little or no effect on the gamma-secretase cleavage at the epsilon-site.

Inhibition kinetics and molecular simulation of p-substituted cinnamic acid derivatives on tyrosinase.

PubMed

Cui, Yi; Hu, Yong-Hua; Yu, Feng; Zheng, Jing; Chen, Lin-Shan; Chen, Qing-Xi; Wang, Qin

2017-02-01

This study was to investigate the inhibition effects of para-substituted cinnamic acid derivatives (4-chlorocinnamic acid, 4-ethoxycinnamic acid and 4-nitrocinnamic acid) on tyrosinase catalyzing the substrates, with the purpose of elucidating the inhibition mechanism of the tested derivatives on tyrosinase by the UV-vis spectrum, fluorescence spectroscopy, copper interacting and molecular docking, respectively. The native-PAGE results showed that 4-chlorocinnamic acid (4-CCA), 4-ethoxycinnamic acid (4-ECA) and 4-nitrocinnamic acid (4-NCA) had inhibitory effects on tyrosinase. Spectrophotometric analysis used to determine the inhibition capabilities of these compounds on tyrosinase catalyzing L-tyrosine (L-Tyr) and L-3,4-Dihydroxyphenylalanine (L-DOPA) as well. The IC 50 values and inhibition constants were further determined. Moreover, quenching mechanisms of tested compounds to tyrosinase belonged to static type and a red shift on fluorescence emission peak occurred when 4-NCA added. Copper interacting and molecular docking demonstrated that 4-CCA could not bind directly to the copper, but it could interact with residues in the active center of tyrosinase. Meanwhile, 4-ECA and 4-NCA could chelate a copper ion of tyrosinase. Anti-tyrosinase activities of para-substituted cinnamic acid derivatives would lay scientific foundation for their utilization in designing of novel tyrosinase inhibitors. Copyright © 2016 Elsevier B.V. All rights reserved.

Gene-Specific Substitution Profiles Describe the Types and Frequencies of Amino Acid Changes during Antibody Somatic Hypermutation.

PubMed

Sheng, Zizhang; Schramm, Chaim A; Kong, Rui; Mullikin, James C; Mascola, John R; Kwong, Peter D; Shapiro, Lawrence

2017-01-01

Somatic hypermutation (SHM) plays a critical role in the maturation of antibodies, optimizing recognition initiated by recombination of V(D)J genes. Previous studies have shown that the propensity to mutate is modulated by the context of surrounding nucleotides and that SHM machinery generates biased substitutions. To investigate the intrinsic mutation frequency and substitution bias of SHMs at the amino acid level, we analyzed functional human antibody repertoires and developed mGSSP (method for gene-specific substitution profile), a method to construct amino acid substitution profiles from next-generation sequencing-determined B cell transcripts. We demonstrated that these gene-specific substitution profiles (GSSPs) are unique to each V gene and highly consistent between donors. We also showed that the GSSPs constructed from functional antibody repertoires are highly similar to those constructed from antibody sequences amplified from non-productively rearranged passenger alleles, which do not undergo functional selection. This suggests the types and frequencies, or mutational space, of a majority of amino acid changes sampled by the SHM machinery to be well captured by GSSPs. We further observed the rates of mutational exchange between some amino acids to be both asymmetric and context dependent and to correlate weakly with their biochemical properties. GSSPs provide an improved, position-dependent alternative to standard substitution matrices, and can be utilized to developing software for accurately modeling the SHM process. GSSPs can also be used for predicting the amino acid mutational space available for antigen-driven selection and for understanding factors modulating the maturation pathways of antibody lineages in a gene-specific context. The mGSSP method can be used to build, compare, and plot GSSPs; we report the GSSPs constructed for 69 common human V genes (DOI: 10.6084/m9.figshare.3511083) and provide high-resolution logo plots for each (DOI: 10

Emergence of fluoroquinolone-resistant Propionibacterium acnes caused by amino acid substitutions of DNA gyrase but not DNA topoisomerase IV.

PubMed

Nakase, Keisuke; Sakuma, Yui; Nakaminami, Hidemasa; Noguchi, Norihisa

2016-12-01

With the aim of elucidating the mechanisms of fluoroquinolones resistance in Propionibacterium acnes, we determined the susceptibility of fluoroquinolones in 211 isolates from patients with acne vulgaris. We identified five isolates (2.4%) with reduced susceptibility to nadifloxacin (minimum inhibitory concentration ≥ 4 μg/ml). Determination of the sequences of the DNA gyrase (gyrA and gyrB) and DNA topoisomerase (parC and parE) genes showed the amino acid substitutions Ser101Leu and Asp105Gly of GyrA in four and one of the isolates, respectively. In vitro mutation experiments showed that low-level fluoroquinolone-resistant mutants with the Ser101Leu or Asp105Gly substitution in GyrA could be obtained from selection with ciprofloxacin and levofloxacin. The pattern of substitution (Ser101Trp in GyrA) caused by nadifloxacin selection was different from that induced by the other fluoroquinolones. In the isolation of further high-level resistant mutants, acquisition of another amino acid substitution of GyrB in addition to those of GyrA was detected, but there were no substitutions of ParC and ParE. In addition, the mutant prevention concentration and mutation frequency of nadifloxacin were lowest among the tested fluoroquinolones. The growth of the Ser101Trp mutant was lower than that of the other mutants. Our findings suggest that the Ser101Trp mutant of P. acnes emerges rarely and disappears immediately, and the risk for the prevalence of fluoroquinolones-resistant P. acnes differs according to the GyrA mutation type. To our knowledge, this study is the first to demonstrate the mechanisms of resistance to fluoroquinolones in P. acnes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Thermodynamics of axial substitution and kinetics of reactions with amino acids for the paddlewheel complex tetrakis(acetato)chloridodiruthenium(II,III).

PubMed

Santos, Rodrigo L S R; van Eldik, Rudi; de Oliveira Silva, Denise

2012-06-18

The known paddlewheel, tetrakis(acetato)chloridodiruthenium(II,III), offers a versatile synthetic route to a novel class of antitumor diruthenium(II,III) metallo drugs, where the equatorial ligands are nonsteroidal anti-inflammatory carboxylates. This complex was studied here as a soluble starting prototype model for antitumor analogues to elucidate the reactivity of the [Ru(2)(CH(3)COO)(4)](+) framework. Thermodynamic studies on equilibration reactions for axial substitution of water by chloride and kinetic studies on reactions of the diaqua complexes with the amino acids glycine, cysteine, histidine, and tryptophan were performed. The standard thermodynamic reaction parameters ΔH°, ΔS°, and ΔV° were determined and showed that both of the sequential axial substitution reactions are enthalpy driven. Kinetic rate laws and rate constants were determined for the axial substitution reactions of coordinated water by the amino acids that gave the corresponding aqua(amino acid)-Ru(2) substituted species. The results revealed that the [Ru(2)(CH(3)COO)(4)](+) paddlewheel framework remained stable during the axial ligand substitution reactions and was also mostly preserved in the presence of the amino acids.

Pervasive adaptive protein evolution apparent in diversity patterns around amino acid substitutions in Drosophila simulans.

PubMed

Sattath, Shmuel; Elyashiv, Eyal; Kolodny, Oren; Rinott, Yosef; Sella, Guy

2011-02-10

In Drosophila, multiple lines of evidence converge in suggesting that beneficial substitutions to the genome may be common. All suffer from confounding factors, however, such that the interpretation of the evidence-in particular, conclusions about the rate and strength of beneficial substitutions-remains tentative. Here, we use genome-wide polymorphism data in D. simulans and sequenced genomes of its close relatives to construct a readily interpretable characterization of the effects of positive selection: the shape of average neutral diversity around amino acid substitutions. As expected under recurrent selective sweeps, we find a trough in diversity levels around amino acid but not around synonymous substitutions, a distinctive pattern that is not expected under alternative models. This characterization is richer than previous approaches, which relied on limited summaries of the data (e.g., the slope of a scatter plot), and relates to underlying selection parameters in a straightforward way, allowing us to make more reliable inferences about the prevalence and strength of adaptation. Specifically, we develop a coalescent-based model for the shape of the entire curve and use it to infer adaptive parameters by maximum likelihood. Our inference suggests that ∼13% of amino acid substitutions cause selective sweeps. Interestingly, it reveals two classes of beneficial fixations: a minority (approximately 3%) that appears to have had large selective effects and accounts for most of the reduction in diversity, and the remaining 10%, which seem to have had very weak selective effects. These estimates therefore help to reconcile the apparent conflict among previously published estimates of the strength of selection. More generally, our findings provide unequivocal evidence for strongly beneficial substitutions in Drosophila and illustrate how the rapidly accumulating genome-wide data can be leveraged to address enduring questions about the genetic basis of adaptation.

40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

Code of Federal Regulations, 2011 CFR

2011-07-01

... subject to this section: P-84-310, triethanolamine salt of a substituted organic acid. (b) Definitions... used in or as a metalworking fluid, which includes as one of its components P-84-310, is prohibited... metalworking fluid a product containing P-84-310 is prohibited from adding any nitrosating agent to the product...

D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP.

PubMed

Jia, Fengjing; Wang, Jiayi; Peng, Jinxiu; Zhao, Ping; Kong, Ziqing; Wang, Kairong; Yan, Wenjin; Wang, Rui

2017-10-01

With the increasing emergence of resistant microbes toward conventional antimicrobial agents, there is an urgent need for the development of antimicrobial agents with novel action mode. Antimicrobial peptides (AMPs) are believed to be one kind of ideal alternatives. However, AMPs can be easily degraded by protease, which limited their therapeutic use. In the present study, D-amino acid substitution strategy was employed to enhance the stability of polybia-CP. We investigated the stability of peptides against the degradation of trypsin and chymotrypsin by determining the antimicrobial activity or determining the HPLC profile of peptides after incubation with proteases. Our results showed that both the all D-amino acid derivative (D-CP) and partial D-lysine substitution derivative (D-lys-CP) have an improved stability against trypsin and chymotrypsin. Although D-CP takes left-hand α-helical conformation and D-lys-CP loses some α-helical content, both of the D-amino acid-substituted derivatives maintain their parental peptides' membrane active action mode. In addition, D-lys-CP showed a slight weaker antimicrobial activity than polybia-CP, but the hemolytic activity decreased greatly. These results suggest that D-CP and D-lys-CP can offer strategy to improve the property of AMPs and may be leading compounds for the development of novel antimicrobial agents. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Amino Acid Substitutions in PB1 of Avian Influenza Viruses Influence Pathogenicity and Transmissibility in Chickens

PubMed Central

Suzuki, Yasushi; Uchida, Yuko; Tanikawa, Taichiro; Maeda, Naohiro; Takemae, Nobuhiro

2014-01-01

ABSTRACT Amino acid substitutions were introduced into avian influenza virus PB1 in order to characterize the interaction between polymerase activity and pathogenicity. Previously, we used recombinant viruses containing the hemagglutinin (HA) and neuraminidase (NA) genes from the highly pathogenic avian influenza virus (HPAIV) H5N1 strain and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken and wild-bird hosts (LP and WB, respectively) to demonstrate that the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in chickens is regulated by the PB1 gene (Y. Uchida et al., J. Virol. 86:2686–2695, 2012, doi:http://dx.doi.org/10.1128/JVI.06374-11). In the present study, we introduced a C38Y substitution into WB PB1 and demonstrated that this substitution increased both polymerase activity in DF-1 cells in vitro and the pathogenicity of the recombinant viruses in chickens. The V14A substitution in LP PB1 reduced polymerase activity but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increased polymerase activity correlates directly with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis. IMPORTANCE We identified 2 novel amino acid substitutions in the avian influenza virus PB1 gene that affect the characteristics of highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype, such as viral replication and polymerase activity in vitro and pathogenicity and transmissibly in chickens. An amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens and was associated with changes in polymerase activity in vitro. A substitution at residue 14 reduced polymerase activity in vitro, while its effects on pathogenicity and transmissibility depended on the constellation of

Classification of group B streptococci with reduced β-lactam susceptibility (GBS-RBS) based on the amino acid substitutions in PBPs.

PubMed

Kimura, Kouji; Nagano, Noriyuki; Arakawa, Yoshichika

2015-01-01

All clinical isolates of group B Streptococcus (GBS; Streptococcus agalactiae) are considered uniformly susceptible to β-lactams, including penicillins. However, GBS with reduced penicillin susceptibility (PRGBS) were first identified by our group in Japan and have also been reported from North America. PRGBS are non-susceptible to penicillin because of acquisition of amino acid substitutions near the conserved active-site motifs in PBP2X. In particular, V405A and Q557E are considered the key amino acid substitutions responsible for penicillin non-susceptibility. We revealed that in addition to the substitutions in PBP2X, an amino acid substitution in PBP1A confers high-level cephalosporin resistance in GBS. As the number of publications on GBS with reduced β-lactam susceptibility (GBS-RBS), especially PRGBS, and concomitantly the need for a systematic classification of GBS-RBS is increasing, we propose here a classification of GBS-RBS based on the amino acid substitutions in their PBPs. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Nucleic acid detection kits

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

2005-03-29

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

Evaluating EDTA as a substitute for phosphoric acid-etching of enamel and dentin.

PubMed

Imbery, Terence A; Kennedy, Matthew; Janus, Charles; Moon, Peter C

2012-01-01

Matrix metalloproteinases (MMPs) are proteolytic enzymes released when dentin is acid-etched. The enzymes are capable of destroying unprotected collagen fibrils that are not encapsulated by the dentin adhesive. Chlorhexidine applied after etching inhibits the activation of released MMPs, whereas neutral ethylenediamine tetra-acetic acid (EDTA) prevents the release of MMPs. The purpose of this study was to determine if conditioning enamel and dentin with EDTA can be a substitute for treating acid-etching enamel and dentin with chlorhexidine. A column of composite resin was bonded to enamel and dentin after conditioning. Shear bond strengths were evaluated after 48 hours and after accelerated aging for three hours in 12% sodium hypochlorite. Shear bond strengths ranged from 15.6 MP a for accelerated aged EDTA enamel specimens to 26.8 MPa for dentin conditioned with EDTA and tested after 48 hours. A three-way ANOVA and a Tukey HSD test found statistically significant differences among the eight groups and the three independent variables (P < 0.05). EDTA was successfully substituted for phosphoric acid-etched enamel and dentin treated with chlorhexidine. Interactions of conditioning agent and aging were significant for dentin but not for enamel. In an effort to reduce the detrimental effects of MMPs, conditioning enamel and dentin with EDTA is an alternative to treating acid-etched dentin and enamel with chlorhexidine.

Identification of Amino Acid Substitutions Supporting Antigenic Change of Influenza A(H1N1)pdm09 Viruses

PubMed Central

Koel, Björn F.; Mögling, Ramona; Chutinimitkul, Salin; Fraaij, Pieter L.; Burke, David F.; van der Vliet, Stefan; de Wit, Emmie; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Smith, Derek J.; Fouchier, Ron A. M.

2015-01-01

ABSTRACT The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity. IMPORTANCE Influenza A viruses can cause significant morbidity and mortality in humans. Amino acid substitutions in the hemagglutinin protein can result in escape from antibody-mediated neutralization. This allows the virus to reinfect individuals that have acquired immunity to previously circulating strains through infection or vaccination. To date, the vast majority of A(H1N1)pdm09 strains remain antigenically similar to the virus

Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids

PubMed Central

2015-01-01

Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies. PMID:26505212

Efficient synthesis of hydroxystyrenes via biocatalytic decarboxylation/deacetylation of substituted cinnamic acids by newly isolated Pantoea agglomerans strains.

PubMed

Sharma, Upendra K; Sharma, Nandini; Salwan, Richa; Kumar, Rakesh; Kasana, Ramesh C; Sinha, Arun K

2012-02-01

Decarboxylation of substituted cinnamic acids is a predominantly followed pathway for obtaining hydroxystyrenes-one of the most extensively explored bioactive compounds in the food and flavor industry (e.g. FEMA GRAS approved 4-vinylguaiacol). For this, mild and green strategies providing good yields with high product selectivity are needed. Two newly isolated bacterial strains, i.e. Pantoea agglomerans KJLPB4 and P. agglomerans KJPB2, are reported for mild and effective decarboxylation of substituted cinnamic acids into corresponding hydroxystyrenes. Key operational parameters for the process, such as incubation temperature, incubation time, substrate concentration and effect of co-solvent, were optimized using ferulic acid as a model substrate. With strain KJLPB4, 1.51 g L⁻¹ 4-vinyl guaiacol (98% yield) was selectively obtained from 2 g L⁻¹ ferulic acid at 28 °C after 48 h incubation. However, KJPB2 provided vanillic acid in 85% yield after 72 h following the oxidative decarboxylation pathway. In addition, KJLPB4 was effectively exploited for the deacetylation of acetylated α-phenylcinnamic acids, providing corresponding compounds in 65-95% yields. Two newly isolated microbial strains are reported for the mild and selective decarboxylation of substituted cinnamic acids into hydroxystyrenes. Preparative-scale synthesis of vinyl guaiacol and utilization of renewable feedstock (ferulic acid extracted from maize bran) have been demonstrated to enhance the practical utility of the process. Copyright © 2011 Society of Chemical Industry.

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Synthesis and stereochemical analysis of β-nitromethane substituted γ-amino acids and peptides.

PubMed

Ganesh Kumar, Mothukuri; Mali, Sachitanand M; Gopi, Hosahudya N

2013-02-07

The high diastereoselectivity in the Michael addition of nitromethane to α,β-unsaturated γ-amino esters, crystal conformations of β-nitromethane substituted γ-amino acids and peptides are studied. Results suggest that the N-Boc protected amide NH, conformations of α,β-unsaturated γ-amino esters and alkyl side chains play a crucial role in dictating the high diastereoselectivity of nitromethane addition to E-vinylogous amino esters. Investigation of the crystal conformations of both α,β-unsaturated γ-amino esters and the Michael addition products suggests that an H-C(γ)-C(β)=C(α) eclipsed conformer of the unsaturated amino ester leads to the major (anti) product compared to that of an N-C(γ)-C(β)=C(α) eclipsed conformer. The major diastereomers were separated and subjected to the peptide synthesis. The single crystal analysis of the dipeptide containing β-nitromethane substituted γ-amino acids reveals a helical type of folded conformation with an isolated H-bond involving a nine-atom pseudocycle.

Synthesis of ω-Oxo Amino Acids and trans-5-Substituted Proline Derivatives Using Cross-Metathesis of Unsaturated Amino Acids.

PubMed

Salih, Nabaz; Adams, Harry; Jackson, Richard F W

2016-09-16

A range of 7-oxo, 8-oxo, and 9-oxo amino acids, analogues of 8-oxo-2-aminodecanoic acid, one of the key components of the cyclic tetrapeptide apicidin, have been prepared by a three-step process involving copper-catalyzed allylation of serine-, aspartic acid-, and glutamic acid-derived organozinc reagents, followed by cross-metathesis of the resulting terminal alkenes with unsaturated ketones and hydrogenation. The intermediate 7-oxo-5-enones underwent a highly diastereoselective (dr ≥96:4) acid-catalyzed aza-Michael reaction to give trans-2,5-disubstituted pyrrolidines, 5-substituted proline derivatives. The aza-Michael reaction was first observed when the starting enones were allowed to stand in solution in deuterochloroform but can be efficiently promoted by catalytic amounts of dry HCl.

Acidic Fluids Across Mars: Detections of Magnesium-Nickel Sulfates

NASA Technical Reports Server (NTRS)

Yen, A. S.; Ming, D. W.; Gellert, R.; Mittlefehldt, D. W.; Rampe, E. B.; Vaniman, D. T.; Thompson, L. M.; Morris, R. V.; Clark, B. C.; VanBommel, S. J.

2017-01-01

Calcium, magnesium and ferric iron sulfates have been detected by the instrument suites on the Mars rovers. A subset of the magnesium sulfates show clear associations with nickel. These associations indicate Ni(2+) co-precipitation with or substitution for Mg(2+) from sulfate-saturated solutions. Nickel is ex-tracted from primary rocks almost exclusively at pH values less than 6, constraining the formation of these Mg-Ni sulfates to mildly to strongly acidic conditions. There is clear evidence for aqueous alteration at the rim of Endeavour Crater (Meridiani Planum), in the Murray formation mudstone (Gale Crater), and near Home Plate (Gusev Crater). The discovery of Mg-Ni sulfates at these locations indicates a history of fluid-rock interactions at low pH.

Nucleophilic Aromatic Substitution.

ERIC Educational Resources Information Center

Avila, Walter B.; And Others

1990-01-01

Described is a microscale organic chemistry experiment which demonstrates one feasible route in preparing ortho-substituted benzoic acids and provides an example of nucleophilic aromatic substitution chemistry. Experimental procedures and instructor notes for this activity are provided. (CW)

Substitutions in position 222 of haemagglutinin of pandemic influenza A (H1N1) 2009 viruses in Spain.

PubMed

Ledesma, Juan; Pozo, Francisco; Pérez Ruiz, Mercedes; Navarro, Jose María; Piñeiro, Luis; Montes, Milagros; Pérez Castro, Sonia; Suárez Fernández, Jonathan; García Costa, Juan; Fernández, Mirian; Galán, Juan Carlos; Cuevas, María Teresa; Casas, Inmaculada; Pérez Breña, Pilar

2011-05-01

A change of aspartic acid (D) to glycine (G) at position 222 in the haemagglutinin (HA) protein of pandemic influenza A (H1N1) 2009 viruses was described in Norway on November 2009 with considerable frequency in fatal and severe cases. This change was detected in other countries and was related only with severe disease. Other substitutions to glutamic acid (E) or asparagine (N) at position 222 were detected among pandemic viruses but it is unclear what implications might have in terms of severity. To analyse the appearance of amino acid substitutions at position 222 in the HA protein of circulating viruses in Spain and to determine their relationships with the disease symptoms observed. Pandemic influenza A (H1N1) 2009 viruses detected in respiratory samples of 273 severe and 533 non-severe cases from different Spanish regions were selected for sequencing of a partial segment of HA1 subunit and studied to monitor substitutions at position 222. D222G substitution was only detected in viruses from 14 severe cases (5.12%). D222E was found in viruses from 47 severe (17.21%) and from 52 non-severe cases (9.75%). D222N occurred in viruses from 3 additional severe cases (0.37%). Appearance of D222G and D222E substitution in HA of pandemic influenza A (H1N1) viruses circulating in Spain might be related with severe respiratory disease. Copyright © 2011 Elsevier B.V. All rights reserved.

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

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

Zhang, Xinsheng, E-mail: xzhang@iavi.org; Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY; Wallace, Olivia L.

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions,more » which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.« less

Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

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

Xi, T; Jones, I M; Mohrenweiser, H W

2003-11-03

Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of themore » variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.« less

40 CFR 721.5262 - 2,7-Naphthalenedisulfonic acid, 5-[[4-chloro-6-[substituted] amino]-1,3,5-triazin-2-yl]amino]-4...

Code of Federal Regulations, 2011 CFR

2011-07-01

... identified generically as 2,7-Naphthalenedisulfonic acid, 5-[[4-chloro-6-[substituted] amino]-1,3,5-triazin-2... 40 Protection of Environment 31 2011-07-01 2011-07-01 false 2,7-Naphthalenedisulfonic acid, 5-[[4-chloro-6-[substituted] amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(1-sulfo-2-naphthalenyl)azo...

40 CFR 721.5262 - 2,7-Naphthalenedisulfonic acid, 5-[[4-chloro-6-[substituted] amino]-1,3,5-triazin-2-yl]amino]-4...

Code of Federal Regulations, 2010 CFR

2010-07-01

... identified generically as 2,7-Naphthalenedisulfonic acid, 5-[[4-chloro-6-[substituted] amino]-1,3,5-triazin-2... 40 Protection of Environment 30 2010-07-01 2010-07-01 false 2,7-Naphthalenedisulfonic acid, 5-[[4-chloro-6-[substituted] amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(1-sulfo-2-naphthalenyl)azo...

Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

PubMed

Donmez, Soner; Arslan, Fatma; Arslan, Halit

2016-05-01

In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor.

Identification by random forest method of HLA class I amino acid substitutions associated with lower survival at day 100 in unrelated donor hematopoietic cell transplantation.

PubMed

Marino, S R; Lin, S; Maiers, M; Haagenson, M; Spellman, S; Klein, J P; Binkowski, T A; Lee, S J; van Besien, K

2012-02-01

The identification of important amino acid substitutions associated with low survival in hematopoietic cell transplantation (HCT) is hampered by the large number of observed substitutions compared with the small number of patients available for analysis. Random forest analysis is designed to address these limitations. We studied 2107 HCT recipients with good or intermediate risk hematological malignancies to identify HLA class I amino acid substitutions associated with reduced survival at day 100 post transplant. Random forest analysis and traditional univariate and multivariate analyses were used. Random forest analysis identified amino acid substitutions in 33 positions that were associated with reduced 100 day survival, including HLA-A 9, 43, 62, 63, 76, 77, 95, 97, 114, 116, 152, 156, 166 and 167; HLA-B 97, 109, 116 and 156; and HLA-C 6, 9, 11, 14, 21, 66, 77, 80, 95, 97, 99, 116, 156, 163 and 173. In all 13 had been previously reported by other investigators using classical biostatistical approaches. Using the same data set, traditional multivariate logistic regression identified only five amino acid substitutions associated with lower day 100 survival. Random forest analysis is a novel statistical methodology for analysis of HLA mismatching and outcome studies, capable of identifying important amino acid substitutions missed by other methods.

Amino Acid Substitution in Trichophyton rubrum Squalene Epoxidase Associated with Resistance to Terbinafine

PubMed Central

Osborne, Colin S.; Leitner, Ingrid; Favre, Bertrand; Ryder, Neil S.

2005-01-01

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE. PMID:15980358

Fourier transform infrared spectroscopic imaging parameters describing acid phosphate substitution in biologic hydroxyapatite.

PubMed

Spevak, Lyudmila; Flach, Carol R; Hunter, Tracey; Mendelsohn, Richard; Boskey, Adele

2013-05-01

Acid phosphate substitution into mineralized tissues is an important determinant of their mechanical properties and their response to treatment. This study identifies and validates Fourier transform infrared spectroscopic imaging (FTIRI) spectral parameters that provide information on the acid phosphate (HPO4) substitution into hydroxyapatite in developing mineralized tissues. Curve fitting and Fourier self-deconvolution were used to identify subband positions in model compounds (with and without HPO4). The intensity of subbands at 1127 and 1110 cm(-1) correlated with the acid phosphate content in these models. Peak height ratios of these subbands to the ν3 vibration at 1096 cm(-1) found in stoichiometric apatite were evaluated in the model compounds and mixtures thereof. FTIRI spectra of bones and teeth at different developmental ages were analyzed using these spectral parameters. Factor analysis (a chemometric technique) was also conducted on the tissue samples and resulted in factor loadings with spectral features corresponding to the HPO4 vibrations described above. Images of both factor correlation coefficients and the peak height ratios 1127/1096 and 1112/1096 cm(-1) demonstrated higher acid phosphate content in younger vs. more mature regions in the same specimen. Maps of the distribution of acid phosphate content will be useful for characterizing the extent of new bone formation, the areas of potential decreased strength, and the effects of therapies such as those used in metabolic bone diseases (osteoporosis, chronic kidney disease) on mineral composition. Because of the wider range of values obtained with the 1127/1096 cm(-1) parameter compared to the 1110/1096 cm(-1) parameter and the smaller scatter in the slope, it is suggested that this ratio should be the parameter of choice.

Facile on-site detection of substituted aromatic pollutants in water using thin layer chromatography combined with surface-enhanced Raman spectroscopy.

PubMed

Li, Dawei; Qu, Lulu; Zhai, Wenlei; Xue, Jinqun; Fossey, John S; Long, Yitao

2011-05-01

A novel facile method for on-site detection of substituted aromatic pollutants in water using thin layer chromatography (TLC) combined with surface-enhanced Raman spectroscopy (SERS) was explored. Various substituted aromatics in polluted water were separated by a convenient TLC protocol and then detected using a portable Raman spectrometer with the prepared silver colloids serving as SERS-active substrates. The effects of operating conditions on detection efficacy were evaluated, and the application of TLC-SERS to on-site detection of artificial and real-life samples of aromatics/polluted water was systematically investigated. It was shown that commercially available Si 60-F(254) TLC plates were suitable for separation and displayed low SERS background and good separation efficiency, 2 mM silver colloids, 20 mM NaCl (working as aggregating agent), 40 mW laser power, and 50 s intergration time were appropriate for the detection regime. Furthermore, qualitative and quantitative detection of most of substituted aromatic pollutants was found to be readily accomplished using the developed TLC-SERS technique, which compared well with GC-MS in terms of identification ability and detection accuracy, and a limit of detection (LOD) less than 0.2 ppm (even at ppb level for some analytes) could be achieved under optimal conditions. The results reveal that the presented convenient method could be used for the effective separation and detection of the substituted aromatic pollutants of water on site, thus reducing possible influences of sample transportation and contamination while shortening the overall analysis time for emergency and routine monitoring of the substituted aromatics/polluted water.

Lewis acid tuned facial stereodivergent HDA reactions using beta-substituted N-vinyloxazolidinones.

PubMed

Gohier, Frédéric; Bouhadjera, Keltoum; Faye, Djibril; Gaulon, Catherine; Maisonneuve, Vincent; Dujardin, Gilles; Dhal, Robert

2007-01-18

The [4 + 2] acido-catalyzed heterocycloaddition between new beta-substituted N-vinyl-1,3-oxazolidin-2-ones (with R' = Me, Ar, CH2 Ar) and beta,gamma-unsaturated alpha-ketoesters (R = Ar) afforded heteroadducts with high levels of endo and facial selectivities. A complete reversal of facial differentiation was achieved by varying the Lewis acid, leading to the stereoselective formation of either endo-alpha or endo-beta adducts. [reaction: see text].

Improved Synthesis of 5-Substituted 1H-Tetrazoles via the [3+2] Cycloaddition of Nitriles and Sodium Azide Catalyzed by Silica Sulfuric Acid

PubMed Central

Du, Zhenting; Si, Changmei; Li, Youqiang; Wang, Yin; Lu, Jing

2012-01-01

A silica supported sulfuric acid catalyzed [3+2] cycloaddition of nitriles and sodium azide to form 5-substituted 1H-tetrazoles is described. The protocol can provide a series of 5-substituted 1H-tetrazoles using silica sulfuric acid from nitriles and sodium azide in DMF in 72%–95% yield. PMID:22606004

Electronic Effects of 11β Substituted 17β-Estradiol Derivatives and Instrumental Effects on the Relative Gas Phase Acidity

NASA Astrophysics Data System (ADS)

Bourgoin-Voillard, Sandrine; Fournier, Françoise; Afonso, Carlos; Zins, Emilie-Laure; Jacquot, Yves; Pèpe, Claude; Leclercq, Guy; Tabet, Jean-Claude

2012-12-01

Numerous studies have highlighted the role of the proton donor characteristics of the phenol group of 17β-estradiol (E2) in its association with the estrogen receptor alpha (ERα). Since the substitutions at position C(11) have been reported to modulate this association, we hypothesized that such substitutions may modify the phenol acidity. Hence, phenol gas-phase acidity of nine C(11)-substituted E2-derivatives were evaluated using the extended Cooks' kinetic method, which is a method widely used to determine thermochemical properties by mass spectrometry. To enhance accuracy in data collection we recorded data from several instruments, including quadrupole ion trap, triple quadrupole, and hybrid QqTOF. Indeed, we report for the first time the use of the QqTOF instrument to provide a novel means to improve data accuracy by giving access to an intermediate effective temperature range. All experimental gas-phase acidity values were supported by theoretical calculations. Our results confirmed the ability of distant substituents at C(11) to modulate the phenol acidity through electrostatic interactions, electron withdrawing inductive effects, and mesomeric effects. However, no relationship was found between the phenol gas-phase acidity of investigated steroids and their binding affinity for ERα assessed in solution. Thus, our results highlight that the intrinsic properties of the hormone do not influence sufficiently the stabilization of the hormone/ERα complex. It is more likely that such stabilization would be more related to factors depending on the environment within the binding pocket such as hydrophobic, steric as well as direct intermolecular electrostatic effects between ERα residues and the substituted steroidal estrogens.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

PubMed

Skoczinski, Pia; Volkenborn, Kristina; Fulton, Alexander; Bhadauriya, Anuseema; Nutschel, Christina; Gohlke, Holger; Knapp, Andreas; Jaeger, Karl-Erich

2017-09-25

Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an

Omega-3 Fatty Acid Formulations in Cardiovascular Disease: Dietary Supplements are Not Substitutes for Prescription Products.

PubMed

Fialkow, Jonathan

2016-08-01

Omega-3 fatty acid products are available as prescription formulations (icosapent ethyl, omega-3-acid ethyl esters, omega-3-acid ethyl esters A, omega-3-carboxylic acids) and dietary supplements (predominantly fish oils). Most dietary supplements and all but one prescription formulation contain mixtures of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Products containing both EPA and DHA may raise low-density lipoprotein cholesterol (LDL-C). In clinical trials, the EPA-only prescription product, icosapent ethyl, did not raise LDL-C compared with placebo. To correct a common misconception, it is important to note that omega-3 fatty acid dietary supplements are not US FDA-approved over-the-counter drugs and are not required to demonstrate safety and efficacy prior to marketing. Conversely, prescription products are supported by extensive clinical safety and efficacy investigations required for FDA approval and have active and ongoing safety monitoring programs. While omega-3 fatty acid dietary supplements may have a place in the supplementation of diet, they generally contain lower levels of EPA and DHA than prescription products and are not approved or intended to treat disease. Perhaps due to the lack of regulation of dietary supplements, EPA and DHA levels may vary widely within and between brands, and products may also contain unwanted cholesterol or fats or potentially harmful components, including toxins and oxidized fatty acids. Accordingly, omega-3 fatty acid dietary supplements should not be substituted for prescription products. Similarly, prescription products containing DHA and EPA should not be substituted for the EPA-only prescription product, as DHA may raise LDL-C and thereby complicate the management of patients with dyslipidemia.

Detection of triterpene acids distribution in loquat (Eriobotrya japonica) leaf using hyperspectral imaging

NASA Astrophysics Data System (ADS)

Shi, Jiyong; Chen, Wu; Zou, Xiaobo; Xu, Yiwei; Huang, Xiaowei; Zhu, Yaodi; Shen, Tingting

2018-01-01

Hyperspectral images (431-962 nm) and partial least squares (PLS) were used to detect the distribution of triterpene acids within loquat (Eriobotrya japonica) leaves. 72 fresh loquat leaves in the young group, mature group and old group were collected for hyperspectral imaging; and triterpene acids content of the loquat leaves was analyzed using high performance liquid chromatography (HPLC). Then the spectral data of loquat leaf hyperspectral images and the triterpene acids content were employed to build calibration models. After spectra pre-processing and wavelength selection, an optimum calibration model (Rp = 0.8473, RMSEP = 2.61 mg/g) for predicting triterpene acids was obtained by synergy interval partial least squares (siPLS). Finally, spectral data of each pixel in the loquat leaf hyperspectral image were extracted and substituted into the optimum calibration model to predict triterpene acids content of each pixel. Therefore, the distribution map of triterpene acids content was obtained. As shown in the distribution map, triterpene acids are accumulated mainly in the leaf mesophyll regions near the main veins, and triterpene acids concentration of young group is less than that of mature and old groups. This study showed that hyperspectral imaging is suitable to determine the distribution of active constituent content in medical herbs in a rapid and non-invasive manner.

Kinetic resolution and stereoselective synthesis of 3-substituted aspartic acids by using engineered methylaspartate ammonia lyases.

PubMed

Raj, Hans; Szymanski, Wiktor; de Villiers, Jandré; Puthan Veetil, Vinod; Quax, Wim J; Shimamoto, Keiko; Janssen, Dick B; Feringa, Ben L; Poelarends, Gerrit J

2013-08-19

Enzymatic amino acid synthesis: Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to >98:2 and enantiomeric excess values of up to >99 %, by using engineered methylaspartate ammonia lyases are described. These biocatalytic methodologies for the selective preparation of aspartic acid derivatives appear to be attractive alternatives for existing chemical methods. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluating mineralogy at terrestrial analogs for early Mars: Detection and characterization of clays with XRD and investigation of iron substitution in natroalunite

NASA Astrophysics Data System (ADS)

Beckerman, Laura Grace

The Mars Science Laboratory (MSL) Curiosity rover is equipped with CheMin, the first x-ray diffraction (XRD) instrument on Mars, for in situ mineralogy as part of its mission to seek evidence of past habitability at Gale Crater. Detection and characterization of hydrated minerals like clays and sulfates provides crucial insight into Mars' early geochemistry. For example, clays are often interpreted as having formed in lacustrine environments at neutral pHs, while sulfates such as jarosite are evidence of acid sulfate alteration. However, CheMin's inability to remove non-clay minerals and to preferentially orient samples may pose significant challenges to clay detection and characterization at Gale Crater. To evaluate the effect of particle size separation (<0.2 microm), removal of non-clay minerals, preferred orientation, and ethylene glycol solvation on XRD analyses of clays, we used both a CheMin analog instrument and a traditional laboratory XRD to identify clays in acid sulfate altered basalt from Mars analog sites in Costa Rica. We detected kaolinite in four of the fourteen samples studied, one of which also contained montmorillonite. Kaolinite was not detected in two samples with the analog instrument prior to clay isolation. These results suggest that CheMin may miss detection of some clays at Gale Crater, which could affect interpretations of early Mars' habitability. Mistaking iron-rich natroalunite (Na[Al,Fe]3(SO4) 2(OH)6) for jarosite (KFe3(SO4) 2(OH)6) could also impact interpretations of early Mars, as natroalunite can form over a broader range of pH, water:rock ratios, and redox conditions than can jarosite. To determine if iron-rich natroalunite is a common alteration product at Mars analog sites, we assessed iron content in natroalunite from Costa Rica. We detected up to 30% iron substitution in natroalunite at diverse geochemical settings. We also evaluated the feasibility of using XRD or Raman spectroscopy for in situ iron-rich natroalunite

β-Dicyanovinyl substituted porphyrinogen: synthesis, a reversible sensor for picric acid among explosives and a unique sensor for cyanide and fluoride ions by switching between various porphyrinoid states.

PubMed

Chahal, Mandeep K; Sankar, Muniappan

2017-09-12

β-Dicyanovinyl substituted porphyrinogen (OxP-MN) was synthesized and utilized as a novel multifunctional sensor for the detection of biologically and environmentally important analytes. OxP-MN (1) acts as a selective and reversible probe for rapid colorimetric detection of picric acid (PA) among other nitroaromatics by switching between two porphyrinoid states. This system displayed a higher β 2 value of 1.7 × 10 8 M -2 and was able to detect PA down to 1.12 ppm (4.99 μM). β-Dicyanovinyl substituted porphyrinogen (OxP-MN) reported here contains a porphyrinogen anion binding site and a dicyanovinyl group as a cyanide-dependent reactive subunit. OxP-MN displayed the first evidence that a β-electron acceptor through a vinyl linker in the case of porphyrinogen results in only an abated shift in the spectrum in contrast to its porphyrin analogues. Porphyrinogen OxP-MN (1) can be switched between a number of porphyrinoid states such as metalloporphodimethene, metalloporphyrin, porphyrinogen, etc. by using CN - , F - and other basic anions. In addition, OxP-MN unveils the unique property of detecting toxic cyanide ions and fluoride ions when "hidden" within a mixture of other anions. Also, OxP-MN behaves as a dual sensor for picric acid and basic anions such as F - , CN - , OAc - , and H 2 PO 4 - via the indicator displacement assay under the unrestricted queue.

Nucleic acid detection assays

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

2005-04-05

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

Substituent and solvent effects on electronic spectra of some substituted phenoxyacetic acids.

PubMed

Shanthi, M; Kabilan, S

2007-06-01

The effects of substituents and solvents have been studied through the absorption spectra of nearly 19 para- and ortho-substituted phenoxyacetic acids in the range of 200-400 nm. The effects of substituent on the absorption spectra of compounds under present investigation are interpreted by correlation of absorption frequencies with simple and extended Hammett equations. Effect of solvent polarity and hydrogen bonding on the absorption spectra are interpreted by means of Kamlet equation and the results are discussed.

Substituent and solvent effects on electronic spectra of some substituted phenoxyacetic acids

NASA Astrophysics Data System (ADS)

Shanthi, M.; Kabilan, S.

2007-06-01

The effects of substituents and solvents have been studied through the absorption spectra of nearly 19 para- and ortho-substituted phenoxyacetic acids in the range of 200-400 nm. The effects of substituent on the absorption spectra of compounds under present investigation are interpreted by correlation of absorption frequencies with simple and extended Hammett equations. Effect of solvent polarity and hydrogen bonding on the absorption spectra are interpreted by means of Kamlet equation and the results are discussed.

Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.

PubMed

Xiang, Kuan-Hui; Michailidis, Eleftherios; Ding, Hai; Peng, Ya-Qin; Su, Ming-Ze; Li, Yao; Liu, Xue-En; Dao Thi, Viet Loan; Wu, Xian-Fang; Schneider, William M; Rice, Charles M; Zhuang, Hui; Li, Tong

2017-02-01

As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These

Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry.

PubMed

Blincoe, William D; Rodriguez-Granillo, Agustina; Saurí, Josep; Pierson, Nicholas A; Joyce, Leo A; Mangion, Ian; Sheng, Huaming

2018-04-01

Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighboring group participation (NGP) effect to differentiate ortho-substituted benzoic acid/ester derivatives with high resolution mass spectrometry (HRMS 1 ). Significant water/alcohol loss (>30% abundance in MS 1 spectra) was observed for ortho-substituted nucleophilic groups; these fragment peaks are not observable for the corresponding para and meta-substituted analogs. Experiments were also extended to the analysis of two intermediates in the synthesis of suvorexant (Belsomra) with additional analysis conducted with nuclear magnetic resonance (NMR), density functional theory (DFT), and ion mobility spectrometry-mass spectrometry (IMS-MS) studies. Significant water/alcohol loss was also observed for 1-substituted 1, 2, 3-triazoles but not for the isomeric 2-substituted 1, 2, 3-triazole analogs. IMS-MS, NMR, and DFT studies were conducted to show that the preferred orientation of the 2-substituted triazole rotamer was away from the electrophilic center of the reaction, whereas the 1-subtituted triazole was oriented in close proximity to the center. Abundance of NGP product was determined to be a product of three factors: (1) proton affinity of the nucleophilic group; (2) steric impact of the nucleophile; and (3) proximity of the nucleophile to carboxylic acid/ester functional groups. Graphical Abstract ᅟ.

Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry

NASA Astrophysics Data System (ADS)

Blincoe, William D.; Rodriguez-Granillo, Agustina; Saurí, Josep; Pierson, Nicholas A.; Joyce, Leo A.; Mangion, Ian; Sheng, Huaming

2018-02-01

Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighboring group participation (NGP) effect to differentiate ortho-substituted benzoic acid/ester derivatives with high resolution mass spectrometry (HRMS1). Significant water/alcohol loss (>30% abundance in MS1 spectra) was observed for ortho-substituted nucleophilic groups; these fragment peaks are not observable for the corresponding para and meta-substituted analogs. Experiments were also extended to the analysis of two intermediates in the synthesis of suvorexant (Belsomra) with additional analysis conducted with nuclear magnetic resonance (NMR), density functional theory (DFT), and ion mobility spectrometry-mass spectrometry (IMS-MS) studies. Significant water/alcohol loss was also observed for 1-substituted 1, 2, 3-triazoles but not for the isomeric 2-substituted 1, 2, 3-triazole analogs. IMS-MS, NMR, and DFT studies were conducted to show that the preferred orientation of the 2-substituted triazole rotamer was away from the electrophilic center of the reaction, whereas the 1-subtituted triazole was oriented in close proximity to the center. Abundance of NGP product was determined to be a product of three factors: (1) proton affinity of the nucleophilic group; (2) steric impact of the nucleophile; and (3) proximity of the nucleophile to carboxylic acid/ester functional groups. [Figure not available: see fulltext.

Immunohistochemical detection of a substituted 1,N(2)-ethenodeoxyguanosine adduct by omega-6 polyunsaturated fatty acid hydroperoxides in the liver of rats fed a choline-deficient, L-amino acid-defined diet.

PubMed

Kawai, Yoshichika; Kato, Yoji; Nakae, Dai; Kusuoka, Osamu; Konishi, Yoichi; Uchida, Koji; Osawa, Toshihiko

2002-03-01

Endogenous lipid peroxidation products react with DNA and form exocyclic DNA adducts. The purpose of this study was to investigate the in vivo formation of 7-(2-oxo-heptyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct (Oxo-heptyl-varepsilondG), one of the major products from the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with dG. The monoclonal antibody specific to Oxo-heptyl-varepsilondG was prepared using a chemically synthesized conjugate of Oxo-heptyl-varepsilondG and carrier protein as immunogen. The characterization showed that the obtained antibody (mAb6A3) is specific to the Oxo-heptyl-varepsilondG moiety. Using the novel antibody, the presence of the Oxo-heptyl-varepsilondG adduct in vivo was immunohistochemically revealed in the liver of rats fed a choline-deficient, L-amino acid-defined diet, an endogenous carcinogenesis model, for 3 days. In addition, the Oxo-heptyl-varepsilondG formation was confirmed in DNAs treated with peroxidized linoleic acid, arachidonic acid and gamma-linolenic acid, respectively, suggesting that the hydroperoxides of omega-6 polyunsaturated fatty acids could be the potential sources of Oxo-heptyl-varepsilondG formation in vivo. Collectively, the results in this study suggest the first evidence that the formation of Oxo-heptyl-varepsilondG, the omega-6 lipid hydroperoxide-mediated DNA adduct, may be a potential biomarker for the early phase of carcinogenesis.

Substitution and addition reactions of •OH with p-substituted-phenols

NASA Astrophysics Data System (ADS)

Albarrán, Guadalupe; Galicia-Jiménez, Eduardo; Mendoza, Edith; Schuler, Robert H.

2017-04-01

The directing effect of a hydroxyl group on the substitution and addition reactions of •OH to the substituted and free positions in aromatic rings of p-substituted-phenols were studied in aqueous solutions containing either K3Fe(CN)6 as an oxidant of the substituted hydroxycyclohexadienyl radical initially formed or using ascorbic acid. The results showed that the attack of the •OH to the substituted position (ipso position) was followed by elimination of the substituent producing hydroquinone. The addition reaction of the •OH to the free position on the ring produced 4-substituent-catechol and 4-substituent-resorcinol derivatives. Identification and quantification of the radiolytic products were carried out using high performance liquid chromatography. The results of the yields are given for the p-halogen-phenols (p-X-Ph) p-F-Ph, p-Cl-Ph, p-Br-Ph and p-I-Ph. Other compounds, p-nitro-Ph, p-OH-benzoic acid, p-OH-benzonitrile, p-OH-benzaldehyde, p-OH-anisole and p-OH-benzyl alcohol (represented as p-Z-Ph), were only studied using K3Fe(CN)6 as the oxidant. The results show that the p-X-Ph are attacked by the •OH at the ipso position to the halogen in the proportion 1:0.53:0.46:0.11 for F>Cl>Br>I. The •OH attacked at the ipso position to the p-Z-Phs through a substitution reaction, which depended on the substituent group. Thus, the strongly deactivating groups produced less hydroquinone, indicating less substitution reaction than the strongly activating groups.

40 CFR 72.41 - Phase I substitution plans.

Code of Federal Regulations, 2010 CFR

2010-07-01

... (CONTINUED) PERMITS REGULATION Acid Rain Compliance Plan and Compliance Options § 72.41 Phase I substitution... section 410 of the Act. (b)(1) The designated representative may include, in the Acid Rain permit... an Acid Rain permit containing the plan, except that if the substitution plan is conditionally...

40 CFR 72.41 - Phase I substitution plans.

Code of Federal Regulations, 2012 CFR

2012-07-01

... (CONTINUED) PERMITS REGULATION Acid Rain Compliance Plan and Compliance Options § 72.41 Phase I substitution... section 410 of the Act. (b)(1) The designated representative may include, in the Acid Rain permit... an Acid Rain permit containing the plan, except that if the substitution plan is conditionally...

40 CFR 72.41 - Phase I substitution plans.

Code of Federal Regulations, 2013 CFR

2013-07-01

... (CONTINUED) PERMITS REGULATION Acid Rain Compliance Plan and Compliance Options § 72.41 Phase I substitution... section 410 of the Act. (b)(1) The designated representative may include, in the Acid Rain permit... an Acid Rain permit containing the plan, except that if the substitution plan is conditionally...

40 CFR 72.41 - Phase I substitution plans.

Code of Federal Regulations, 2011 CFR

2011-07-01

... (CONTINUED) PERMITS REGULATION Acid Rain Compliance Plan and Compliance Options § 72.41 Phase I substitution... section 410 of the Act. (b)(1) The designated representative may include, in the Acid Rain permit... an Acid Rain permit containing the plan, except that if the substitution plan is conditionally...

40 CFR 72.41 - Phase I substitution plans.

Code of Federal Regulations, 2014 CFR

2014-07-01

... (CONTINUED) PERMITS REGULATION Acid Rain Compliance Plan and Compliance Options § 72.41 Phase I substitution... section 410 of the Act. (b)(1) The designated representative may include, in the Acid Rain permit... an Acid Rain permit containing the plan, except that if the substitution plan is conditionally...

High temperature dissolution of chromium substituted nickel ferrite in nitrilotriacetic acid medium

NASA Astrophysics Data System (ADS)

Sathyaseelan, V. S.; Chandramohan, P.; Velmurugan, S.

2016-12-01

High temperature (HT) dissolution of chromium substituted nickel ferrite was carried out with relevance to the decontamination of nuclear reactors by way of chemical dissolution of contaminated corrosion product oxides present on stainless steel coolant circuit surfaces. Chromium substituted nickel ferrites of composition, NiFe(2-x)CrxO4 (x ≤ 1), was synthetically prepared and characterized. HT dissolution of these oxides was carried out in nitrilotriacetic acid medium at 160 °C. Dissolution was remarkably increased at 160 °C when compared to at 85 °C in a reducing decontamination formulation. Complete dissolution could be achieved for the oxides with chromium content 0 and 0.2. Increasing the chromium content brought about a marked reduction in the dissolution rate. About 40 fold decrease in rate of dissolution was observed when chromium was increased from 0 to 1. The rate of dissolution was not very significantly reduced in the presence of N2H4. Dissolution of oxide was found to be stoichiometric.

Uric acid detection using uv-vis spectrometer

NASA Astrophysics Data System (ADS)

Norazmi, N.; Rasad, Z. R. Abdul; Mohamad, M.; Manap, H.

2017-10-01

The aim of this research is to detect uric acid (UA) concentration using Ultraviolet-Visible (UV-Vis) spectrometer in the Ultraviolet (UV) region. Absorption technique was proposed to detect different uric acid concentrations and its UV absorption wavelength. Current practices commonly take a lot of times or require complicated structures for the detection process. By this proposed spectroscopic technique, every concentration can be detected and interpreted into an absorbance value at a constant wavelength peak in the UV region. This is due to the chemical characteristics belong to the uric acid since it has a particular absorption cross-section, σ which can be calculated using Beer’s Lambert law formula. The detection performance was displayed using Spectrasuite sofware. It showed fast time response about 3 seconds. The experiment proved that the concentrations of uric acid were successfully detected using UV-Vis spectrometer at a constant absorption UV wavelength, 294.46 nm in a low time response. Even by an artificial sample of uric acid, it successfully displayed a close value as the ones reported with the use of the medical sample. It is applicable in the medical field and can be implemented in the future for earlier detection of abnormal concentration of uric acid.

Equilibrium Acidities and Homolytic Bond Dissociation Enthalpies of the Acidic C-H Bonds in P-(Para-substituted benzyl)triphenylphosphonium Cations and Related Cations.

PubMed

Zhang, Xian-Man; Fry, Albert J.; Bordwell, Frederick G.

1996-06-14

Equilibrium acidities (pK(HA)) of six P-(para-substituted benzyl)triphenylphosphonium (p-GC(6)H(4)CH(2)PPh(3)(+)) cations, P-allyltriphenylphosphonium cation, P-cinnamyltriphenylphosphonium cation, and As-(p-cyanobenzyl)triphenylarsonium cation, together with the oxidation potentials [E(ox)(A(-))] of their conjugate anions (ylides) have been measured in dimethyl sulfoxide (DMSO) solution. The acidifying effects of the alpha-triphenylphosphonium groups on the acidic C-H bonds in toluene and propene were found to be ca 25 pK(HA) units (34 kcal/mol). Introduction of an electron-withdrawing group such as 4-NO(2), 4-CN, or 4-Br into the para position of the benzyl ring in p-GC(6)H(4)CH(2)PPh(3)(+) cations resulted in an additional acidity increase, but introduction of the 4-OEt electron-donating group decreases the acidity. The equilibrium acidities of p-GC(6)H(4)CH(2)PPh(3)(+) cations were nicely linearly correlated with the Hammett sigma(-) constants of the substituents (G) with a slope of 4.78 pK(HA) units (R(2) = 0.992) (Figure 1). Reversible oxidation potentials of the P-(para-substituted benzyl)triphenylphosphonium ylides were obtained by fast scan cyclic voltammetry. The homolytic bond dissociation enthalpies (BDEs) of the acidic C-H bonds in these cations, estimated by combining their equilibrium acidities with the oxidation potentials of their corresponding conjugate anions, showed that the alpha-Ph(3)P(+) groups have negligible stabilizing or destabilizing effects on the adjacent radicals. The equilibrium acidity of As-(p-cyanobenzyl)triphenylarsonium cation is 4 pK(HA) units weaker than that of P-(p-cyanobenzyl)triphenylphosphonium cation, but the BDE of the acidic C-H bond in As-(p-cyanobenzyl)triphenylarsonium cation is ca 2 kcal/mol higher than that in P-(p-cyanobenzyl)triphenylphosphonium cation.

Dipole moment and solvatochromism of benzoic acid liquid crystals: Tuning the dipole moment and molecular orbital energies by substituted Au under external electric field

NASA Astrophysics Data System (ADS)

Sıdır, Yadigar Gülseven; Sıdır, İsa; Demiray, Ferhat

2017-06-01

The optical absorption and steady-state fluorescence spectra of 4-heptyloxybenzoic acid (4hoba), 4-octyloxybenzoic acid (4ooba) and 4-nonyloxybenzoic acid (4noba) liquid crystals have been measured in a series of different polarity organic solvents. The ground state (μg) and excited state (μe) dipole moments of the monomeric and dimeric 4-alkyloxybenzoic acid liquid crystals have been obtained by means of different solvatochromic shift methods. HOMO-LUMO gaps (HLG) and dipole moments have been tuned by applying external electric (EF) field on monomer, dimer and Au substituted monomer and dimer liquid crystal structures. By applying external electric field, Au substituted monomer liquid crystals display semiconductor character, while Au substituted dimer liquid crystals gain metallic character under E = 0.04 V/Å. Eventuated specific and non-specific interactions between solvent and solute in solvent medium have been expounded by using LSER (Linear Solvation Energy Relationships).

Conformational properties of a pyridyl-substituted cinnamic acid studied by NMR measurements and computations

NASA Astrophysics Data System (ADS)

Csankó, K.; Forgo, P.; Boros, K.; Hohmann, J.; Sipos, P.; Pálinkó, I.

2013-07-01

Following a preliminary exploration of the conformational space by the PM3 and HF/6-31 G*ab initio methods the conformational characteristics of the scarcely available Z isomer of an α-pyridyl-substituted cinnamic acid dimer [Z-2(3‧-pyridyl)-3-phenylpropanoic acid] was studied by NMR spectroscopy (NOESY measurements) in DMSO(d6), methanol(d4) and chloroform(d1). Calculations predicted that full conjugation was overruled by steric interactions and the rotation of the pyridyl ring was not restricted. NOESY measurements verified indeed that in all three solvents the pyridyl group was virtually freely rotating, while some restriction applied for that of the phenyl group.

The computational analysis and modelling of substitution effects on hydrolysis of formanilides in acidic aqueous solutions

NASA Astrophysics Data System (ADS)

Lukeš, Vladimír; Škorňa, Peter; Michalík, Martin; Klein, Erik

2017-11-01

Various para, meta and ortho substituted formanilides have been theoretically studied. For trans and cis-isomers of non-substituted formanilide, the calculated B3LYP vibration normal modes were analyzed. Substituent effect on the selected normal modes was described and the comparison with the available experimental data is presented. The calculated B3LYP proton affinities were correlated with Hammett constants, Fujita-Nishioka equation and the rate constants of the hydrolysis in 1 M HCl. Found linear dependences allow predictions of dissociation constants (pKBH+) and hydrolysis rate constants. Obtained results indicate that protonation of amide group may represent the rate determining step of acid catalyzed hydrolysis.

Development of amino acid substituted gemini surfactant-based mucoadhesive gene delivery systems for potential use as noninvasive vaginal genetic vaccination.

PubMed

Singh, Jagbir; Michel, Deborah; Getson, Heather M; Chitanda, Jackson M; Verrall, Ronald E; Badea, Ildiko

2015-02-01

Recently, we synthesized amino acid- and peptide-substituted gemini surfactants, 'biolipids' that exhibited high transfection efficiency in vitro. In this study, we developed these plasmid DNA and gemini surfactant lipid particles for noninvasive administration in vaginal cavity. Novel formulations of these gene delivery systems were prepared with poloxamer 407 to induce in situ gelling of the formulation and diethylene glycol monoethyl ether to improve their penetration across mucosal tissue. Poloxamer at 16% w/v concentration in diethylene glycol monoethyl ether aqueous solution produced dispersions that gelled near body temperature and had a high yield value, preventing leakage of the formulation from the vaginal cavity. Intravaginal administration in rabbits showed that the glycyl-lysine-substituted gemini surfactant led to a higher gene expression compared with the parent unsubstituted gemini surfactant. This provides a proof-of-concept that amino acid substituted gemini surfactants can be used as noninvasive mucosal (vaginal) gene delivery systems to treat diseases associated with mucosal epithelia.

Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

NASA Astrophysics Data System (ADS)

Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

2013-11-01

Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses

PubMed Central

Harvey, William T.; Benton, Donald J.; Gregory, Victoria; Hall, James P. J.; Daniels, Rodney S.; Bedford, Trevor; Haydon, Daniel T.; Hay, Alan J.; McCauley, John W.; Reeve, Richard

2016-01-01

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens. PMID:27057693

Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid.

PubMed

Wester, Anita; Devocelle, Marc; Tallant, E Ann; Chappell, Mark C; Gallagher, Patricia E; Paradisi, Francesca

2017-10-01

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.

α-Amino Acid Derived Benzimidazole-Linked Rhodamines: A Case of Substitution Effect at the Amino Acid Site toward Spiro Ring Opening for Selective Sensing of Al3+ Ions.

PubMed

Majumdar, Anupam; Mondal, Subhendu; Daniliuc, Constantin G; Sahu, Debashis; Ganguly, Bishwajit; Ghosh, Sourav; Ghosh, Utpal; Ghosh, Kumaresh

2017-08-07

α-Amino acid derived benzimidazole-linked rhodamines have been synthesized, and their metal ion sensing properties have been evaluated. Experimentally, l-valine- and l-phenylglycine-derived benzimidazole-based rhodamines 1 and 2 selectively recognize Al 3+ ion in aqueous CH 3 CN (CH 3 CN/H 2 O 4/1 v/v, 10 mM tris HCl buffer, pH 7.0) over the other cations by exhibiting color and "turn-on" emission changes. In contrast, glycine-derived benzimidazole 3 remains silent in the recognition event and emphasizes the role of α-substitution of amino acid undertaken in the design. The fact has been addressed on the basis of the single-crystal X-ray structures and theoretical calculations. Moreover, pink 1·Al 3+ and 2·Al 3+ ensembles selectively sensed F - ions over other halides through a discharge of color. Importantly, compounds 1 and 2 are cell permeable and have been used as imaging reagents for the detection of Al 3+ uptake in human lung carcinoma cell line A549.

Biodegradation of chloro- and bromobenzoic acids: effect of milieu conditions and microbial community analysis.

PubMed

Gaza, Sarah; Felgner, Annika; Otto, Johannes; Kushmaro, Ariel; Ben-Dov, Eitan; Tiehm, Andreas

2015-04-28

Monohalogenated benzoic acids often appear in industrial wastewaters where biodegradation can be hampered by complex mixtures of pollutants and prevailing extreme milieu conditions. In this study, the biodegradation of chlorinated and brominated benzoic acids was conducted at a pH range of 5.0-9.0, at elevated salt concentrations and with pollutant mixtures including fluorinated and iodinated compounds. In mixtures of the isomers, the degradation order was primarily 4-substituted followed by 3-substituted and then 2-substituted halogenated benzoic acids. If the pH and salt concentration were altered simultaneously, long adaptation periods were required. Community analyses were conducted in liquid batch cultures and after immobilization on sand columns. The Alphaproteobacteria represented an important fraction in all of the enrichment cultures. On the genus level, Afipia sp. was detected most frequently. In particular, Bacteroidetes were detected in high numbers with chlorinated benzoic acids. Copyright © 2015 Elsevier B.V. All rights reserved.

Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes.

PubMed

Yuan, Yuan; Wang, Zhouyong; Jiang, Chao; Wang, Xumin; Huang, Luqi

2014-01-25

Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species. Copyright © 2012. Published by Elsevier B.V.

Molecular cloning and expression of the hyu genes from Microbacterium liquefaciens AJ 3912, responsible for the conversion of 5-substituted hydantoins to alpha-amino acids, in Escherichia coli.

PubMed

Suzuki, Shun'ichi; Takenaka, Yasuhiro; Onishi, Norimasa; Yokozeki, Kenzo

2005-08-01

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to alpha-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl alpha-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.

Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

PubMed Central

Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

2015-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

The association of substituting carbohydrates with total fat and different types of fatty acids with mortality and weight change among diabetes patients.

PubMed

Campmans-Kuijpers, Marjo J; Sluijs, Ivonne; Nöthlings, Ute; Freisling, Heinz; Overvad, Kim; Boeing, Heiner; Masala, Giovanna; Panico, Salvatore; Tumino, Rosario; Sieri, Sabina; Johansson, Ingegerd; Winkvist, Anna; Katzke, Verena A; Kuehn, Tilman; Nilsson, Peter M; Halkjær, Jytte; Tjønneland, Anne; Spijkerman, Annemieke M; Arriola, Larraitz; Sacerdote, Carlotta; Barricarte, Aurelio; May, Anne M; Beulens, Joline W

2016-10-01

Substitution of carbohydrates with fat in a diet for type 2 diabetes patients is still debated. This study aimed to investigate the association between dietary carbohydrate intake and isocaloric substitution with (i) total fat, (ii) saturated fatty acids (SFA), (iii) mono-unsaturated fatty acids (MUFA) and (iv) poly-unsaturated fatty acids (PUFA) with all-cause and cardiovascular (CVD) mortality risk and 5-year weight change in patients with type 2 diabetes. The study included 6192 patients with type 2 diabetes from 15 cohorts of the European Prospective Investigation into Cancer and Nutrition (EPIC). Dietary intake was assessed at recruitment with country-specific food-frequency questionnaires. Cox and linear regression were used to estimate the associations with (CVD) mortality and weight change, adjusting for confounders and using different methods to adjust for energy intake. After a mean follow-up of 9.2 y ± SD 2.3 y, 791 (13%) participants had died, of which 268 (4%) due to CVD. Substituting 10 g or 5 energy% of carbohydrates by total fat was associated with a higher all-cause mortality risk (HR 1.07 [1.02-1.13]), or SFAs (HR 1.25 [1.11-1.40]) and a lower risk when replaced by MUFAs (HR 0.89 [0.77-1.02]). When carbohydrates were substituted with SFAs (HR 1.22 [1.00-1.49]) or PUFAs (HR 1.29 [1.02-1.63]) CVD mortality risk increased. The 5-year weight was lower when carbohydrates were substituted with total fat or MUFAs. These results were consistent over different energy adjustment methods. In diabetes patients, substitution of carbohydrates with SFAs was associated with a higher (CVD) mortality risk and substitution by total fat was associated with a higher all-cause mortality risk. Substitution of carbohydrates with MUFAs may be associated with lower mortality risk and weight reduction. Instead of promoting replacement of carbohydrates by total fat, dietary guideline should continue focusing on replacement by fat-subtypes; especially SFAs by MUFAs

Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

PubMed

Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

2015-06-15

Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of hyperthermia on the repair of sublethal radiation damage in normal and membrane fatty acid substituted fibroblasts

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

Wolters, H.; Kelholt, D.; Konings, A.W.

1987-02-01

The interaction of heat and X irradiation was studied in normal and polyunsaturated fatty acid (PUFA) substituted mouse fibroblast LM cells. As a result of the substitution the membranes of the PUFA cells were more fluid than the membranes of the normal cells. Three different heat doses were applied (60 min 42 degrees C, 20 min 43 degrees C, and 10 min 44 degrees C) in combination with single or split doses of X rays. Heat radiosensitization was the largest for the 60 min 42 degrees C treatment. Heat radiosensitization and the heat-induced inhibition of the rate of sublethal damagemore » repair were the same for the normal and the PUFA cells. It is concluded from the experiments reported that the processes of hyperthermic inhibition of SLD repair and hyperthermic radiosensitization are independent of membrane fluidity and membrane fatty acid composition.« less

Multiple Genetic Pathways Involving Amino Acid Position 143 of HIV-1 Integrase Are Preferentially Associated with Specific Secondary Amino Acid Substitutions and Confer Resistance to Raltegravir and Cross-Resistance to Elvitegravir

PubMed Central

Frantzell, Arne; Fransen, Signe; Petropoulos, Christos J.

2013-01-01

Y143C,R substitutions in HIV-1 integrase define one of three primary raltegravir (RAL) resistance pathways. Here we describe clinical isolates with alternative substitutions at position 143 (Y143A, Y143G, Y143H, and Y143S [Y143A,G,H,S]) that emerge less frequently, and we compare the genotypic and phenotypic profiles of these viruses to Y143C,R viruses to reconcile the preferential selection of Y143C,R variants during RAL treatment. Integrase amino acid sequences and RAL susceptibility were characterized in 117 patient isolates submitted for drug resistance testing and contained Y143 amino acid changes. The influence of specific Y143 substitutions on RAL susceptibility and their preferential association with particular secondary substitutions were further defined by evaluating the composition of patient virus populations along with a large panel of site-directed mutants. Our observations demonstrate that the RAL resistance profiles of Y143A,G,H,S viruses and their association with specific secondary substitutions are similar to the well-established Y143C profile but distinct from the Y143R profile. Y143R viruses differ from Y143A,C,G,H,S viruses in that Y143R confers a greater reduction in RAL susceptibility as a single substitution, consistent with a lower resistance barrier. Among Y143A,C,G,H,S viruses, the higher prevalence of Y143C viruses is the result of a lower genetic barrier than that of the Y143A,G,S viruses and a lower resistance barrier than that of the Y143H viruses. In addition, Y143A,C,G,H,S viruses require multiple secondary substitutions to develop large reductions in RAL susceptibility. Patient-derived viruses containing Y143 substitutions exhibit cross-resistance to elvitegravir. PMID:23733474

Binding of ring-substituted indole-3-acetic acids to human serum albumin.

PubMed

Soskić, Milan; Magnus, Volker

2007-07-01

The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

Substitution of Linoleic Acid for Other Macronutrients and the Risk of Ischemic Stroke.

PubMed

Venø, Stine K; Schmidt, Erik B; Jakobsen, Marianne U; Lundbye-Christensen, Søren; Bach, Flemming W; Overvad, Kim

2017-12-01

Ischemic stroke is a major health problem worldwide, but the influence of dietary factors on stroke risk is not well known. This study aimed to investigate the risk of ischemic stroke and its subtypes with a higher intake from linoleic acid and a concomitant lower intake from saturated fatty acids, monounsaturated fatty acids, or glycemic carbohydrates. In the Danish prospective Diet, Cancer, and Health Study of 57 053 participants aged 50 to 64 years at baseline, information on diet was collected using a validated semiquantitative food frequency questionnaire. Information on ischemic stroke was obtained from the Danish National Patient Register, and cases were all validated and subclassified according to the TOAST (Trial of ORG 10172 in Acute Stroke Treatment) classification. Substitution of linoleic acid for saturated fatty acid, monounsaturated fatty acid, or glycemic carbohydrates was investigated in relation to the risk of ischemic stroke and subtypes. Cox proportional hazards regression was used to estimate the associations with ischemic stroke adjusting for appropriate confounders. During 13.5 years of follow-up 1879 participants developed ischemic stroke. A slightly lower risk of ischemic stroke was found with a 5% higher intake of linoleic acid and a concomitant lower intake of saturated fatty acid (hazard ratio, 0.98; 95% confidence interval, 0.83-1.16), monounsaturated fatty acid (hazard ratio, 0.80; 95% confidence interval, 0.63-1.02), and glycemic carbohydrates (hazard ratio, 0.92; 95% confidence interval, 0.78-1.09), although not statistically significant. Similar patterns of association were found for large-artery atherosclerosis and small-vessel occlusions. This study suggests that replacing saturated fatty acid, glycemic carbohydrate, or monounsaturated fatty acid with linoleic acid may be associated with a lower risk of ischemic stroke. © 2017 American Heart Association, Inc.

A Diastereoselective Multicomponent Reaction for Construction of Alkynylamide-Substituted α,β-Diamino Acid Derivatives To Hunt Hits.

PubMed

Lei, Ruirui; Wu, Yong; Dong, Suzhen; Jia, Kaili; Liu, Shunying; Hu, Wenhao

2017-03-17

A highly diasetereoselective Mannich-type multicomponent reaction was developed to rapidly construct alkynylamide-substituted α,β-diamino acid derivatives from simple starting materials under mild conditions in moderate to good yields for hit hunting. Most of the resulting products 4 exhibited good anticancer activity in HCT116, BEL7402, and SMMC7721 cells.

Nucleic Acid Detection Methods

DOEpatents

Smith, Cassandra L.; Yaar, Ron; Szafranski, Przemyslaw; Cantor, Charles R.

1998-05-19

The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.

Nucleic acid detection methods

DOEpatents

Smith, C.L.; Yaar, R.; Szafranski, P.; Cantor, C.R.

1998-05-19

The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3{prime}-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated. 18 figs.

Rapid acquisition adaptive amino acid substitutions involved in the virulence enhancement of an H1N2 avian influenza virus in mice.

PubMed

Yu, Zhijun; Sun, Weiyang; Zhang, Xinghai; Cheng, Kaihui; Zhao, Chuqi; Xia, Xianzhu; Gao, Yuwei

2017-08-01

Although H1N2 avian influenza virus (AIV) only infect birds, documented cases of swine infection with H1N2 influenza viruses suggest this subtype AIV may pose a potential threat to mammals. Here, we generated mouse-adapted variants of a H1N2 AIV to identify adaptive changes that increased virulence in mammals. MLD 50 of the variants were reduced >1000-fold compared to the parental virus. Variants displayed enhanced replication in vitro and in vivo, and replicate in extrapulmonary organs. These data show that enhanced replication capacity and expanded tissue tropism may increase the virulence of H1N2 AIV in mice. Sequence analysis revealed multiple amino acid substitutions in the PB2 (L134H, I647L, and D701N), HA (G228S), and M1 (D231N) proteins. These results indicate that H1N2 AIV can rapidly acquire adaptive amino acid substitutions in mammalian hosts, and these amino acid substitutions collaboratively enhance the ability of H1N2 AIV to replicate and cause severe disease in mammals. Copyright © 2017 Elsevier B.V. All rights reserved.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

PubMed

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-10-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

Crystal Structure of a Novel N-Substituted L-Amino Acid Dioxygenase from Burkholderia ambifaria AMMD

PubMed Central

Qin, Hui-Min; Miyakawa, Takuya; Jia, Min Ze; Nakamura, Akira; Ohtsuka, Jun; Xue, You-Lin; Kawashima, Takashi; Kasahara, Takuya; Hibi, Makoto; Ogawa, Jun; Tanokura, Masaru

2013-01-01

A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids. PMID:23724013

Substituted naphthoquinones as novel amino acid sensitive reagents for the detection of latent fingermarks on paper surfaces.

PubMed

Jelly, R; Lewis, S W; Lennard, C; Lim, K F; Almog, J

2010-10-15

In this paper, we present our preliminary studies into naphthoquinones as novel reagents for the detection of latent fingermarks on paper. Latent fingermarks deposited on paper substrates were treated with solutions of selected naphthoquinones in ethyl acetate/HFE-7100, with subsequent heating. The selected compounds were 1,4-dihydroxy-2-naphthoic acid, 1,2-naphthoquinone-4-sulfonate, 2-methoxy-1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone. All of the tested compounds yielded purple-brown visible fingermarks, which also exhibited photoluminescence when illuminated with a high intensity filtered light source at 555 nm and viewed through red goggles. Indirect heat using an oven at 150°C for 1h was found to be superior to direct heat with an iron, which while providing faster development lead to increased levels of background colouration. Luminescence spectrophotometry revealed differences in photoluminescence characteristics for fingermarks developed with the different naphthoquinones, with excitation over the range 530-590 nm. Luminescence spectrophotometry of developed lysine, glycine and serine spots on paper was used to confirm that the naphthoquinones were reacting with amino acids in the latent fingermark. Copyright © 2010 Elsevier B.V. All rights reserved.

Photooxidation of 3-substituted pyrroles:  a postcolumn reaction detection system for singlet molecular oxygen in HPLC.

PubMed

Denham, K; Milofsky, R E

1998-10-01

A postcolumn photochemical reaction detection scheme, based on the reaction of 3-substituted pyrroles with singlet molecular oxygen ((1)O(2)), has been developed. The method is selective and sensitive for the determination of a class of organic compounds called (1)O(2)-sensitizers and is readily coupled to HPLC. Following separation by HPLC, analytes ((1)O(2)-sensitizers) are excited by a Hg pen-ray lamp. Analytes that are efficient (1)O(2)-sensitizers promote ground-state O(2) ((3)Σ(g)(-)) to an excited state ((1)Σ(g)(+) or (1)Δ(g)), which reacts rapidly with tert-butyl-3,4,5-trimethylpyrrolecarboxylate (BTMPC) or N-benzyl-3-methoxypyrrole-2-tert-carboxylate (BMPC), which is added to the mobile phase. Detection is based on the loss of pyrrole (BTMPC or BMPC). The reaction is catalytic in nature since one analyte molecule may absorb light many times, producing large amounts of (1)O(2). Detection limits for several (1)O(2)-sensitizers were improved by 1-2 orders of magnitude over optimized UV-absorbance detection. This paper discusses the optimization of the reaction conditions for this photochemical reaction detection scheme and its application to the detection of PCBs, nitrogen heterocycles, nitro and chloro aromatics, and other substituted aromatic compounds.

Functional Compensation of a Detrimental Amino Acid Substitution in a Cytotoxic-T-Lymphocyte Epitope of Influenza A Viruses by Comutations

PubMed Central

Rimmelzwaan, G. F.; Berkhoff, E. G. M.; Nieuwkoop, N. J.; Fouchier, R. A. M.; Osterhaus, A. D. M. E.

2004-01-01

Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs. PMID:15280506

Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position.

PubMed

Lin, Du'an; Lam, Hiu Yung; Han, Wenbo; Cotroneo, Nicole; Pandya, Bhaumik A; Li, Xuechen

2017-02-01

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported. Copyright © 2016 Elsevier Ltd. All rights reserved.

What makes lithium substituted polyacrylic acid a better binder than polyacrylic acid for silicon-graphite composite anodes?

DOE PAGES

Hays, Kevin A.; Ruther, Rose E.; Kukay, Alexander J.; ...

2018-03-04

Lithium substituted polyacrylic acid (LiPAA) has previously been demonstrated as a superior binder over polyacrylic acid (PAA) for Si anodes, but from where does this enhanced performance arise? In this paper, full cells are assembled with PAA and LiPAA based Si-graphite composite anodes that dried at temperatures from 100 °C to 200 °C. The performance of full cells containing PAA based Si-graphite anodes largely depend on the secondary drying temperature, as decomposition of the binder is correlated to increased electrode moisture and a rise in cell impedance. Full cells containing LiPAA based Si-graphite composite electrodes display better Coulombic efficiency thanmore » those with PAA, because of the electrochemical reduction of the PAA binder. This is identified by attenuated total reflectance Fourier transform infrared spectrometry and observed gassing during the electrochemical reaction. Finally, Coulombic losses from the PAA and Si SEI, along with depletion of the Si capacity in the anode results in progressive underutilization of the cathode and full cell capacity loss.« less

What makes lithium substituted polyacrylic acid a better binder than polyacrylic acid for silicon-graphite composite anodes?

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

Hays, Kevin A.; Ruther, Rose E.; Kukay, Alexander J.

Lithium substituted polyacrylic acid (LiPAA) has previously been demonstrated as a superior binder over polyacrylic acid (PAA) for Si anodes, but from where does this enhanced performance arise? In this paper, full cells are assembled with PAA and LiPAA based Si-graphite composite anodes that dried at temperatures from 100 °C to 200 °C. The performance of full cells containing PAA based Si-graphite anodes largely depend on the secondary drying temperature, as decomposition of the binder is correlated to increased electrode moisture and a rise in cell impedance. Full cells containing LiPAA based Si-graphite composite electrodes display better Coulombic efficiency thanmore » those with PAA, because of the electrochemical reduction of the PAA binder. This is identified by attenuated total reflectance Fourier transform infrared spectrometry and observed gassing during the electrochemical reaction. Finally, Coulombic losses from the PAA and Si SEI, along with depletion of the Si capacity in the anode results in progressive underutilization of the cathode and full cell capacity loss.« less

What makes lithium substituted polyacrylic acid a better binder than polyacrylic acid for silicon-graphite composite anodes?

NASA Astrophysics Data System (ADS)

Hays, Kevin A.; Ruther, Rose E.; Kukay, Alexander J.; Cao, Pengfei; Saito, Tomonori; Wood, David L.; Li, Jianlin

2018-04-01

Lithium substituted polyacrylic acid (LiPAA) has previously been demonstrated as a superior binder over polyacrylic acid (PAA) for Si anodes, but from where does this enhanced performance arise? In this study, full cells are assembled with PAA and LiPAA based Si-graphite composite anodes that dried at temperatures from 100 °C to 200 °C. The performance of full cells containing PAA based Si-graphite anodes largely depend on the secondary drying temperature, as decomposition of the binder is correlated to increased electrode moisture and a rise in cell impedance. Full cells containing LiPAA based Si-graphite composite electrodes display better Coulombic efficiency than those with PAA, because of the electrochemical reduction of the PAA binder. This is identified by attenuated total reflectance Fourier transform infrared spectrometry and observed gassing during the electrochemical reaction. Coulombic losses from the PAA and Si SEI, along with depletion of the Si capacity in the anode results in progressive underutilization of the cathode and full cell capacity loss.

Waste-free synthesis of condensed heterocyclic compounds by rhodium-catalyzed oxidative coupling of substituted arene or heteroarene carboxylic acids with alkynes.

PubMed

Shimizu, Masaki; Hirano, Koji; Satoh, Tetsuya; Miura, Masahiro

2009-05-01

The direct oxidative coupling of 2-amino- and 2-hydroxybenzoic acids with internal alkynes proceeds efficiently in the presence of a rhodium/copper catalyst system under air to afford the corresponding 8-substituted isocoumarin derivatives, some of which exhibit solid-state fluorescence. Depending on conditions, 4-ethenylcarbazoles can be synthesized selectively from 2-(arylamino)benzoic acids. The oxidative coupling reactions of heteroarene carboxylic acids as well as aromatic diacids with an alkyne are also described.

Optical Sensors for Detection of Amino Acids.

PubMed

Pettiwala, Aafrin M; Singh, Prabhat K

2017-11-06

Amino acids are crucially involved in a myriad of biological processes. Any aberrant changes in physiological level of amino acids often manifest in common metabolic disorders, serious neurological conditions and cardiovascular diseases. Thus, devising methods for detection of trace amounts of amino acids becomes highly elemental to their efficient clinical diagnosis. Recently, the domain of developing optical sensors for detection of amino acids has witnessed significant activity which is the focus of the current review article. We undertook a detailed search of the peer-reviewed literature that primarily deals with optical sensors for amino acids and focuses on the use of different type of materials as a sensing platform. Ninety-five papers have been included in the review, majority of which deals with optical sensors. We attempt to systematically classify these contributions based on applications of various chemical and biological scaffolds such as polymers, supramolecular assemblies, nanoparticles, DNA, heparin etc. for the sensing of amino acids. This review identifies that supramolecular assemblies and nanomaterial continue to be commonly used materials to devise sensors for amino acids followed by surfactant assemblies. The broad implications of amino acids in human health and diagnosis have stirred a lot of interest to develop optimized optical detection systems for amino acids in recent years, using different materials based on chemical and biological scaffolds. We have also attempted to highlight the merits and demerits of some of the noteworthy sensor systems to instigate further efforts for constructing amino acids sensor based on unconventional concepts. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

PubMed

Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

2014-09-01

The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

40 CFR 721.5286 - Benzenediazonium, [[[[(substituted) azo]phenyl]sulfonyl]amino]-, coupled with aminophenol...

Code of Federal Regulations, 2010 CFR

2010-07-01

...) azo]phenyl]sulfonyl]amino]-, coupled with aminophenol, diazotized aminobenzoic acid, diazotized..., [[[[(substituted) azo]phenyl]sulfonyl]amino]-, coupled with aminophenol, diazotized aminobenzoic acid, diazotized..., [[[[(substituted)azo]phenyl]sulfonyl]amino]-, coupled with aminophenol, diazotized aminobenzoic acid, diazotized...

40 CFR 721.5286 - Benzenediazonium, [[[[(substituted) azo]phenyl]sulfonyl]amino]-, coupled with aminophenol...

Code of Federal Regulations, 2011 CFR

2011-07-01

...) azo]phenyl]sulfonyl]amino]-, coupled with aminophenol, diazotized aminobenzoic acid, diazotized..., [[[[(substituted) azo]phenyl]sulfonyl]amino]-, coupled with aminophenol, diazotized aminobenzoic acid, diazotized..., [[[[(substituted)azo]phenyl]sulfonyl]amino]-, coupled with aminophenol, diazotized aminobenzoic acid, diazotized...

Background of the Hammett equation as observed for isolated molecules: meta- and para-substituted benzoic acids.

PubMed

Exner, Otto; Böhm, Stanislav

2002-09-06

Fundamental model compounds for the Hammett equation, meta- and para-substituted benzoic acids, were investigated by the density functional theory at the B3LYP/6-311+G(d,p) level. Energies of 25 acids and of their anions were calculated in all possible conformations and from them the energies of the assumed mixture of conformers. Relative acidities correlated with the experimental gas-phase acidities almost within the experimental uncertainty, much more precisely than in the case of previous calculations at lower levels. Dissection of the substituent effects into those operating in the acid molecule and in the anion was carried out by means of isodesmic reactions starting from monosubstituted benzenes. Both effects are cooperating in the resulting effect on the acidity; those in the acid molecule are smaller but not negligible. They are also responsible for some deviations from the Hammett equation (through-resonance of para donor substituents) and for the weaker resonance in the acid molecule in meta derivatives; in the anions the inductive and resonance effects are almost equal. On the other hand, the cooperation of effects in the acid and in the anion makes the relative acidity more sensitive to electron withdrawing and is probably one of the reasons why the Hammett equation is so generally valid.

A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

PubMed Central

Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

2013-01-01

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses. PMID:23874963

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives.

PubMed

Takebayashi, Jun; Kaji, Hiroaki; Ichiyama, Kenji; Makino, Kazutaka; Gohda, Eiichi; Yamamoto, Itaru; Tai, Akihiro

2007-10-15

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.

Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues.

PubMed

Mei, Hui; Liao, Jen-Yu; Jimenez, Randi M; Wang, Yajun; Bala, Saikat; McCloskey, Cailen; Switzer, Christopher; Chaput, John C

2018-05-02

In vitro selection experiments carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixture leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogues in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-phenyl tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library containing the 7-deaza-7-phenyl guanine analogue was used to evolve a biologically stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.

PON-Sol: prediction of effects of amino acid substitutions on protein solubility.

PubMed

Yang, Yang; Niroula, Abhishek; Shen, Bairong; Vihinen, Mauno

2016-07-01

Solubility is one of the fundamental protein properties. It is of great interest because of its relevance to protein expression. Reduced solubility and protein aggregation are also associated with many diseases. We collected from literature the largest experimentally verified solubility affecting amino acid substitution (AAS) dataset and used it to train a predictor called PON-Sol. The predictor can distinguish both solubility decreasing and increasing variants from those not affecting solubility. PON-Sol has normalized correct prediction ratio of 0.491 on cross-validation and 0.432 for independent test set. The performance of the method was compared both to solubility and aggregation predictors and found to be superior. PON-Sol can be used for the prediction of effects of disease-related substitutions, effects on heterologous recombinant protein expression and enhanced crystallizability. One application is to investigate effects of all possible AASs in a protein to aid protein engineering. PON-Sol is freely available at http://structure.bmc.lu.se/PON-Sol The training and test data are available at http://structure.bmc.lu.se/VariBench/ponsol.php mauno.vihinen@med.lu.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts.

PubMed

Yamamoto, Haruki; Kusumi, Junko; Yamakawa, Hisanori; Fujita, Yuichi

2017-05-24

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

PubMed Central

Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

1986-01-01

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme. Images PMID:2876430

Sulfenic acid chemistry, detection and cellular lifetime☆

PubMed Central

Gupta, Vinayak; Carroll, Kate S.

2014-01-01

Background Reactive oxygen species-mediated cysteine sulfenic acid modification has emerged as an important regulatory mechanism in cell signaling. The stability of sulfenic acid in proteins is dictated by the local microenvironment and ability of antioxidants to reduce this modification. Several techniques for detecting this cysteine modification have been developed, including direct and in situ methods. Scope of review This review presents a historical discussion of sulfenic acid chemistry and highlights key examples of this modification in proteins. A comprehensive survey of available detection techniques with advantages and limitations is discussed. Finally, issues pertaining to rates of sulfenic acid formation, reduction, and chemical trapping methods are also covered. Major conclusions Early chemical models of sulfenic acid yielded important insights into the unique reactivity of this species. Subsequent pioneering studies led to the characterization of sulfenic acid formation in proteins. In parallel, the discovery of oxidant-mediated cell signaling pathways and pathological oxidative stress has led to significant interest in methods to detect these modifications. Advanced methods allow for direct chemical trapping of protein sulfenic acids directly in cells and tissues. At the same time, many sulfenic acids are short-lived and the reactivity of current probes must be improved to sample these species, while at the same time, preserving their chemical selectivity. Inhibitors with binding scaffolds can be rationally designed to target sulfenic acid modifications in specific proteins. General significance Ever increasing roles for protein sulfenic acids have been uncovered in physiology and pathology. A more complete understanding of sulfenic acid-mediated regulatory mechanisms will continue to require rigorous and new chemical insights. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and

Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

PubMed

Singh, R P; Nie, X; Singh, M; Coffin, R; Duplessis, P

2002-01-01

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

Fabrication of calcium phosphate–calcium sulfate injectable bone substitute using hydroxy-propyl-methyl-cellulose and citric acid

PubMed Central

Thai, Van Viet

2010-01-01

In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan, and hydroxyl propyl methyl cellulose (HPMC) as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate dihydrate (DCPD) and calcium sulfate dehydrate (CSD, CaSO4·2H2O) powders as the solid phase, were fabricated. Two groups were classified based on the percent of citric acid in the liquid phase (20, 40 wt%). In each groups, the HPMC percentage was 0, 2, and 4 wt%. An increase in compressive strength due to changes in morphology was confirmed by scanning electron microscopy images. A good conversion rate of HAp at 20% citric acid was observed in the XRD profiles. In addition, HPMC was not obviously affected by apatite formation. However, both HPMC and citric acid increased the compressive strength of IBS. The maximum compressive strength for IBS was with 40% citric acid and 4% HPMC after 14 days of incubation in 100% humidity at 37°C. PMID:20333539

Antiproliferative activity and SARs of caffeic acid esters with mono-substituted phenylethanols moiety.

PubMed

Xie, Jin; Yang, Fengzhi; Zhang, Man; Lam, Celine; Qiao, Yixue; Xiao, Jia; Zhang, Dongdong; Ge, Yuxuan; Fu, Lei; Xie, Dongsheng

2017-01-15

A series of CAPE derivatives with mono-substituted phenylethanols moiety were synthesized and evaluated by MTT assay on growth of 4 human cancer cell lines (Hela, DU-145, MCF-7 and ECA-109). The substituent effects on the antiproliferative activity were systematically investigated for the first time. It was found that electron-donating and hydrophobic substituents at 2'-position of phenylethanol moiety could significantly enhance CAPE's antiproliferative activity. 2'-Propoxyl derivative, as a novel caffeic acid ester, exhibited exquisite potency (IC 50 =0.4±0.02 & 0.6±0.03μM against Hela and DU-145 respectively). Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

1999-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

2012-10-16

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant Enterococcus strains result in high-level vancomycin resistance and low-level teicoplanin resistance.

PubMed

Hashimoto, Y; Tanimoto, K; Ozawa, Y; Murata, T; Ike, Y

2000-04-15

The vancomycin-resistant enterococci GV1, GV2 and GV3, which were isolated from droppings from broiler farms in Japan have been characterized as VanA-type VRE, which express high-level vancomycin resistance (256 or 512 microg ml(-1), MIC) and low-level teicoplanin resistance (1 or 2 microg ml(-1), MIC). The vancomycin resistances were encoded on plasmids. The vancomycin resistance conjugative plasmid pMG2 was isolated from the GV2 strain. The VanA determinant of pMG2 showed the same genetic organization as that of the VanA genes encoded on the representative transposon Tn1546, which comprises vanRSHAXYZ. The nucleotide sequences of all the genes, except the gene related to the vanS gene on Tn1546, were completely identical to the genes encoded on Tn1546. Three amino acid substitutions in the N-terminal region of the deduced VanS were detected in the nucleotide sequence of vanS encoded on pMG2. There were also three amino acid substitutions in the vanS gene of the GV1 and GV3 strains in the same positions as in the vanS gene of pMG2. Vancomycin induced the increased teicoplanin resistance in these strains.

Toluene nitration in irradiated nitric acid and nitrite solutions

NASA Astrophysics Data System (ADS)

Elias, Gracy; Mincher, Bruce J.; Mezyk, Stephen P.; Muller, Jim; Martin, Leigh R.

2011-04-01

The kinetics, mechanisms, and stable products produced for the nitration of aryl alkyl mild ortho-para director toluene in irradiated nitric acid and neutral nitrite solutions were investigated using γ and pulse radiolysis. Electron pulse radiolysis was used to determine the bimolecular rate constants for the reaction of toluene with different transient species produced by irradiation. HPLC with UV detection, GC-MS and LC-MS, were used to assess the stable reaction products. Free-radical based nitration reaction products were found in irradiated acidic and neutral media. In 6.0 M HNO3, ring substitution, side chain substitution, and oxidation, produced different nitrated toluene products. For ring substitution, nitrogen oxide radicals were added mainly to cyclohexadienyl radicals, whereas for side chain substitution, these radicals were added to the carbon-centered benzyl radical produced by H-atom abstraction. In neutral nitrite solutions, radiolytically-induced ring nitration products approached a statistically random distribution, suggesting a direct free-radical reaction involving addition of the rad NO2 radical.

Supramolecular control over recognition and efficient detection of picric acid.

PubMed

Béreau, Virginie; Duhayon, Carine; Sutter, Jean-Pascal

2014-10-18

Bimetallic Schiff-base Al(3+) complexes bearing ester functions at the periphery of the ligands are shown to be efficient fluorescent chemosensors for picric acid detection. The prominent role of an association between the chemosensor and the picric acid in the detection process is demonstrated. The detection of picric acid in water is achieved with the sensor deposited on paper.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

PubMed

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

Biosensors for D-amino acid detection.

PubMed

Sacchi, Silvia; Rosini, Elena; Caldinelli, Laura; Pollegioni, Loredano

2012-01-01

The presence of D-amino acids in foods is promoted by harsh technological processes (e.g., high temperature or extreme pH values) or can be the consequence of adulteration or microbial contamination (D-amino acids are major components of the bacterial cell wall). For this reason, quality control is becoming more and more important both for the industry (as a cost factor) and for consumer protection. For routine food analysis and quality control, simple and easily applicable analytical methods are needed: biosensors can often satisfy these requirements. The use of an enzymatic, stereospecific reaction could confer selectivity to a biosensor for detecting and quantifying D-amino acids in foodstuffs. The flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis is an ideal biocatalyst for this kind of application because of its absolute stereospecificity, very high turnover number with various substrates, tight binding with the FAD cofactor, and broad substrate specificity. Furthermore, alterations in the local brain concentrations of D-serine (predominantly D-amino acid in the mammalian central nervous system) have been related to several neurological and psychiatric diseases. Therefore, quantifying this neuromodulator represents an important task in biological, medical, and pharmaceutical research. Recently, an enzymatic microbiosensor, also using R. gracilis D-amino acid oxidase as biocatalyst, was developed for detecting D-serine in vivo.

Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

PubMed Central

Gross, Christian H.; Parsons, Jonathan D.; Grossman, Trudy H.; Charifson, Paul S.; Bellon, Steven; Jernee, James; Dwyer, Maureen; Chambers, Stephen P.; Markland, William; Botfield, Martyn; Raybuck, Scott A.

2003-01-01

DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli. PMID:12604539

Nucleic acid in-situ hybridization detection of infectious agents

NASA Astrophysics Data System (ADS)

Thompson, Curtis T.

2000-04-01

Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

A Facile Semi-Synthetic Approach towards Halogen-Substituted Aminobenzoic Acid Analogues of Platensimycin.

PubMed

Qiu, Lin; Tian, Kai; Pan, Jian; Jiang, Lin; Yang, Hu; Zhu, Xiangcheng; Shen, Ben; Duan, Yanwen; Huang, Yong

2017-02-09

Platensimycin (PTM), produced by several strains of Streptomyces platensis, is a promising drug lead for infectious diseases and diabetes. The recent pilot-scale production of PTM from S. platensis SB12026 has set the stage for the facile semi-synthesis of a focused library of PTM analogues. In this study, gram-quantity of platensic acid (PTMA) was prepared by the sulfuric acid-catalyzed ethanolysis of PTM, followed by a mild hydrolysis in aqueous lithium hydroxide. Three PTMA esters were also obtained in near quantitative yields in a single step, suggesting a facile route to make PTMA aliphatic esters. 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)-catalyzed coupling of PTMA and 33 aminobenzoates resulted in the synthesis of 28 substituted aminobenzoate analogues of PTM, among which 26 of them were reported for the first time. Several of the PTM analogues showed weak antibacterial activity against methicillin-resistant Staphylococcus aureus. Our study supported the potential utility to integrate natural product biosynthetic and semi-synthetic approaches for structure diversification.

Overdispersion of the Molecular Clock: Temporal Variation of Gene-Specific Substitution Rates in Drosophila

PubMed Central

Hartl, Daniel L.

2008-01-01

Simple models of molecular evolution assume that sequences evolve by a Poisson process in which nucleotide or amino acid substitutions occur as rare independent events. In these models, the expected ratio of the variance to the mean of substitution counts equals 1, and substitution processes with a ratio greater than 1 are called overdispersed. Comparing the genomes of 10 closely related species of Drosophila, we extend earlier evidence for overdispersion in amino acid replacements as well as in four-fold synonymous substitutions. The observed deviation from the Poisson expectation can be described as a linear function of the rate at which substitutions occur on a phylogeny, which implies that deviations from the Poisson expectation arise from gene-specific temporal variation in substitution rates. Amino acid sequences show greater temporal variation in substitution rates than do four-fold synonymous sequences. Our findings provide a general phenomenological framework for understanding overdispersion in the molecular clock. Also, the presence of substantial variation in gene-specific substitution rates has broad implications for work in phylogeny reconstruction and evolutionary rate estimation. PMID:18480070

Substitution determination of Fmoc‐substituted resins at different wavelengths

PubMed Central

Kley, Markus; Bächle, Dirk; Loidl, Günther; Meier, Thomas; Samson, Daniel

2017-01-01

In solid‐phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc‐substituted resins, substitution determination is often performed by suspending the Fmoc‐protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene–piperidine adduct that is quantified by ultraviolet–visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene–piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert–Beer's law, together with the substance‐specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol−1 cm−1 and 8100 l mol−1 cm−1 have been reported for the dibenzofulvene–piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. PMID:28635051

Palladium-Catalyzed Asymmetric Allylic Alkylation of 4-Substituted Isoxazolidin-5-ones: Straightforward Access to β2,2 -Amino Acids.

PubMed

Nascimento de Oliveira, Marllon; Arseniyadis, Stellios; Cossy, Janine

2018-04-03

We report here an unprecedented and highly enantioselective palladium-catalyzed allylic alkylation applied to 4-substituted isoxazolidin-5-ones. Ultimately, the process provides a straightforward access to β 2,2 -amino acids bearing an all-carbon quaternary stereogenic center in great yields and a high degree of enantioselectivity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of naphthenic acids in fish exposed to commercial naphthenic acids and oil sands process-affected water.

PubMed

Young, R F; Orr, E A; Goss, G G; Fedorak, P M

2007-06-01

Naphthenic acids are a complex mixture of carboxylic acids that occur naturally in petroleum. During the extraction of bitumen from the oil sands in northeastern Alberta, Canada, naphthenic acids are released into the aqueous phase and these acids become the most toxic components in the process-affected water. Although previous studies have exposed fish to naphthenic acids or oil sands process-affected waters, there has been no analytical method to specifically detect naphthenic acids in fish. Here, we describe a qualitative method to specifically detect these acids. In 96-h static renewal tests, rainbow trout (Oncorhynchus mykiss) fingerlings were exposed to three different treatments: (1) fed pellets that contained commercial naphthenic acids (1.5mg g(-1) of food), (2) kept in tap water that contained commercial naphthenic acids (3mg l(-1)) and (3) kept in an oil sands process-affected water that contained 15mg naphthenic acids l(-1). Five-gram samples of fish were homogenized and extracted, then the mixture of free fatty acids and naphthenic acids was isolated from the extract using strong anion exchange chromatography. The mixture was derivatized and analyzed by gas chromatography-mass spectrometry. Reconstructed ion chromatograms (m/z=267) selectively detected naphthenic acids. These acids were present in each fish that was exposed to naphthenic acids, but absent in fish that were not exposed to naphthenic acids. The minimum detectable concentration was about 1microg naphthenic acids g(-1) of fish.

Liquid crystal based biosensors for bile acid detection

NASA Astrophysics Data System (ADS)

He, Sihui; Liang, Wenlang; Tanner, Colleen; Fang, Jiyu; Wu, Shin-Tson

2013-03-01

The concentration level of bile acids is a useful indicator for early diagnosis of liver diseases. The prevalent measurement method in detecting bile acids is the chromatography coupled with mass spectrometry, which is precise yet expensive. Here we present a biosensor platform based on liquid crystal (LC) films for the detection of cholic acid (CA). This platform has the advantage of low cost, label-free, solution phase detection and simple analysis. In this platform, LC film of 4-Cyano-4'-pentylbiphenyl (5CB) was hosted by a copper grid supported with a polyimide-coated glass substrate. By immersing into sodium dodecyl sulfate (SDS) solution, the LC film was coated with SDS which induced a homeotropic anchoring of 5CB. Addition of CA introduced competitive adsorption between CA and SDS at the interface, triggering a transition from homeotropic to homogeneous anchoring. The detection limit can be tuned by changing the pH value of the solution from 12uM to 170uM.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

PubMed Central

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

Intake of dietary saturated fatty acids and risk of type 2 diabetes in the European Prospective Investigation into Cancer and Nutrition-Netherlands cohort: associations by types, sources of fatty acids and substitution by macronutrients.

PubMed

Liu, Shengxin; van der Schouw, Yvonne T; Soedamah-Muthu, Sabita S; Spijkerman, Annemieke M W; Sluijs, Ivonne

2018-03-09

The association between dietary saturated fatty acids (SFA) intake and type 2 diabetes (T2D) remains unclear. This study aimed at investigating the association between SFA intake and T2D risk based on (1) individual SFA (differing in carbon chain length), (2) food sources of SFA and (3) the substituting macronutrients. 37,421 participants from the European Prospective Investigation into Cancer and Nutrition-Netherlands (EPIC-NL) cohort were included in this study. Baseline dietary intake was assessed by a validated food frequency questionnaire. T2D risks were estimated by Cox regression models adjusted for non-dietary and dietary covariates. 893 incident T2D cases were documented during 10.1-year follow-up. We observed no association between total SFA and T2D risk. Marginally inverse associations were found for lauric acid (HR per 1 SD of energy%, 95% CI 0.92, 0.85-0.99), myristic acid (0.89, 0.79-0.99), margaric acid (0.84, 0.73-0.97), odd-chain SFA (pentadecylic plus margaric acids; 0.88, 0.79-0.99), and cheese derived SFA (0.90, 0.83-0.98). Soft and liquid fats derived SFA was found related to higher T2D risk (1.08, 1.01-1.17). When substituting SFA by proteins, carbohydrates and polyunsaturated fatty acids, significantly higher risks of T2D were observed (HRs per 1 energy% ranging from 1.05 to 1.15). In this Dutch population, total SFA does not relate to T2D risk. Rather, the association may depend on the types and food sources of SFA. Cheese-derived SFA and individual SFA that are commonly found in cheese, were significantly related to lower T2D risks. We cannot exclude the higher T2D risks found for soft and liquid fats derived SFA and for substituting SFA with other macronutrients are influenced by residual confounding by trans fatty acids or limited intake variation in polyunsaturated fatty acids and vegetable protein.

Development of calcium phosphate cement using chitosan and citric acid for bone substitute materials.

PubMed

Yokoyama, Atsuro; Yamamoto, Satoru; Kawasaki, Takao; Kohgo, Takao; Nakasu, Masanori

2002-02-01

We developed a calcium phosphate cement that could be molded into any desired shape due to its chewing-gum-like consistency after mixing. The powder component of the cement consists of alpha-tricalcium phosphate and tetracalcium phosphate, which were made by decomposition of hydroxyapatite ceramic blocks. The liquid component consists of citric acid, chitosan and glucose solution. In this study, we used 20% citric acid (group 20) and 45% citric acid (group 45). The mechanical properties and biocompatibility of this new cement were investigated. The setting times of cements were 5.5 min, in group 20 and 6.4 min, in group 45. When incubated in physiological saline, the cements were transformed to hydroxyapatite at 3, and 6 weeks, the compressive strengths were 15.6 and 20.7 MPa, in group 45 and group 20, respectively. The inflammatory response around the cement implanted on the bone and in the subcutaneous tissue in rats was more prominent in group 45 than in group 20 at 1 week after surgery. After 4 weeks, the inflammation disappeared and the cement had bound to bone in both groups. These results indicate that this new calcium phosphate cement is a suitable bone substitute material and that the concentration of citric acid in the liquid component affects its mechanical properties and biocompatibility.

Amino acid substitution converts WEREWOLF function from an activator to a repressor of Arabidopsis non-hair cell development.

PubMed

Tominaga-Wada, Rumi; Nukumizu, Yuka; Wada, Takuji

2012-02-01

Root hair cell or non-hair cell fate determination in Arabidopsis thaliana root epidermis is model system for plant cell development. Two types of MYB transcription factors, the R2R3-type MYB, WEREWOLF (WER), and an R3-type MYB, CAPRICE (CPC), are involved in this cell fate determination process. To study the molecular basis of this process, we analyzed the functional relationship of WER and CPC. WER-CPC chimeric constructs were made from WER where all or parts of the MYB R3 region were replaced with the corresponding regions from CPC R3, and the constructs were introduced into the cpc-2 mutant. Although, the WER gene did not rescue the cpc-2 mutant 'small number of root hairs' phenotype, the WER-CPC chimera with two amino acids substitution (WC6) completely rescued the cpc-2 mutant phenotype. Furthermore, the WER-CPC chimera with 37 amino acids substitution (WC5) excessively rescued the cpc-2 mutant and induced 2.5 times more root hairs than wild-type. Consistent with this phenotype, GL2 gene expression was strongly reduced in WC5 in a cpc-2 background. Our results suggest that swapping at least two amino acids is sufficient to convert WER to CPC function. Therefore, these key residues may have strongly contributed to the selection of these important functions over evolution. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Detection of nucleic acids by multiple sequential invasive cleavages 02

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

2002-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Virulence of an H5N8 highly pathogenic avian influenza is enhanced by the amino acid substitutions PB2 E627K and HA A149V.

PubMed

Wu, Haibo; Peng, Xiuming; Lu, Rufeng; Xu, Lihua; Liu, Fumin; Cheng, Linfang; Lu, Xiangyun; Yao, Hangping; Wu, Nanping

2017-10-01

A novel reassortant H5N8 highly pathogenic avian influenza (HPAI) virus was recently identified in Asia, Europe, and North America. The H5N8 HPAI virus has raised serious concerns regarding the potential risk for human infection. However, the molecular changes responsible for allowing mammalian infection in H5N8 HPAI viruses are not clear. The objective of this study was to identify amino acid substitutions that are potentially associated with the adaptation of H5N8 HPAI viruses to mammals. In this study, an avian-origin H5N8 virus was adapted to mice through serial lung-to-lung passage. The virulence of mouse-adapted virus was increased and adaptive mutations, HA (A149V) and PB2 (E627K), were detected after the ninth passage in each series of mice. Reverse genetics were used to generate reassortants of the wild type and mouse-adapted viruses. Substitutions in the HA (A149V) and PB2 (E627K) proteins led to enhanced viral virulence in mice, the viruses displayed expanded tissue tropism, and increased replication kinetics in mammalian cells. Continued surveillance in poultry for amino acid changes that might indicate H5N8 HPAI viruses pose a threat to human health is required. Copyright © 2017. Published by Elsevier B.V.

Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

PubMed

Chen, Ming; Liu, Minghua; Yu, Lili; Cai, Guoru; Chen, Qinghai; Wu, Rong; Wang, Feng; Zhang, Bo; Jiang, Tianlun; Fu, Welling

2005-08-01

A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples.

Kinetics of the substitution of dehydroacetic acid in tris (dehydroacetato) Fe(III) complex by 8-hydroxyquinoline, di- and tetra-hydroxyquinone

NASA Astrophysics Data System (ADS)

Fouad, D. M.; Ismail, N. M.; El-Gahami, M. A.; Ibrahim, S. A.

2007-06-01

The ligand substitution reactions of dehydroacetic acid (Hdha) in [Fe(dha) 3] with second ligand such as 8-hydroxyquinoline (Hquin), 1,4-dihydroxyanthraquinone (H 2dhaq) and 1,4,5,8-tetra-hydroxyanthraquinone (H 4thaq) were investigated spectrophotometrically by in low polarity solvents like benzene, chloroform and dichloromethane. It is deduced that the substitution reaction takes place through one successive step. The reaction was performed at four different temperatures (5-25) °C, and it exhibits a first order dependence on the concentration of the starting complex. The observed rate constant depends on the concentration of both leaving and entering ligands. The evaluation of the kinetic data gives activation parameters which support an associative mechanism in the transition states and the higher rate of substitution of the dha in Fe(dha) 3 complex is due to entropy effect. The solid complexes were synthesized and characterized by elemental analysis, IR and UV-vis spectral techniques.

A unique amino acid substitution, T126I, in human genotype C of hepatitis B virus S gene and its possible influence on antigenic structural change.

PubMed

Ren, Fengrong; Tsubota, Asahito; Hirokawa, Takatsugu; Kumada, Hiromitsu; Yang, Ziheng; Tanaka, Hiroshi

2006-11-15

Amino acid substitutions in the S gene of hepatitis B virus (HBV), especially in the 'a' determinant region, have been suggested to affect the antigenicity of the virus and the clinical outcome of the infected patient. However, no convincing evidence has been presented for this hypothesis, partly because the 3D structure of the S protein has not been determined. Comparative analysis of viral genes offers an approach to testing this hypothesis, as it may reveal signals of natural selection and provide insights into the functional significance of the observed amino acid substitutions. In this study, we analyze HBV S gene sequences obtained from 24 patients infected with HBV genotypes B or C, together with 16 representative viral strains of HBV genotypes A-F retrieved from GenBank. We use phylogenetic methods to infer evolutionary changes among HBV genotypes and to identify amino acid residues potentially under positive selective pressure. Furthermore, we employ the fragment assembly method to predict structural changes in the S protein. The results showed that an amino acid substitution within the 'a' determinant, T126I, was unique to genotype C, may affect the antigenicity of the HBsAg, and may result in poorer clinical outcomes of patients infected with genotype C viral strains. We suggest that an integrated approach of evolutionary comparison and structural prediction is useful in generating hypotheses for further laboratory validation.

Substitution determination of Fmoc-substituted resins at different wavelengths.

PubMed

Eissler, Stefan; Kley, Markus; Bächle, Dirk; Loidl, Günther; Meier, Thomas; Samson, Daniel

2017-10-01

In solid-phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc-substituted resins, substitution determination is often performed by suspending the Fmoc-protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene-piperidine adduct that is quantified by ultraviolet-visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene-piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert-Beer's law, together with the substance-specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol -1  cm -1 and 8100 l mol -1  cm -1 have been reported for the dibenzofulvene-piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.

PubMed

Peng, Tao; Huang, Bingzhen; Sun, Yao; Lu, Yongbo; Bonewald, Lynda; Chen, Shuo; Butler, William T; Feng, Jerry Q; D'Souza, Rena N; Qin, Chunlin

2009-01-01

Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1. Copyright 2008 S. Karger AG, Basel.

A GC-MS method for the detection of toluene and ethylbenzene in volatile substance abuse.

PubMed

El-Haj, B M; Al-Amri, A M; Hassan, M H; Bin-Khadem, R K; Al-Hadi, A A

2000-09-01

The interference of some substances with the gas chromatography-flame ionization detection and gas chromatography-Fourier transform infrared detection of toluene and ethylbenzene in volatile substance abuse poses problems. A gas chromatography-mass spectrometry (GC-MS) method that will overcome such interference has been developed for the detection of toluene and/or ethylbenzene in the headspace of preparations and products containing these substances and in the headspace of blood samples in the cases of volatile substance abuse. The method is based on converting toluene to benzoic acid via the formation of benzotrichloride. The latter compound was obtained upon the reaction of toluene with chlorine gas under direct sunlight conditions. In the presence of water, benzotrichloride was converted to benzoic acid. Ethylbenzene was converted to benzoic acid and two phenylethanols via the formation of side chain chloro-substituted phenylethanes followed by reaction with water. The chloro-substituted phenylethanes were obtained by the reaction of ethylbenzene with chlorine under direct sunlight conditions. The benzoic acid resulting from toluene and/or ethylbenzene and the two phenylethanols resulting from ethylbenzene were detected by GC-MS as their trimethylsilyl (TMS) derivatives. For the method to be viable for the detection of volatile substance abuse, the chlorination reactions were effected in the gaseous state.

Detection and isolation of nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1997-01-01

A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

Detection and isolation of nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1997-04-01

A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

Molecular characterization of amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.

PubMed

Esmaelizad, Majid; Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

2011-12-01

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted C(α) amino acids.

PubMed

Hicks, Rickey P

2016-09-15

Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C(α) amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X=any one of the following residues, A5c, A6c, Tic or Oic and B=any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC50 (<7.5μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC50 (<7.5μM) values against selected cancer cell lines. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and alpha-glucose substitution of poly(glycerophosphate) backbones.

PubMed

Räisänen, Liisa; Draing, Christian; Pfitzenmaier, Markus; Schubert, Karin; Jaakonsaari, Tiina; von Aulock, Sonja; Hartung, Thomas; Alatossava, Tapani

2007-06-01

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of alpha-glucosyl and D-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of D-alanine residues in the LTA backbones. Prior incubation of the LTAs with alpha-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of alpha-glucosyl-substituted, D-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption.

Molecular Interaction between Lipoteichoic Acids and Lactobacillus delbrueckii Phages Depends on d-Alanyl and α-Glucose Substitution of Poly(Glycerophosphate) Backbones▿

PubMed Central

Räisänen, Liisa; Draing, Christian; Pfitzenmaier, Markus; Schubert, Karin; Jaakonsaari, Tiina; von Aulock, Sonja; Hartung, Thomas; Alatossava, Tapani

2007-01-01

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of α-glucosyl and d-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of d-alanine residues in the LTA backbones. Prior incubation of the LTAs with α-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of α-glucosyl-substituted, d-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption. PMID:17416656

Using Phylogenetic Analysis to Detect Market Substitution of Atlantic Salmon for Pacific Salmon: An Introductory Biology Laboratory Experiment

ERIC Educational Resources Information Center

Cline, Erica; Gogarten, Jennifer

2012-01-01

We describe a laboratory exercise developed for the cell and molecular biology quarter of a year-long majors' undergraduate introductory biology sequence. In an analysis of salmon samples collected by students in their local stores and restaurants, DNA sequencing and phylogenetic analysis were used to detect market substitution of Atlantic salmon…

Halogeno-substituted 2-aminobenzoic acid derivatives for negative ion fragmentation studies of N-linked carbohydrates.

PubMed

Harvey, David J

2005-01-01

Negative ion electrospray mass spectra of high-mannose N-linked glycans derivatised with 2-aminobenzoic acids and ionised from solutions containing ammonium hydroxide gave prominent [M-H](-) ions accompanied by weaker [M-2H](2-) ions. Fragmentation of both types of ions gave prominent singly charged glycosidic cleavage ions containing the derivatised reducing terminus and ions from the non-reducing terminus that appeared to be products of cross-ring cleavages. Differentiation of these two groups of ions was conveniently achieved in a single spectrum by use of chloro- or bromo-substituted benzoic acids in order to label ions containing the derivative with an atom with a distinctive isotope pattern. Fragmentation of the doubly charged ions gave more abundant fragments, both singly and doubly charged, than did fragmentation of the singly charged ions, but information of chain branching was masked by the appearance of prominent ions produced by internal cleavages. Copyright (c) 2005 John Wiley & Sons, Ltd.

Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

PubMed Central

Öhrmalm, Christina; Jobs, Magnus; Eriksson, Ronnie; Golbob, Sultan; Elfaitouri, Amal; Benachenhou, Farid; Strømme, Maria; Blomberg, Jonas

2010-01-01

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes. PMID:20864443

Individual Substitution Mutations in the AID C Terminus That Ablate IgH Class Switch Recombination

PubMed Central

Kadungure, Tatenda; Ucher, Anna J.; Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

2015-01-01

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The C terminus of AID is required for CSR but not for SHM, but the reason for this is not entirely clear. By retroviral transduction of mutant AID proteins into aid -/- mouse splenic B cells, we show that 4 amino acids within the C terminus of mouse AID, when individually mutated to specific amino acids (R190K, A192K, L196S, F198S), reduce CSR about as much or more than deletion of the entire C terminal 10 amino acids. Similar to ΔAID, the substitutions reduce binding of UNG to Ig Sμ regions and some reduce binding of Msh2, both of which are important for introducing S region DNA breaks. Junctions between the IgH donor switch (S)μ and acceptor Sα regions from cells expressing ΔAID or the L196S mutant show increased microhomology compared to junctions in cells expressing wild-type AID, consistent with problems during CSR and the use of alternative end-joining, rather than non-homologous end-joining (NHEJ). Unlike deletion of the AID C terminus, 3 of the substitution mutants reduce DNA double-strand breaks (DSBs) detected within the Sμ region in splenic B cells undergoing CSR. Cells expressing these 3 substitution mutants also have greatly reduced mutations within unrearranged Sμ regions, and they decrease with time after activation. These results might be explained by increased error-free repair, but as the C terminus has been shown to be important for recruitment of NHEJ proteins, this appears unlikely. We hypothesize that Sμ DNA breaks in cells expressing these C terminus substitution mutants are poorly repaired, resulting in destruction of Sμ segments that are deaminated by these mutants. This could explain why these mutants cannot undergo CSR. PMID:26267846

Whole-Gene Positive Selection, Elevated Synonymous Substitution Rates, Duplication, and Indel Evolution of the Chloroplast clpP1 Gene

PubMed Central

Erixon, Per; Oxelman, Bengt

2008-01-01

of whether negative or positive selection is to be expected after gene duplications by providing evidence for the latter alternative. The observed increase in synonymous substitution rates in some of the lineages indicates that the detection of positive selection may be obscured under such circumstances. Future studies are required to explore the functional significance of the large inserted repeated amino acid motifs, as well as the possibility that synonymous substitution rates may be affected by positive selection. PMID:18167545

New gene cluster from the thermophile Bacillus fordii MH602 in the conversion of DL-5-substituted hydantoins to L-amino acids.

PubMed

Mei, Yan-Zhen; Wan, Yong-Min; He, Bing-Fang; Ying, Han-Jie; Ouyang, Ping-Kai

2009-12-01

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction at higher temperature, the advantageous for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile was firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2 kb DNA fragment by Restriction Site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein hyuP, hyperprotein hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 comparing respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.

Radiation chemistry of salicylic and methyl substituted salicylic acids: Models for the radiation chemistry of pharmaceutical compounds

NASA Astrophysics Data System (ADS)

Ayatollahi, Shakiba; Kalnina, Daina; Song, Weihua; Turks, Maris; Cooper, William J.

2013-11-01

Salicylic acid and its derivatives are components of many medications and moieties found in numerous pharmaceutical compounds. They have been used as models for various pharmaceutical compounds in pharmacological studies, for the treatment of pharmaceuticals and personal care products (PPCPs), and, reactions with natural organic matter (NOM). In this study, the radiation chemistry of benzoic acid, salicylic acid and four methyl substituted salicylic acids (MSA) is reported. The absolute bimolecular reaction rate constants for hydroxyl radical reaction with benzoic and salicylic acids as well as 3-methyl-, 4-methyl-, 5-methyl-, and 6-methyl-salicylic acid were determined (5.86±0.54)×109, (1.07±0.07)×1010, (7.48±0.17)×109, (7.31±0.29)×109, (5.47±0.25)×109, (6.94±0.10)×109 (M-1 s-1), respectively. The hydrated electron reaction rate constants were measured (3.02±0.10)×109, (8.98±0.27)×109, (5.39±0.21)×109, (4.33±0.17)×109, (4.72±0.15)×109, (1.42±0.02)×109 (M-1 s-1), respectively. The transient absorption spectra for the six model compounds were examined and their role as model compounds for the radiation chemistry of pharmaceuticals investigated.

A single beta-amino acid substitution to angiotensin II confers AT2 receptor selectivity and vascular function.

PubMed

Jones, Emma S; Del Borgo, Mark P; Kirsch, Julian F; Clayton, Daniel; Bosnyak, Sanja; Welungoda, Iresha; Hausler, Nicholas; Unabia, Sharon; Perlmutter, Patrick; Thomas, Walter G; Aguilar, Marie-Isabel; Widdop, Robert E

2011-03-01

Novel AT(2)R ligands were designed by substituting individual β-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT(2)R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT(1)R- or AT(2)R- transfected HEK-293 cells, only β-Asp(1)-Ang II and Ang II fully displaced [(125)I]-Ang II from AT(1)R. In contrast, β-substitutions at each position of Ang II exhibited AT(2)R affinity, with β-Tyr(4)-Ang II and β-Ile(5)-Ang II exhibiting ≈ 1000-fold AT(2)R selectivity. In mouse aortic rings, β-Tyr(4)-Ang II and β-Ile(5)-Ang II evoked vasorelaxation that was sensitive to blockade by the AT(2)R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT(1)R blockade, β-Ile(5)-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (β-Ile(5)-Ang II plus candesartan, -24 ± 4 mm Hg) to a greater extent than candesartan alone (-11 ± 3 mm Hg, n=7, P<0.05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, β-Tyr(4)-Ang II had no influence on BP (n=10), and it was less stable than β-Ile(5)-Ang II in plasma stability assays. Thus, this study demonstrated that a single β-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT(2)R. This approach to the design and synthesis of novel AT(2)R-selective peptidomimetics shows great potential to provide insight into AT(2)R function.

Effect of Thenardite on the Direct Detection of Aromatic Amino Acids: Implications for the Search for Life in the Solar System

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

C. Doc Richardson; Nancy W. Hinman; Jill R. Scott

2009-10-01

With the discovery of Na-sulfate minerals on Mars and Europa, recent studies using these minerals have focused on their ability to assist in the detection of bio/organic signatures. This study further investigates the ability of thenardite (Na2SO4) to effectively facilitate the ionization and identification of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) using a technique called geomatrix-assisted laser desorption/ionization (GALDI) in conjunction with a Fourier transform mass spectrometry (FTICR-MS). This technique is based on the ability of a mineral host to facilitate the ionization and detection of bio/organic molecules. Spectra obtained from each aromatic amino acid alone and in combinationmore » with thenardite show differences in ionization mechanism and fragmentation patterns. These differences are due to chemical and structural differences between the aromatic side chains of their respective amino acid. Tyrosine and tryptophan when combined with thenardite were observed to undergo cation-attachment ([M+Na]+), due to the high alkali affinity of their aromatic side chains. Subsequent cation substitution of the carboxyl group led to formation double cation-attached peaks ([M-H+Na]Na+). In contrast, phenylalanine mixed with thenardite showed no evidence of Na+ interaction. Understanding how codeposition of amino acids with thenardite can affect the observed mass spectra is important for future exploration missions that are likely to use laser desorption mass spectrometry to search for bio/organic compounds in extraterrestrial environments.« less

Effect of thenardite on the direct detection of aromatic amino acids: implications for the search for life in the solar system

NASA Astrophysics Data System (ADS)

Doc Richardson, C.; Hinman, Nancy W.; Scott, Jill R.

2009-10-01

With the discovery of Na-sulphate minerals on Mars and Europa, recent studies using these minerals have focused on their ability to assist in the detection of bio/organic signatures. This study further investigates the ability of thenardite (Na2SO4) to effectively facilitate the ionization and identification of aromatic amino acids (phenylalanine, tyrosine and tryptophan) using a technique called geomatrix-assisted laser desorption/ionization in conjunction with a Fourier transform ion cyclotron resonance mass spectrometry. This technique is based on the ability of a mineral host to facilitate desorption and ionization of bio/organic molecules for detection. Spectra obtained from each aromatic amino acid alone and in combination with thenardite show differences in ionization mechanism and fragmentation patterns. These differences are due to chemical and structural differences between the aromatic side chains of their respective amino acid. Tyrosine and tryptophan when combined with thenardite were observed to undergo cation-attachment ([M+Na]+), due to the high alkali ion affinity of their aromatic side chains. In addition, substitution of the carboxyl group hydrogen by sodium led to formation of [M-H+Na]Na+ peaks. In contrast, phenylalanine mixed with thenardite showed no evidence of Na+ attachment. Understanding how co-deposition of amino acids with thenardite can affect the observed mass spectra is important for future exploration missions that are likely to use laser desorption mass spectrometry to search for bio/organic compounds in extraterrestrial environments.

Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

PubMed

Mu, Bingnan; Xu, Helan; Yang, Yiqi

2015-11-01

In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. Copyright © 2015 Elsevier Ltd. All rights reserved.

40 CFR 721.981 - Substituted naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex.

Code of Federal Regulations, 2014 CFR

2014-07-01

... naphthalenyl-substituted azonaphthol chromium complex. 721.981 Section 721.981 Protection of Environment...-substituted naphthalenyl-substituted azonaphthol chromium complex. (a) Chemical substance and significant new... naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex (PMN P-93-1631) is subject to...

40 CFR 721.981 - Substituted naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex.

Code of Federal Regulations, 2013 CFR

2013-07-01

... naphthalenyl-substituted azonaphthol chromium complex. 721.981 Section 721.981 Protection of Environment...-substituted naphthalenyl-substituted azonaphthol chromium complex. (a) Chemical substance and significant new... naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex (PMN P-93-1631) is subject to...

40 CFR 721.981 - Substituted naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex.

Code of Federal Regulations, 2012 CFR

2012-07-01

... naphthalenyl-substituted azonaphthol chromium complex. 721.981 Section 721.981 Protection of Environment...-substituted naphthalenyl-substituted azonaphthol chromium complex. (a) Chemical substance and significant new... naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex (PMN P-93-1631) is subject to...

40 CFR 721.981 - Substituted naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex.

Code of Federal Regulations, 2011 CFR

2011-07-01

... naphthalenyl-substituted azonaphthol chromium complex. 721.981 Section 721.981 Protection of Environment...-substituted naphthalenyl-substituted azonaphthol chromium complex. (a) Chemical substance and significant new... naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex (PMN P-93-1631) is subject to...

Effect of chemical substitutions on photo-switching properties of 3-hydroxy-picolinic acid studied by ab initio methods

NASA Astrophysics Data System (ADS)

Rode, Michał F.; Sobolewski, Andrzej L.

2014-02-01

Effect of chemical substitutions to the molecular structure of 3-hydroxy-picolinic acid on photo-switching properties of the system operating on excited-state intramolecular double proton transfer (d-ESIPT) process [M. F. Rode and A. L. Sobolewski, Chem. Phys. 409, 41 (2012)] was studied with the aid of electronic structure theory methods. It was shown that simultaneous application of electron-donating and electron-withdrawing substitutions at certain positions of the molecular frame increases the height of the S0-state tautomerization barrier (ensuring thermal stability of isomers) and facilitates a barrierless access to the S1/S0 conical intersection from the Franck-Condon region of the S1 potential-energy surface. Results of study point to the conclusion that the most challenging issue for practical design of a fast molecular photoswitch based on d-ESIPT phenomenon are to ensure a selectivity of optical excitation of a given tautomeric form of the system.

Establishment of an evaluation model for human milk fat substitutes.

PubMed

Wang, Yong-Hua; Mai, Qing-Yun; Qin, Xiao-Li; Yang, Bo; Wang, Zi-Lian; Chen, Hai-Tian

2010-01-13

Fatty acid composition and distribution of human milk fat (HMF), from mothers over different lactating periods in Guangzhou, China, were analyzed. The universal characteristics were consistent with previously reported results although the fatty acid content was within a different range and dependent on the local population (low saturated fatty acid and high oleic acid for Guangdong mothers' milk fat). Based on the composition of the total and sn-2 fatty acids of mature milk fat, an efficient evaluation model was innovatively established by adopting the "deducting score" principle. The model showed good agreement between the scores and the degree of similarity by assessing 15 samples from different sources including four samples of HMF, eight samples of human milk fat substitutes (HMFSs) and infant formulas, and three samples of fats and oils. This study would allow for the devolvement of individual human milk fat substitutes with different and specific fatty acid compositions for local infants.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of

Large differences in proportions of harmful and benign amino acid substitutions between proteins and diseases.

PubMed

Schaafsma, Gerard C P; Vihinen, Mauno

2017-07-01

Genes and proteins are known to have differences in their sensitivity to alterations. Despite numerous sequencing studies, proportions of harmful and harmless substitutions are not known for proteins and groups of proteins. To address this question, we predicted the outcome for all possible single amino acid substitutions (AASs) in nine representative protein groups by using the PON-P2 method. The effects on 996 proteins were studied and vast differences were noticed. Proteins in the cancer group harbor the largest proportion of harmful variants (42.1%), whereas the non-disease group of proteins not known to have a disease association and not involved in the housekeeping functions had the lowest number of harmful variants (4.2%). Differences in the proportions of the harmful and benign variants are wide within each group, but they still show clear differences between the groups. Frequently appearing protein domains show a wide spectrum of variant frequencies, whereas no major protein structural class-specific differences were noticed. AAS types in the original and variant residues showed distinctive patterns, which are shared by all the protein groups. The observations are relevant for understanding genetic bases of diseases, variation interpretation, and for the development of methods for that purpose. © 2017 Wiley Periodicals, Inc.

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

PubMed

Xu, Changping; Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Cao, Zengguo; Li, Ling; Wang, Jianzhong; Yan, Feihu; Wang, Lina; Chi, Hang; Gai, Weiwei; Wang, Chong; Zhao, Yongkun; Feng, Yan; Wang, Tiecheng; Gao, Yuwei; Lu, Yiyu; Yang, Songtao; Xia, Xianzhu

2016-05-01

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

Nucleic acid sequence detection using multiplexed oligonucleotide PCR

DOEpatents

Nolan, John P [Santa Fe, NM; White, P Scott [Los Alamos, NM

2006-12-26

Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

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

Wallis, G.A.; Rash, B.; Sweetman, W.A.

1994-02-01

Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified twomore » individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.« less

Thin layer chromatographic method for the detection of uric acid: collaborative study.

PubMed

Thrasher, J J; Abadie, A

1978-07-01

A collaborative study has been completed on an improved method for the detection and confirmation of uric acid from bird and insect excreta. The proposed method involves the lithium carbonate solubilization of the suspect excreta material, followed by butanol-methanol-water-acetic acid thin layer chromatography, and trisodium phosphate-phosphotungstic acid color development. The collaborative tests resulted in 100% detection of uric acid standard at the 50 ng level and 75% detection at the 20-25 ng level. No false positives were reported during tests of compounds similar to uric acid. The proposed method has been adopted official first action; the present official final action method, 44.161, will be retained for screening purposes.

Arsenic scavenging by aluminum-substituted ferrihydrites in a circumneutral pH river impacted by acid mine drainage.

PubMed

Adra, Areej; Morin, Guillaume; Ona-Nguema, Georges; Menguy, Nicolas; Maillot, Fabien; Casiot, Corinne; Bruneel, Odile; Lebrun, Sophie; Juillot, Farid; Brest, Jessica

2013-11-19

Ferrihydrite (Fh) is a nanocrystalline ferric oxyhydroxide involved in the retention of pollutants in natural systems and in water-treatment processes. The status and properties of major chemical impurities in natural Fh is however still scarcely documented. Here we investigated the structure of aluminum-rich Fh, and their role in arsenic scavenging in river-bed sediments from a circumneutral river (pH 6-7) impacted by an arsenic-rich acid mine drainage (AMD). Extended X-ray absorption fine structure (EXAFS) spectroscopy at the Fe K-edge shows that Fh is the predominant mineral phase forming after neutralization of the AMD, in association with minor amount of schwertmannite transported from the AMD. TEM-EDXS elemental mapping and SEM-EDXS analyses combined with EXAFS analysis indicates that Al(3+) substitutes for Fe(3+) ions into the Fh structure in the natural sediment samples, with local aluminum concentration within the 25-30 ± 10 mol %Al range. Synthetic aluminous Fh prepared in the present study are found to be less Al-substituted (14-20 ± 5 mol %Al). Finally, EXAFS analysis at the arsenic K-edge indicates that As(V) form similar inner-sphere surface complexes on the natural and synthetic Al-substituted Fh studied. Our results provide direct evidence for the scavenging of arsenic by natural Al-Fh, which emphasize the possible implication of such material for scavenging pollutants in natural or engineered systems.

Detection of Rickettsia helvetica and Candidatus R. tarasevichiae DNA in Ixodes persulcatus ticks collected in Northeastern European Russia (Komi Republic).

PubMed

Kartashov, Mikhail Yu; Glushkova, Ludmila I; Mikryukova, Tamara P; Korabelnikov, Igor V; Egorova, Yulia I; Tupota, Natalia L; Protopopova, Elena V; Konovalova, Svetlana N; Ternovoi, Vladimir A; Loktev, Valery B

2017-06-01

The number of tick-borne infections in the northern European regions of Russia has increased considerably in the last years. In the present study, 676 unfed adult Ixodes persulcatus ticks were collected in the Komi Republic from 2011 to 2013 to study tick-borne rickettsioses. Rickettsia spp. DNA was detected by PCR in 51 (7.6%) ticks. The nucleotide sequence analysis of gltA fragments (765bp) from 51 ticks indicated that 60.8% and 39.2% of the ticks were infected with Rickettsia helvetica and Candidatus R. tarasevichiae, respectively. The gltA fragments showed 100% identity with those of Candidatus R. tarasevichiae previously discovered in Siberia and China, whereas R. helvetica showed 99.9% sequence identity with European isolates. The ompB had 8 nucleotide substitutions, 6 of which resulted in amino acid substitutions. In the sca9 gene, 3 nucleotide substitutions were detected, and only one resulted in amino acid substitution. The smpA, ompW, and β-lactamase genes of R. helvetica also showed a high level of sequence identity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex.

PubMed Central

Marcelli, M; Zoppi, S; Wilson, C M; Griffin, J E; McPhaul, M J

1994-01-01

We have investigated the basis of androgen resistance in seven unrelated individuals with complete testicular feminization or Reifenstein syndrome caused by single amino acid substitutions in the hormone-binding domain of the androgen receptor. Monolayer-binding assays of cultured genital skin fibroblasts demonstrated absent ligand binding, qualitative abnormalities of androgen binding, or a decreased amount of qualitatively normal receptor. The consequences of these mutations were examined by introducing the mutations by site-directed mutagenesis into the androgen receptor cDNA sequence and expressing the mutant cDNAs in mammalian cells. The effects of the amino acid substitutions on the binding of different androgens and on the capacity of the ligand-bound receptors to activate a reporter gene were investigated. Substantial differences were found in the responses of the mutant androgen receptors to incubation with testosterone, 5 alpha-dihydrotestosterone, and mibolerone. In several instances, increased doses of hormone or increased frequency of hormone addition to the incubation medium resulted in normal or near normal activation of a reporter gene by cells expressing the mutant androgen receptors. These studies suggest that the stability of the hormone receptor complex is a major determinant of receptor function in vivo. Images PMID:7929841

Computer-assisted automatic synthesis II. Development of a fully automated apparatus for preparing substituted N–(carboxyalkyl)amino acids

PubMed Central

Hayashi, Nobuyoshi; Sugawara, Tohru; Shintani, Motoaki; Kato, Shinji

1989-01-01

A versatile automated apparatus, equipped with an artificial intelligence has been developed which may be used to prepare and isolate a wide variety of compounds. The prediction of the optimum reaction conditions and the reaction control in real time, are accomplished using novel kinetic equations and substituent effects in an artificial intelligence software which has already reported [1]. This paper deals with the design and construction of the fully automated system, and its application to the synthesis of a substituted N-(carboxyalkyl)amino acid. The apparatus is composed of units for perfoming various tasks, e.g. reagent supply, reaction, purification and separation, each linked to a control system. All synthetic processes including washing and drying of the apparatus after each synthetic run were automatically performed from the mixing of the reactants to the isolation of the products as powders with purities of greater than 98%. The automated apparatus has been able to run for 24 hours per day, and the average rate of synthesis of substituted N-(carboxyalkyl)amino acids has been three compounds daily. The apparatus is extremely valuable for synthesizing many derivatives of one particular compound structure. Even if the chemical yields are low under the optimum conditions, it is still possible to obtain a sufficient amount of the desired product by repetition of the reaction. Moreover it was possible to greatly reduce the manual involvement of the many syntheses which are a necessary part of pharmaceutical research. PMID:18924679

Detection of nucleic acid sequences by invader-directed cleavage

DOEpatents

Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

1999-01-01

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

Association of Dietary Proportions of Macronutrients with Visceral Adiposity Index: Non-Substitution and Iso-Energetic Substitution Models in a Prospective Study.

PubMed

Moslehi, Nazanin; Ehsani, Behnaz; Mirmiran, Parvin; Hojjat, Parvane; Azizi, Fereidoun

2015-10-26

We aimed to investigate associations between dietary macronutrient proportions and prospective visceral adiposity index changes (ΔVAI). The study included 1254 adults (18-74 years), from the Tehran Lipid and Glucose Study (TLGS), who were followed for three years. Dietary intakes were assessed twice using food frequency questionnaires. Associations of dietary macronutrient with ΔVAI and risk of visceral adiposity dysfunction (VAD) after three years were investigated. The percentage of energy intake from protein in the total population, and from fat in women, were associated with higher increases in VAI. A 5% higher energy intake from protein substituted for carbohydrate, monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) was associated with higher ΔVAI. Higher energy intake from animal protein substituted for PUFAs was positively associated with ΔVAI. Substituting protein and PUFAs with MUFAs were related to higher ΔVAI. The associations were similar in men and women, but reached significance mostly among women. Risk of VAD was increased when 1% of energy from protein was replaced with MUFAs. Substituting protein for carbohydrate and fat, and fat for carbohydrate, resulted in increased risk of VAD in women. Higher dietary proportions of protein and animal-derived MUFA may be positively associated with ΔVAI and risk of VAD.

Unraveling the effects of amino acid substitutions enhancing lipase resistance to an ionic liquid: a molecular dynamics study.

PubMed

Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Fulton, Alexander; Zhu, Leilei; Davari, Mehdi D; Jaeger, Karl-Erich; Schwaneberg, Ulrich; Bocola, Marco

2018-04-04

Understanding of the structural and dynamic properties of enzymes in non-aqueous media (e.g., ionic liquids, ILs) is highly attractive for protein engineers and synthetic biochemists. Despite a growing number of molecular dynamics (MD) simulation studies on the influence of different ILs on wild-type enzymes, the effects of various amino acid substitutions on the stability and activity of enzymes in ILs remain to be unraveled at the molecular level. Herein, we selected fifty previously reported Bacillus subtilis lipase A (BSLA) variants with increased resistance towards an IL (15 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate; [Bmim][TfO]), and also ten non-resistant BSLA variants for a MD simulation study to identify the underlying molecular principles. Some important properties differentiating resistant and non-resistant BSLA variants from wild-type were elucidated. Results show that, in 15 vol% [Bmim][TfO] aqueous solution, 40% and 60% of non-resistant variants have lower and equal probabilities to form a catalytically important hydrogen bond between S77 and H156 compared to wild-type, whereas 36% and 56% of resistant variants show increased and equal probabilities, respectively. Introducing positively charged amino acids close to the substrate-binding cleft for instance I12R is beneficial for the BSLA resistance towards 15 vol% [Bmim][TfO], likely due to the reduced probability of [Bmim]+ cations clustering near the cleft. In contrast, substitution with a large hydrophobic residue like I12F can block the cleft through hydrophobic interaction with a neighboring nonpolar loop 134-137 or/and an attractive π-π interaction with [Bmim]+ cations. In addition, the resistant variants having polar substitutions on the surface show higher ability to stabilize the surface water molecule network in comparison to non-resistant variants. This study can guide experimentalists to rationally design promising IL-resistant enzymes, and contribute to a deeper

Effects of a naturally occurring amino acid substitution in bovine PrP: a model for inherited prion disease in a natural host species

USDA-ARS?s Scientific Manuscript database

The most common hereditary prion disease is human Creutzfeldt-Jakob disease (CJD) associated with a mutation in the prion gene (PRNP) resulting in a glutamic acid to lysine substitution at position 200 (E200K) in the prion protein. Models of E200K CJD in transgenic mice have proven interesting but h...

Tandem SN2' nucleophilic substitution/oxidative radical cyclization of aryl substituted allylic alcohols with 1,3-dicarbonyl compounds.

PubMed

Zhang, Zhen; Li, Cheng; Wang, Shao-Hua; Zhang, Fu-Min; Han, Xue; Tu, Yong-Qiang; Zhang, Xiao-Ming

2017-04-11

A novel and efficient tandem S N 2' nucleophilic substitution/oxidative radical cyclization reaction of aryl substituted allylic alcohols with 1,3-dicarbonyl compounds has been developed by using Mn(OAc) 3 as an oxidant, which enables the expeditious synthesis of polysubstituted dihydrofuran (DHF) derivatives in moderate to high yields. The use of weakly acidic hexafluoroisopropanol (HFIP) as the solvent rather than AcOH has successfully improved the yields and expanded the substrate scope of this type of radical cyclization reactions. Mechanistic studies confirmed the cascade reaction process involving a final radical cyclization.

Nonsynonymous substitution rate heterogeneity in the peptide-binding region among different HLA-DRB1 lineages in humans.

PubMed

Yasukochi, Yoshiki; Satta, Yoko

2014-05-02

An extraordinary diversity of amino acid sequences in the peptide-binding region (PBR) of human leukocyte antigen [HLA; human major histocompatibility complex (MHC)] molecules has been maintained by balancing selection. The process of accumulation of amino acid diversity in the PBR for six HLA genes (HLA-A, B, C, DRB1, DQB1, and DPB1) shows that the number of amino acid substitutions in the PBR among alleles does not linearly correlate with the divergence time of alleles at the six HLA loci. At these loci, some pairs of alleles show significantly less nonsynonymous substitutions at the PBR than expected from the divergence time. The same phenomenon was observed not only in the HLA but also in the rat MHC. To identify the cause for this, DRB1 sequences, a representative case of a typical nonlinear pattern of substitutions, were examined. When the amino acid substitutions in the PBR were placed with maximum parsimony on a maximum likelihood tree based on the non-PBR substitutions, heterogeneous rates of nonsynonymous substitutions in the PBR were observed on several branches. A computer simulation supported the hypothesis that allelic pairs with low PBR substitution rates were responsible for the stagnation of accumulation of PBR nonsynonymous substitutions. From these observations, we conclude that the nonsynonymous substitution rate at the PBR sites is not constant among the allelic lineages. The deceleration of the rate may be caused by the coexistence of certain pathogens for a substantially long time during HLA evolution. Copyright © 2014 Yasukochi and Satta.

Electron-rich triphenylamine-based sensors for picric acid detection.

PubMed

Chowdhury, Aniket; Mukherjee, Partha Sarathi

2015-04-17

This paper demonstrates the role of solvent in selectivity and sensitivity of a series of electron-rich compounds for the detection of trace amounts of picric acid. Two new electron-rich fluorescent esters (6, 7) containing a triphenylamine backbone as well as their analogous carboxylic acids (8, 9) have been synthesized and characterized. Fluorescent triphenylamine coupled with an ethynyl moiety constitutes π-electron-rich selective and sensitive probes for electron-deficient picric acid (PA). In solution, the high sensitivity of all the sensors toward PA can be attributed to a combined effect of the ground-state charge-transfer complex formation and resonance energy transfer between the sensor and analyte. The acids 8 and 9 also showed enhanced sensitivity for nitroaromatics in the solid state, and their enhanced sensitivity could be attributed to exciton migration due to close proximity of the neighboring acid molecules, as evident from the X-ray diffraction study. The compounds were found to be quite sensitive for the detection of trace amount of nitroaromatics in solution, solid, and contact mode.

Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots

PubMed Central

Missihoun, Tagnon D.; Kotchoni, Simeon O.; Bartels, Dorothea

2016-01-01

Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species. PMID:27798665

Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots.

PubMed

Missihoun, Tagnon D; Kotchoni, Simeon O; Bartels, Dorothea

2016-01-01

Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species Arabidopsis thaliana then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the ALDH genes in Brachypodium distachyon, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that B. distachyon, like other grasses, contains more ALDH2C/REF1 isoforms than A. thaliana and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species.

A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti.

PubMed

Chang, Cheng; Shen, Wen-Kai; Wang, Tzu-Ting; Lin, Ying-Hsi; Hsu, Err-Lieh; Dai, Shu-Mei

2009-04-01

To identify pertinent mutations associated with knockdown resistance to permethrin, the entire coding sequence of the voltage-gated sodium channel gene Aa-para was sequenced and analyzed from a Per-R strain with 190-fold resistance to permethrin and two susceptible strains of Aedes aegypti. The longest transcript, a 6441bp open reading frame, encodes 2147 amino acid residues with an estimated molecular mass of 241kDa. A total of 33 exons were found in the Aa-para gene over 293kb of genomic DNA. Three previously unreported optional exons were identified. The first two exons, m and n, were located within the intracellular domain I/II, and the third, f', was found within the II/III linkers. The two mutually exclusive exons, d and l, were the only alternative exons in all the cDNA clones sequenced in this study. The most distinct finding was a novel amino acid substitution mutation, D1794Y, located within the extracellular linker between IVS5 and IVS6, which is concurrent with the known V1023G mutation in Aa-para of the Per-R strain. The high frequency and coexistence of the two mutations in the Per-R strain suggest that they might exert a synergistic effect to provide the knockdown resistance to permethrin. Furthermore, both cDNA and genomic DNA data from the same individual mosquitoes have demonstrated that RNA editing was not involved in amino acid substitutions of the Per-R strain.

The nucleotide sequence of HLA-B{sup *}2704 reveals a new amino acid substitution in exon 4 which is also present in HLA-B{sup *}2706

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

Rudwaleit, M.; Bowness, P.; Wordsworth, P.

1996-12-31

The HLA-B27 subtype HLA-B{sup *}2704 is virtually absent in Caucasians but common in Orientals, where it is associated with ankylosing spondylitis. The amino acid sequence of HLA-B{sup *}2704 has been established by peptide mapping and was shown to differ by two amino acids from HLA-B{sup *}2705, HLA-B{sup *}2704 is characterized by a serine for aspartic acid substitution at position 77 and glutamic acid for valine at position 152. To date, however, no nucleotide sequence confirming these changes at the DNA level has been published. 13 refs., 2 figs.

Hybridization-based detection of Helicobacter pylori at human body temperature using advanced locked nucleic acid (LNA) probes.

PubMed

Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina; Figueiredo, Céu; Wengel, Jesper; Filipe Azevedo, Nuno

2013-01-01

The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.

Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

PubMed Central

Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina; Figueiredo, Céu; Wengel, Jesper; Filipe Azevedo, Nuno

2013-01-01

The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2’-O-methyl RNAs (2’OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others. PMID:24278398

Natural variation in a single amino acid substitution underlies physiological responses to topoisomerase II poisons.

PubMed

Zdraljevic, Stefan; Strand, Christine; Seidel, Hannah S; Cook, Daniel E; Doench, John G; Andersen, Erik C

2017-07-01

Many chemotherapeutic drugs are differentially effective from one patient to the next. Understanding the causes of this variability is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate sensitivity to a group of anti-neoplastic drugs that target topoisomerase II using the model organism Caenorhabditis elegans. We show that wild strains of C. elegans vary in their sensitivity to these drugs, and we use an unbiased genetic approach to demonstrate that this natural variation is explained by a methionine-to-glutamine substitution in topoisomerase II (TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and its poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms (methionine in hTOPIIα and glutamine in hTOPIIβ). We go on to show that this amino acid difference between the two human topoisomerase isoforms influences cytotoxicity of topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIβ are differentially affected by various poisons and demonstrate the utility of C. elegans in understanding the genetics of drug responses.

Multiple adaptive amino acid substitutions increase the virulence of a wild waterfowl-origin reassortant H5N8 avian influenza virus in mice.

PubMed

Yu, Zhijun; Cheng, Kaihui; Sun, Weiyang; Zhang, Xinghai; Xia, Xianzhu; Gao, Yuwei

2018-01-15

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) caused poultry outbreaks in the Republic of Korea in 2014. The novel H5N8 HPAIV has spread to Asia, Europe, and North America and caused great public concern from then on. Here, we generated mouse-adapted variants of a wild waterfowl-origin H5N8 HPAIV to identify adaptive mutants that confer enhanced pathogenicity in mammals. The mouse lethal doses (MLD 50 ) of the mouse-adapted variants were reduced 31623-fold compared to the wild-type (WT) virus. Mouse-adapted variants displayed enhanced replication in vitro and in vivo, and expanded tissue tropism in mice. Sequence analysis revealed four amino acid substitutions in the PB2 (E627K), PA (F35S), HA (R227H), and NA (I462V) proteins. These data suggest that multiple amino acid substitutions collaboratively increase the virulence of a wild bird-origin reassortant H5N8 HPAIV and cause severe disease in mice. Copyright © 2017 Elsevier B.V. All rights reserved.

Emergence of Avian Influenza Viruses with Enhanced Transcription Activity by a Single Amino Acid Substitution in the Nucleoprotein during Replication in Chicken Brains ▿

PubMed Central

Tada, Tatsuya; Suzuki, Koutaro; Sakurai, Yu; Kubo, Masanori; Okada, Hironao; Itoh, Toshihiro; Tsukamoto, Kenji

2011-01-01

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP I109T). By analyzing viruses constructed by the reverse-genetic method, we established that the NP I109T substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP I109T substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP I109T substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP I109T mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains. PMID:21795332

Structure-performance relationships of phenyl cinnamic acid derivatives as MALDI-MS matrices for sulfatide detection.

PubMed

Tambe, Suparna; Blott, Henning; Fülöp, Annabelle; Spang, Nils; Flottmann, Dirk; Bräse, Stefan; Hopf, Carsten; Junker, Hans-Dieter

2017-02-01

A key aspect for the further development of matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (MS) is a better understanding of the working principles of MALDI matrices. To address this issue, a chemical compound library of 59 structurally related cinnamic acid derivatives was synthesized. Potential MALDI matrices were evaluated with sulfatides, a class of anionic lipids which are abundant in complex brain lipid mixtures. For each matrix relative mean S/N ratios of sulfatides were determined against 9-aminoacridine as a reference matrix using negative ion mass spectrometry with 355 and 337 nm laser systems. The comparison of matrix features with their corresponding relative mean S/N ratios for sulfatide detection identified correlations between matrix substitution patterns, their chemical functionality, and their MALDI-MS performance. Crystal structures of six selected matrices provided structural insight in hydrogen bond interactions in the solid state. Principal component analysis allowed the additional identification of correlation trends between structural and physical matrix properties like number of exchangeable protons at the head group, MW, logP, UV-Vis, and sulfatide detection sensitivity. Graphical abstract Design, synthesis and mass spectrometric evaluation of MALDI-MS matrix compound libraries allows the identification of matrix structure - MALDI-MS performance relationships using multivariate statistics as a tool.

Amino acid substitutions in low pathogenic avian influenza virus strains isolated from wild birds in Korea.

PubMed

Oh, Kwang-Hyun; Mo, Jong-Suk; Bae, Yeon-Ji; Lee, Seung-Baek; Lai, Van Dam; Wang, Seung-Jun; Mo, In-Pil

2018-06-01

Wild birds are natural hosts and reservoirs for influenza A viruses. However, many species, such as many waterfowl, are asymptomatic when infected and so facilitate the generation of viral genetic diversity. Mutations of key genes affect the replicability, pathogenicity, transmissibility, and antiviral resistance of influenza A viruses. In this study, we isolated avian influenza (AI) viruses from wild bird fecal samples and analyzed changes in amino acids over time and geographic region to monitor the biological change of the AI virus. Between 2014 and 2016, we collected 38,921 fresh fecal samples from major wild bird habitats located throughout Korea and isolated 123 AI viruses. We subsequently selected 22 amino acid sites to analyze for changes. These sites included ten sites associated with replication, ten sites associated with pathogenicity, three sites associated with transmission, and seven sites associated with antiviral resistance. We found substitution rates of 71.7% at the C38Y amino acid site within the polymerase basic protein 1 (PB1) gene, 66.7% at the D222G site within the hemagglutinin (HA) 1 gene, and 75.6% at the A184 site within the nucleoprotein (NP) gene. Alterations of the PB1, HA1, and NP genes are closely associated with increased pathogenicity in chickens and mammals. The remaining sites of interest exhibited few modifications. In this study, we confirmed that AI viruses circulating among wild birds in Korea consistently exhibit modifications at amino acid sites linked with replication and pathogenicity.

Detection of folic acid protein in human serum using reduced graphene oxide electrodes modified by folic-acid.

PubMed

He, Lijie; Wang, Qian; Mandler, Daniel; Li, Musen; Boukherroub, Rabah; Szunerits, Sabine

2016-01-15

The detection of disease markers is considered an important step for early diagnosis of cancer. We design in this work a novel electrochemical sensing platform for the sensitive and selective detection of folic acid protein (FP). The platform is fabricated by electrophoretic deposition (EPD) of reduced graphene oxide (rGO) onto a gold electrode and post-functionalization of rGO with folic acid. Upon FP binding, a significant current decrease can be measured using differential pulse voltammetry (DPV). Using this scheme, a detection limit of 1pM is achieved. Importantly, the method also allows the detection of FP in serum being thus an appealing approach for the sensitive detection of biomarkers in clinical samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Trifluoromethyl-substituted polymers

NASA Technical Reports Server (NTRS)

1993-01-01

Current work sponsored by the grant at Southwest Texas State University is directed toward the synthesis and characterization of: (1) N-alkylated polyamides derived from o-fluorinated diacids; (2) highly fluorinated polyethers; (3) polyesters derived from 2-hydroxy-2-propyl substituted arenes and/or 2,5-difluoroterephthalic acid; and (4) silicon-containing fluoropolymers. Work during the period from 1 July to 31 Dec. 1993 focused primarily on items 3 and 4 and on the development of a phosphorus containing modification of '12F-PEK.'

Synthetic versatility of 2-substituted-6-methyl 2,3-dihydropyridinones in the synthesis of polyfunctional piperidine-based compounds and related β amino acid derivatives.

PubMed

Yang, Yang; Hardman, Clayton

2017-10-18

Chiral 2-substituted-6-methyl 2,3-dihydropyidinones 9, which can be facilely obtained from an asymmetric vinylogous Mannich reaction (VMR) with 1,3-bis-trimethysily enol ether, were used as versatile intermediates in constructing chiral polyfunctional piperidine-based compounds. The 6-methyl group of such compounds can be conveniently functionalized via alkylation and acylation reactions to provide efficient entries to the synthesis of a variety of chiral multi-substituted piperidine-based compounds. Further elaboration of the corresponding intermediates also provided access to polyfunctional indolizidine-based compounds. These methods were showcased in an asymmetric synthesis of 2,6-di-substituted piperidine compound 13, reported as the key intermediate in the synthesis of (+)-calvine and a natural alkaloid (-)-indolizidine 209D. Furthermore, selective C5 iodination of compound 9 enabled the installation of additional functional groups at this position. Finally, we demonstrated that the oxidative cleavage of 2-substituted-6-methyl-2,3-dihydropyidinones is a practical and efficient method for the enantioselective synthesis of β-amino acids, which can undergo further intra-molecular cyclization to give the corresponding chiral four-membered β-lactam derivatives.

Kit for detecting nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

2001-01-01

A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the

Detection of soybean in soy-based meat substitutes.

PubMed

Abd Allah, M A; Foda, Y H; el-Dashlouty, S; el-Sanafiry, N Y; Abu Salem, F M

1986-01-01

The statistical analysis of the available data indicated that the straight line equations of protein, fat, fibre, calcium, methionine, and lysine could successively be used for forecasting the added soy percent in a given recipe. On the other hand, the areas of the identified bands in the electropherograms of the investigated samples were considered a reasonable tool for the quantitative determination of whole soybean in soy-based meat substitutes.

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

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

Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.

E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adoptedmore » a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.« less

Zn-site Substitution Effect in YbCo2Zn20

NASA Astrophysics Data System (ADS)

Kobayashi, Riki; Takamura, Haruki; Higa, Yasuyuki; Ikeda, Yoichi; Matsubayashi, Kazuyuki; Uwatoko, Yoshiya; Yoshizawa, Hideki; Aso, Naofumi

2017-04-01

We have investigated the substitution effect of YbCo2(Zn1-xTx)20 (T = Cu, Ga, and Cd) systems by using the experiments of X-ray powder diffraction (XRPD), specific heat, magnetic susceptibility, magnetization, and electrical resistivity in order to find out a material that approaches a quantum critical point by chemical pressure. The XRPD and electrical resistivity measurements clarify that the Cu-substitution makes the lattice constants shrink and keeps the magnetic electrical resistivity high, while the Ga- and the Cd-substitution show opposite relation of the Cu-substitution. However, we could not detect clear substitution effect in the specific heat, magnetic susceptibility, and magnetization measurements of Cu-substitution system within our experiments. It is necessary that to study the Cu-substitution samples that have higher x value at lower temperature.

Application of ChemDraw NMR Tool: Correlation of Program-Generated 13C Chemical Shifts and pKa Values of para-Substituted Benzoic Acids

NASA Astrophysics Data System (ADS)

Wang, Hongyi

2005-09-01

An application of ChemDraw NMR Tool was demonstrated by correlation of program-generated 13 C NMR chemical shifts and p K a values of para-substituted benzoic acids. Experimental 13 C NMR chemical shifts were analyzed in the same way for comparison. The project can be used as an assignment at the end of the first-year organic chemistry course to review topics or explore new techniques: Hammett equation, acid base equilibrium theory, electronic nature of functional groups, inductive and resonance effects, structure reactivity relationship, NMR spectroscopy, literature search, database search, and ChemDraw software.

Oxidation of Alkyl-substituted Cyclic Hydrocarbons by a Nocardia during Growth on n-Alkanes

PubMed Central

Davis, J. B.; Raymond, R. L.

1961-01-01

Nocardia 107-332, a soil isolate, oxidizes short-chain alkyl-substituted cyclic hydrocarbons to cyclic acids while growing on n-alkanes. Cyclic acids are produced also from relatively long-chain alkyl-substituted cyclics such as n-nonylbenzene or n-dodecylbenzene which alone support growth in a mineral-salts medium. ω-Oxidation of the alkyl substituents is followed by β-oxidation. It is of particular interest that cyclic acids such as cyclohexaneacetic and phenylacetic with C2 residual carboxylic acid substituents are resistant to further oxidation by the nocardia but cyclic acids with C1 or C3 substituents are readily oxidized and utilized for growth. The specificity of microbial oxidations is demonstrated by the conversion of p-isopropyltoluene (p-cymene) to p-isopropylbenzoic acid in n-alkane, growth-supported nocardia cultures. PMID:13720182

Synthesis of [.sup.13C] and [.sup.2H] substituted methacrylic acid, [.sup.13C] and [.sup.2H] substituted methyl methacrylate and/or related compounds

DOEpatents

Alvarez, Marc A [Santa Fe, NM; Martinez, Rodolfo A [Santa Fe, NM; Unkefer, Clifford J [Los Alamos, NM

2008-01-22

The present invention is directed to labeled compounds of the formulae ##STR00001## wherein Q is selected from the group consisting of --S--, --S(.dbd.O)--, and --S(.dbd.O).sub.2--, Z is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group selected from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each independently selected from the group consisting of a C.sub.1-C.sub.4 lower alkyl, an aryl, and an alkoxy group, and X is selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl group, and a fully-deuterated C.sub.1-C.sub.4 lower alkyl group. The present invention is also directed to a process of preparing labeled compounds, e.g., process of preparing [.sup.13C]methacrylic acid by reacting a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13CH.sub.2)-- aryl sulfone precursor with .sup.13CHI to form a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate, and, reacting the (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate with sodium hydroxide, followed by acid to form [.sup.13C]methacrylic acid. The present invention is further directed to a process of preparing [.sup.2H.sub.8]methyl methacrylate by reacting a (HOOC--C(C.sup.2H.sub.3).sub.2-- aryl sulfinyl intermediate with CD.sub.3I to form a (.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate, and heating the(.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate at temperatures and for time sufficient to form [.sup.2H.sub.8]methyl methacrylate.

Integrated sample-to-detection chip for nucleic acid test assays.

PubMed

Prakash, R; Pabbaraju, K; Wong, S; Tellier, R; Kaler, K V I S

2016-06-01

Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes.

40 CFR 721.981 - Substituted naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex.

Code of Federal Regulations, 2010 CFR

2010-07-01

... ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.981 Substituted naphtholoazo-substituted naphthalenyl-substituted azonaphthol chromium complex. (a) Chemical substance and significant new...

Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

2000-01-01

A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

Ultrasensitive Genotypic Detection of Antiviral Resistance in Hepatitis B Virus Clinical Isolates▿ †

PubMed Central

Fang, Jie; Wichroski, Michael J.; Levine, Steven M.; Baldick, Carl J.; Mazzucco, Charles E.; Walsh, Ann W.; Kienzle, Bernadette K.; Rose, Ronald E.; Pokornowski, Kevin A.; Colonno, Richard J.; Tenney, Daniel J.

2009-01-01

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy. PMID:19433559

Structure-activity studies of lGnRH-III through rational amino acid substitution and NMR conformational studies.

PubMed

Pappa, Eleni V; Zompra, Aikaterini A; Diamantopoulou, Zoi; Spyranti, Zinovia; Pairas, George; Lamari, Fotini N; Katsoris, Panagiotis; Spyroulias, George A; Cordopatis, Paul

2012-01-01

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp⁶ for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys⁸ or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp⁶ and Lys⁸ is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined β-turn conformation of GnRH-I. Copyright © 2012 Wiley Periodicals, Inc.

Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

PubMed

Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2014-05-01

The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands. © 2014 FEBS.

Amino acid substitutions affecting aspartic acid 605 and valine 606 decrease the interaction strength between the influenza virus RNA polymerase PB2 '627' domain and the viral nucleoprotein.

PubMed

Hsia, Ho-Pan; Yang, Yin-Hua; Szeto, Wun-Chung; Nilsson, Benjamin E; Lo, Chun-Yeung; Ng, Andy Ka-Leung; Fodor, Ervin; Shaw, Pang-Chui

2018-01-01

The influenza virus RNA genome is transcribed and replicated in the context of the viral ribonucleoprotein (vRNP) complex by the viral RNA polymerase. The nucleoprotein (NP) is the structural component of the vRNP providing a scaffold for the viral RNA. In the vRNP as well as during transcription and replication the viral polymerase interacts with NP but it is unclear which parts of the polymerase and NP mediate these interactions. Previously the C-terminal '627' domain (amino acids 538-693) of PB2 was shown to interact with NP. Here we report that a fragment encompassing amino acids 146-185 of NP is sufficient to mediate this interaction. Using NMR chemical shift perturbation assays we show that amino acid region 601 to 607 of the PB2 '627' domain interacts with this fragment of NP. Substitutions of these PB2 amino acids resulted in diminished RNP activity and surface plasmon resonance assays showed that amino acids D605 was essential for the interaction with NP and V606 may also play a partial role in the interaction. Collectively these results reveal a possible interaction surface between NP and the PB2 subunit of the RNA polymerase complex.

Deoxyribonucleic Acid Probes Analyses for the Detection of Periodontal Pathogens.

PubMed

Al Yahfoufi, Zoubeida; Hadchiti, Wahib; Berberi, Antoine

2015-09-01

In clinical microbiology several techniques have been used to identify bacteria. Recently, Deoxyribonucleic acid (DNA)-based techniques have been introduced to detect human microbial pathogens in periodontal diseases. Deoxyribonucleic acid probes can detect bacteria at a very low level if we compared with the culture methods. These probes have shown rapid and cost-effective microbial diagnosis, good sensitivity and specificity for some periodontal pathogens in cases of severe periodontitis. Eighty-five patients were recruited for the study. Twenty-one subjects ranging between 22 and 48 years of age fulfilled the inclusion and exclusion criteria. Seventy-eight samples became available for DNA probe analysis from the deepest pockets in each quadrant. All 21 patients showed positive results for Prevotella intermedia; also, Prevotella gingivalis was identified in 19 subjects, Aggregatibacter actinomycetemcomitans in 6 subjects. P. intermedia was diagnosed positive in 82% of the subgingival samples taken, 79% for P. gingivalis, and 23% for A. actinomycetemcomitans. This study shows a high frequency of putative periodontal pathogens by using DNA probe technology, which is semi-quantitative in this study. Deoxyribonucleic acid probes can detect bacteria at very low level about 10(3) which is below the detection level of culture methods. The detection threshold of cultural methods. The three types of bacteria can be detected rapidly with high sensitivity by using the DNA probe by general practitioners, and thus can help in the diagnosis process and the treatment.

Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.

PubMed

Kawabata, Yutaka; Murata, Kousaku; Kawai, Shigeyuki

2015-12-25

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescence Visual Detection of Herbal Product Substitutions at Terminal Herbal Markets by CCP-based FRET technique.

PubMed

Jiang, Chao; Yuan, Yuan; Yang, Guang; Jin, Yan; Liu, Libing; Zhao, Yuyang; Huang, Luqi

2016-10-21

Inaccurate labeling of materials used in herbal products may compromise the therapeutic efficacy and may pose a threat to medicinal safety. In this paper, a rapid (within 3 h), sensitive and visual colorimetric method for identifying substitutions in terminal market products was developed using cationic conjugated polymer-based fluorescence resonance energy transfer (CCP-based FRET). Chinese medicinal materials with similar morphology and chemical composition were clearly distinguished by the single-nucleotide polymorphism (SNP) genotyping method. Assays using CCP-based FRET technology showed a high frequency of adulterants in Lu-Rong (52.83%) and Chuan-Bei-Mu (67.8%) decoction pieces, and patented Chinese drugs (71.4%, 5/7) containing Chuan-Bei-Mu ingredients were detected in the terminal herbal market. In comparison with DNA sequencing, this protocol simplifies procedures by eliminating the cumbersome workups and sophisticated instruments, and only a trace amount of DNA is required. The CCP-based method is particularly attractive because it can detect adulterants in admixture samples with high sensitivity. Therefore, the CCP-based detection system shows great potential for routine terminal market checks and drug safety controls.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

PubMed

Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2017-07-01

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Boronic acid based imprinted electrochemical sensor for rutin recognition and detection.

PubMed

Wang, Chunlei; Wang, Qi; Zhong, Min; Kan, Xianwen

2016-10-21

Multi-walled carbon nanotubes (MWNTs) and boronic acid based molecular imprinting polymer (MIP) were successively modified on a glassy carbon electrode surface to fabricate a novel electrochemical sensor for rutin recognition and detection. 3-Aminophenylboronic acid (APBA) was chosen as a monomer for the electropolymerization of MIP film in the presence of rutin. In addition to the imprinted cavities in MIP film to complement the template molecule in shape and functional groups, the high affinity between the boronic acid group of APBA and vicinal diols of rutin also enhanced the selectivity of the sensor, which made the sensor display a good selectivity to rutin. Moreover, the modified MWNTs improved the sensitivity of the sensor for rutin detection. The mole ratios of rutin and APBA, electropolymerized scan cycles and rates, and pH value of the detection solution were optimized. Under optimal conditions, the sensor was used to detect rutin in a linear range from 4.0 × 10 -7 to 1.0 × 10 -5 mol L -1 with a detection limit of 1.1 × 10 -7 mol L -1 . The sensor has also been applied to assay rutin in tablets with satisfactory results.

LASER BIOLOGY AND MEDICINE: Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual photophysical parameters of a fluorescent form of a fluorescent protein mRFP1

NASA Astrophysics Data System (ADS)

Banishev, A. A.; Vrzheshch, E. P.; Shirshin, E. A.

2009-03-01

Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima).

STRUCTURAL AND FUNCTIONAL INTERACTION OF FATTY ACIDS WITH HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT

PubMed Central

Huang, Huan; McIntosh, Avery L.; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P.; Kier, Ann B.; Schroeder, Friedhelm

2014-01-01

The human liver fatty acid binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride (TG) levels. How this amino acid substitution elicits these effects is not known. This issue was addressed with human recombinant wild-type (WT, T94T) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC, and CC). T94A substitution did not or only slightly alter L-FABP binding affinities for saturated, monounsaturated, or polyunsaturated long chain fatty acids (LCFA), nor did it change the affinity for intermediates in TG synthesis. Nevertheless, T94A substitution markedly altered the secondary structural response of L-FABP induced by binding LCFA or intermediates of TG synthesis. Finally, T94A substitution markedly diminished polyunsaturated fatty acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), induction of peroxisome proliferator-activated receptor alpha (PPARα) - regulated proteins such as L-FABP, fatty acid transport protein 5 (FATP5), and PPARα itself in cultured primary human hepatocytes. Thus, while T94A substitution did not alter the affinity of human L-FABP for LCFAs, it significantly altered human L-FABP structure and stability as well as conformational and functional response to these ligands. PMID:24628888

DNA barcoding detects contamination and substitution in North American herbal products

PubMed Central

2013-01-01

Background Herbal products available to consumers in the marketplace may be contaminated or substituted with alternative plant species and fillers that are not listed on the labels. According to the World Health Organization, the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product integrity and authenticity with the goal of protecting consumers from health risks associated with product substitution and contamination. Methods We used DNA barcoding to conduct a blind test of the authenticity for (i) 44 herbal products representing 12 companies and 30 different species of herbs, and (ii) 50 leaf samples collected from 42 herbal species. Our laboratory also assembled the first standard reference material (SRM) herbal barcode library from 100 herbal species of known provenance that were used to identify the unknown herbal products and leaf samples. Results We recovered DNA barcodes from most herbal products (91%) and all leaf samples (100%), with 95% species resolution using a tiered approach (rbcL + ITS2). Most (59%) of the products tested contained DNA barcodes from plant species not listed on the labels. Although we were able to authenticate almost half (48%) of the products, one-third of these also contained contaminants and or fillers not listed on the label. Product substitution occurred in 30/44 of the products tested and only 2/12 companies had products without any substitution, contamination or fillers. Some of the contaminants we found pose serious health risks to consumers. Conclusions Most of the herbal products tested were of poor quality, including considerable product substitution, contamination and use of fillers. These activities dilute the effectiveness of otherwise useful remedies, lowering the perceived value of all related products because of a lack of consumer confidence in them. We suggest that the herbal industry should embrace DNA barcoding for authenticating herbal products through

Oxidation of Naphthenoaromatic and Methyl-Substituted Aromatic Compounds by Naphthalene 1,2-Dioxygenase

PubMed Central

Selifonov, S. A.; Grifoll, M.; Eaton, R. W.; Chapman, P. J.

1996-01-01

Oxidation of acenaphthene, acenaphthylene, and fluorene was examined with recombinant strain Pseudomonas aeruginosa PAO1(pRE695) expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone and cis- and trans-acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Nonspecific dehydrogenase activities present in the host strain led to the conversion of both of the acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was oxidized spontaneously to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. The action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in the formation of benzylic alcohols, i.e., methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially nonspecific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene. PMID:16535238

Imidazoline phosphonic acids

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

Redmore, D.

1972-07-04

Nitrogen-heterocyclic phosphonic acids and derivatives are characterized by aminomethyl (or substituted methyl) phosphonic acids or derivatives thereof bonded directly or indirectly, i.e., through a N-side chain to the nitrogen atom in the heterocyclic ring, for example those containing in the molecule at least one of the following units: ..pi..Equation/sup -/ where represents a heterocyclic ring having a nitrogen atom on the ring; -R'N- represents an amino- terminated side chain attached directly to the ring nitrogen (which side chain may or may not be present); and ..pi..Equation/sup -/ represents a methyl (or substituted methyl) phosphonic acid group where M is hydrogen,more » an alcohol or a salt moiety, and X and Y are hydrogen or a substituted group such as alkyl, aryl, etc., of which one or 2 units may be present depending on the available nitrogen bonded by hydrogens, and to uses for these compounds, for example, as scale inhibitors, corrosion inhibitors, etc. (5 claims)« less

Microbial activity in an acid resin deposit: biodegradation potential and ecotoxicology in an extremely acidic hydrocarbon contamination.

PubMed

Kloos, Karin; Schloter, Michael; Meyer, Ortwin

2006-11-01

Acid resins are residues produced in a recycling process for used oils that was in use in the forties and fifties of the last century. The resin-like material is highly contaminated with mineral oil hydrocarbons, extremely acidic and co-contaminated with substituted and aromatic hydrocarbons, and heavy metals. To determine the potential for microbial biodegradation the acid resin deposit and its surroundings were screened for microbial activity by soil respiration measurements. No microbial activity was found in the core deposit. However, biodegradation of hydrocarbons was possible in zones with a lower degree of contamination surrounding the deposit. An extreme acidophilic microbial community was detected close to the core deposit. With a simple ecotoxicological approach it could be shown that the pure acid resin that formed the major part of the core deposit, was toxic to the indigenous microflora due to its extremely low pH of 0-1.

Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

PubMed Central

de Vries, Ronald P.; Gruppen, Harry; Kabel, Mirjam A.

2015-01-01

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. PMID:26237450

A fluorescent organic cage for picric acid detection.

PubMed

Acharyya, Koushik; Mukherjee, Partha Sarathi

2014-12-25

Dynamic covalent imine chemistry has been utilized to synthesize a fluorescent [3+2] self-assembled nanoscopic organic cage. The fluorescent nature of the reduced analogue of the cage was further exploited for the highly selective detection of the explosive picric acid (PA).

Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells.

PubMed

Mänz, Benjamin; de Graaf, Miranda; Mögling, Ramona; Richard, Mathilde; Bestebroer, Theo M; Rimmelzwaan, Guus F; Fouchier, Ron A M

2016-07-01

A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts. Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions-most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2-have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Rh(III)-Catalyzed Decarboxylative Coupling of Acrylic Acids with Unsaturated Oxime Esters: Carboxylic Acids Serve as Traceless Activators

PubMed Central

2015-01-01

α,β-Unsaturated carboxylic acids undergo Rh(III)-catalyzed decarboxylative coupling with α,β-unsaturated O-pivaloyl oximes to provide substituted pyridines in good yield. The carboxylic acid, which is removed by decarboxylation, serves as a traceless activating group, giving 5-substituted pyridines with very high levels of regioselectivity. Mechanistic studies rule out a picolinic acid intermediate, and an isolable rhodium complex sheds further light on the reaction mechanism. PMID:24512241

Lipozyme RM IM-catalyzed acidolysis of Cinnamomum camphora seed oil with oleic acid to produce human milk fat substitutes enriched in medium-chain fatty acids.

PubMed

Zou, Xian-Guo; Hu, Jiang-Ning; Zhao, Man-Li; Zhu, Xue-Mei; Li, Hong-Yan; Liu, Xiao-Ru; Liu, Rong; Deng, Ze-Yuan

2014-10-29

In the present study, a human milk fat substitute (HMFS) enriched in medium-chain fatty acids (MCFAs) was synthesized through acidolysis reaction from Cinnamomum camphora seed oil (CCSO) with oleic acid in a solvent-free system. A commercial immobilized lipase, Lipozyme RM IM, from Rhizomucor miehei, was facilitated as a biocatalyst. Effects of different reaction conditions, including substrate molar ratio, enzyme concentration, reaction temperature, and reaction time were investigated using response surface methodology (RSM) to obtain the optimal oleic acid incorporation. After optimization, results showed that the maximal incorporation of oleic acid into HMFS was 59.68%. Compared with CCSO, medium-chain fatty acids at the sn-2 position of HMFS accounted for >70%, whereas oleic acid was occupied predominantly at the sn-1,3 position (78.69%). Meanwhile, triacylglycerol (TAG) components of OCO (23.93%), CCO (14.94%), LaCO (13.58%), OLaO (12.66%), and OOO (11.13%) were determined as the major TAG species in HMFS. The final optimal reaction conditions were carried out as follows: substrate molar ratio (oleic acid/CCSO), 5:1; enzyme concentration, 12.5% (w/w total reactants); reaction temperature, 60 °C; and reaction time, 28 h. The reusability of Lipozyme RM IM in the acidolysis reaction was also evaluated, and it was found that it could be reused up to 9 times without significant loss of activities. Urea inclusion method was used to separate and purify the synthetic product. As the ratio of HMFS/urea increased to 1:2, the acid value lowered to the minimum. In a scale-up experiment, the contents of TAG and total tocopherols in HMFS (modified CCSO) were 77.28% and 12.27 mg/100 g, respectively. All of the physicochemical indices of purified product were within food standards. Therefore, such a MCFA-enriched HMFS produced by using the acidolysis method might have potential application in the infant formula industry.

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids.

PubMed

Ardhaoui, M; Falcimaigne, A; Ognier, S; Engasser, J M; Moussou, P; Pauly, G; Ghoul, M

2004-06-10

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and omega-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on omega-substituted fatty acid chain were unfavourable to these reactions. The 1H NMR and 13C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4"'-OH of the rhamnose residue of rutin. Copyright 2004 Elsevier B.V.

Label-free amino acid detection based on nanocomposites of graphene oxide hybridized with gold nanoparticles.

PubMed

Zhang, Qian; Zhang, Diming; Lu, Yanli; Xu, Gang; Yao, Yao; Li, Shuang; Liu, Qingjun

2016-03-15

Nanocomposites of graphene oxide and gold nanoparticles (GO/GNPs) were synthesized for label-free detections of amino acids. Interactions between the composites and amino acids were investigated by both naked-eye observation and optical absorption spectroscopy. The GO/GNPs composites displayed apparent color changes and absorption spectra changes in presences of amino acids including glutamate, aspartate, and cysteine. The interaction mechanisms of the composites and amino acids were discussed and explored with sulfhydryl groups and non-α-carboxylic groups on the amino acids. Sensing properties of the composites were tested, while pure gold particles were used as the control. The results suggested that the GO/GNPs composites had better linearity and stability in dose-dependent responses to the amino acids than those of the particles, especially in detections for acidic amino acids. Therefore, the nanocomposites platform can provide a convenient and efficient approach for label-free optical detections of important molecules such as amino acids. Copyright © 2015 Elsevier B.V. All rights reserved.

Electrochemical detection of uric acid via uricase-immobilized graphene oxide.

PubMed

Omar, Muhamad Nadzmi; Salleh, Abu Bakar; Lim, Hong Ngee; Ahmad Tajudin, Asilah

2016-09-15

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 μM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced osteoconductivity of sodium-substituted hydroxyapatite by system instability.

PubMed

Sang Cho, Jung; Um, Seung-Hoon; Su Yoo, Dong; Chung, Yong-Chae; Hye Chung, Shin; Lee, Jeong-Cheol; Rhee, Sang-Hoon

2014-07-01

The effect of substituting sodium for calcium on enhanced osteoconductivity of hydroxyapatite was newly investigated. Sodium-substituted hydroxyapatite was synthesized by reacting calcium hydroxide and phosphoric acid with sodium nitrate followed by sintering. As a control, pure hydroxyapatite was prepared under identical conditions, but without the addition of sodium nitrate. Substitution of calcium with sodium in hydroxyapatite produced the structural vacancies for carbonate ion from phosphate site and hydrogen ion from hydroxide site of hydroxyapatite after sintering. The total system energy of sodium-substituted hydroxyapatite with structural defects calculated by ab initio methods based on quantum mechanics was much higher than that of hydroxyapatite, suggesting that the sodium-substituted hydroxyapatite was energetically less stable compared with hydroxyapatite. Indeed, sodium-substituted hydroxyapatite exhibited higher dissolution behavior of constituent elements of hydroxyapatite in simulated body fluid (SBF) and Tris-buffered deionized water compared with hydroxyapatite, which directly affected low-crystalline hydroxyl-carbonate apatite forming capacity by increasing the degree of apatite supersaturation in SBF. Actually, sodium-substituted hydroxyapatite exhibited markedly improved low-crystalline hydroxyl-carbonate apatite forming capacity in SBF and noticeably higher osteoconductivity 4 weeks after implantation in calvarial defects of New Zealand white rabbits compared with hydroxyapatite. In addition, there were no statistically significant differences between hydroxyapatite and sodium-substituted hydroxyapatite on cytotoxicity as determined by BCA assay. Taken together, these results indicate that sodium-substituted hydroxyapatite with structural defects has promising potential for use as a bone grafting material due to its enhanced osteoconductivity compared with hydroxyapatite. © 2013 Wiley Periodicals, Inc.

Vibrational spectra for uric acid and its D- and 15N-substituted analogues. Assignments for its normal modes from ab initio 3-21G force field.

NASA Astrophysics Data System (ADS)

Majoube, M.; Vergoten, G.

1993-03-01

FTR, Raman, FTIR spectra are obtained for polycrystalline uric acid and seven of its D-and 15N-substituted analogues. Assignments are given from a normal coordinate analysis carried out using a 3-21G ab initio force field. These are discussed by considering observed and calculated frequencies and D- and 15N-isotopic shifts.

Plasmonics-Based Detection of Virus Using Sialic Acid Functionalized Gold Nanoparticles.

PubMed

Lee, Changwon; Wang, Peng; Gaston, Marsha A; Weiss, Alison A; Zhang, Peng

2017-01-01

Biosensor for the detection of virus was developed by utilizing plasmonic peak shift phenomenon of the gold nanoparticles and viral infection mechanism of hemagglutinin on virus and sialic acid on animal cells. The plasmonic peak of the colloidal gold nanoparticles changes with the aggregation of the particles due to the plasmonic interaction between nearby particles and the color of the colloidal nanoparticle solution changes from wine red to purple. Sialic acid reduced and stabilized colloidal gold nanoparticle aggregation is induced by the addition of viral particles in the solution due to the hemagglutinin-sialic acid interaction. In this work, sialic acid reduced and stabilized gold nanoparticles (d = 20.1 ± 1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B virus species. The detection limit of the virus dilution (hemagglutinination assay titer, 512) was shown to be 0.156 vol% and the upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.

Isothermal amplification detection of nucleic acids by a double-nicked beacon.

PubMed

Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

2016-03-01

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. Copyright © 2015 Elsevier Inc. All rights reserved.

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.

PubMed

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L; Levin, Henry L

2008-06-06

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

Odor detection of mixtures of homologous carboxylic acids and coffee aroma compounds by humans.

PubMed

Miyazawa, Toshio; Gallagher, Michele; Preti, George; Wise, Paul M

2009-11-11

Mixture summation among homologous carboxylic acids, that is, the relationship between detection probabilities for mixtures and detection probabilities for their unmixed components, varies with similarity in carbon-chain length. The current study examined detection of acetic, butyric, hexanoic, and octanoic acids mixed with three other model odorants that differ greatly from the acids in both structure and odor character, namely, 2-hydroxy-3-methylcyclopent-2-en-1-one, furan-2-ylmethanethiol, and (3-methyl-3-sulfanylbutyl) acetate. Psychometric functions were measured for both single compounds and binary mixtures (2 of 5, forced-choice method). An air dilution olfactometer delivered stimuli, with vapor-phase calibration using gas chromatography-mass spectrometry. Across the three odorants that differed from the acids, acetic and butyric acid showed approximately additive (or perhaps even supra-additive) summation at low perithreshold concentrations, but subadditive interactions at high perithreshold concentrations. In contrast, the medium-chain acids showed subadditive interactions across a wide range of concentrations. Thus, carbon-chain length appears to influence not only summation with other carboxylic acids but also summation with at least some unrelated compounds.

Aspartic acid substitutions in monoamine oxidase-A reveal both catalytic-dependent and -independent influences on cell viability and proliferation.

PubMed

Wei, Zelan; Satram-Maharaj, Tamara; Chaharyn, Bradley; Kuski, Kelly; Pennington, Paul R; Cao, Xia; Chlan, Jennifer; Mousseau, Darrell D

2012-11-01

Post-translational influences could underlie the ambiguous roles of monoamine oxidase-A (MAO-A) in pathologies such as depression, cancer and Alzheimer disease. In support of this, we recently demonstrated that the Ca²⁺-sensitive component of MAO-A catalytic activity is inhibited by a pro-survival p38 (MAPK)-dependent mechanism. We substituted three aspartic acid (D) residues in human MAO-A that reside in putative Ca²⁺-binding motifs and overexpressed the individual proteins in the human HEK293 cell line. We assayed the overexpressed proteins for catalytic activity and for their influence on cell viability (using MTT conversion and trypan blue exclusion) and proliferation/DNA synthesis [using bromodeoxyuridine (BrdU) incorporation]. Innate MAO-A catalytic activity (and the capacity for generating hydrogen peroxide) was unaffected by the D61A substitution, but inhibited moderately or completely by the D248A and D328G substitutions, respectively. The Ca²⁺-sensitive activities of wild-type and D248A MAO-A proteins were enhanced by treatment with the selective p38(MAPK) inhibitor, SB203580, but was completely abrogated by the D61A substitution. Monoamine oxidase-A(D61A) was toxic to cells and exerted no effect on cell proliferation, while MAO-A(D248A) was generally comparable to wild-type MAO-A. As expected, the catalytic-dead MAO-A(D328G) was not cytotoxic, but unexpectedly enhanced both MTT conversion and BrdU staining. Variant-dependent changes in Bax and Bcl-2/Bcl-XL protein expression were observed. A different pattern of effects in N2-a cells suggests cell line-dependent roles for MAO-A. A catalytic-dependent mechanism influences MAO-A-mediated cytotoxicity, whereas a catalytic-independent mechanism contributes to proliferation. Context-dependent inputs by either mechanism could underlie the ambiguous pathological contributions of MAO-A.

Development of a highly enantioselective capacitive immunosensor for the detection of alpha-amino acids.

PubMed

Zhang, Song; Ding, Jingjing; Liu, Ying; Kong, Jilie; Hofstetter, Oliver

2006-11-01

This work describes a highly enantioselective and sensitive immunosensor for the detection of chiral amino acids based on capacitive measurement. The sensor was prepared by first binding mercaptoacetic acid to the surface of a gold electrode, followed by modification with tyramine utilizing carbodiimide activation. The hapten 4-amino-D-phenylalanine was then covalently immobilized onto the electrode by diazotization. Stereoselective binding of an anti-D-amino acid antibody to the hapten-modified sensor surface resulted in capacitance changes that were detected with high sensitivity by a potentiostatic step method. Using capacitance measurement, detection limits of 5 pg of antibody/mL were attained. The exquisite stereoselectivity of the antibody was also utilized in a competitive setup to quantitatively determine the concentration of the analyte d-phenylalanine in nonracemic samples containing both enantiomers of this amino acid. Trace impurities of d-phenylalanine as low as 0.001% could be detected.

Rapid detection method for fusaric acid-producing species of Fusarium by PCR

USDA-ARS?s Scientific Manuscript database

Fusaric acid is a mycotoxin produced by species of the fungus Fusarium and can act synergistically with other Fusarium toxins. In order to develop a specific detection method for fusaric acid-producing fungus, PCR prim¬ers were designed to amplify FUB10, a transcription factor gene in fusaric acid ...

Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein.

PubMed

Tsai, Ting-Yueh; Yeh, Teng-Kuang; Chen, Xin; Hsu, Tsu; Jao, Yu-Chen; Huang, Chih-Hsiang; Song, Jen-Shin; Huang, Yu-Chen; Chien, Chia-Hui; Chiu, Jing-Huai; Yen, Shih-Chieh; Tang, Hung-Kuan; Chao, Yu-Sheng; Jiaang, Weir-Torn

2010-09-23

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

PubMed Central

Zanoli, Laura Maria; Spoto, Giuseppe

2012-01-01

Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

pKa Modulation in rhodamine based probes for colorimetric detection of picric acid.

PubMed

Nagarajan, V; Bag, Bamaprasad

2014-12-21

Tuning the pKa in acid sensitive rhodamine spirolactam derivatives as a function of the solvent medium resulted in the selective detection of picric acid from its lower nitro phenolic analogues and a few other carboxylic acids.

Comparative studies on the interaction of caffeic acid, chlorogenic acid and ferulic acid with bovine serum albumin

NASA Astrophysics Data System (ADS)

Li, Shuang; Huang, Kelong; Zhong, Ming; Guo, Jun; Wang, Wei-zheng; Zhu, Ronghua

2010-10-01

The substitution of the hydrogen on aromatic and esterification of carboxyl group of the phenol compounds plays an important role in their bio-activities. In this paper, caffeic acid (CaA), chlorogenic acid (ChA) and ferulic acid (FA) were selected to investigate the binding to bovine serum albumin (BSA) using UV absorption spectroscopy, fluorescence spectroscopy and synchronous fluorescence spectroscopy. It was found that the methoxyl group substituting for the 3-hydroxyl group of CaA decreased the affinity for BSA and the esterification of carboxyl group of CaA with quinic acid increased the affinities. The affinities of ChA and FA with BSA were more sensitive to the temperature than that of CaA with BSA. Synchronous fluorescence spectroscopy and time-resolved fluorescence indicated that the Stern-Volmer plots largely deviated from linearity at high concentrations and were caused by complete quenching of the tyrosine fluorescence of BSA.

Indirect electrochemical detection for total bile acids in human serum.

PubMed

Zhang, Xiaoqing; Zhu, Mingsong; Xu, Biao; Cui, Yue; Tian, Gang; Shi, Zhenghu; Ding, Min

2016-11-15

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00μM to 400μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective. Copyright © 2016. Published by Elsevier B.V.

Hydroxyl radical mediated degradation of phenylarsonic acid.

PubMed

Xu, Tielian; Kamat, Prashant V; Joshi, Sachin; Mebel, Alexander M; Cai, Yong; O'Shea, Kevin E

2007-08-16

Phenyl-substituted arsonic acids have been widely used as feed additives in the poultry industry. While very few studies have been reported on the environmental impact of these compounds, they have been introduced into the environment through land application of poultry litter in large quantities (about 10(6) kg/year). Phenylarsonic acid (PA) was used as a model for problematic arsonic acids. Dilute aqueous solutions of PA were subjected to gamma radiolysis under hydroxyl radical generating conditions, which showed rapid degradation of PA. Product studies indicate addition of (.)OH to the phenyl ring forms the corresponding phenols as the primary products. Arsenite, H3As(III)O3, and arsenate, H3As(V)O4, were also identified as products. The optimized structures and relative calculated energies (using GAUSSIAN 98, the B3LYP/6-31G(d) method) of the various transient intermediates are consistent with the product studies. Pulse radiolysis was used to determine the rate constants of PA with (.)OH (k = 3.2 x 10(9) M(-1) s(-1)) and SO4(.-) (k = 1.0 x 10(9) M(-1) s(-1)). PA reacts slower toward O(.-) (k = 1.9 x 10(7) M(-1) s(-1)) and N3(.) (no detectable transient), due to the lower oxidation potential of these two radicals. Our results indicate advanced oxidative processes employing (.)OH and SO4(.-) can be effective for the remediation of phenyl-substituted arsonic acids.

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein*S⃞

PubMed Central

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L.; Levin, Henry L.

2008-01-01

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the γ-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. PMID:18397885

Size-Selective Detection of Picric Acid by Fluorescent Palladium Macrocycles.

PubMed

Kumar, Sushil; Kishan, Ram; Kumar, Pramod; Pachisia, Sanya; Gupta, Rajeev

2018-02-19

This work presents the synthesis and characterization of two palladium-based fluorescent macrocycles offering hydrogen-bonding cavities of contrasting dimensions. Both palladium macrocycles function as chemosensors for the detection of nitroaromatics, whereas the larger macrocycle not only illustrates nanomolar detection of picric acid but also transports its significant amount from an aqueous to an organic phase.

Effect of leucine-to-methionine substitutions on the diffraction quality of histone chaperone SET/TAF-Ibeta/INHAT crystals.

PubMed

Senda, Miki; Muto, Shinsuke; Horikoshi, Masami; Senda, Toshiya

2008-10-01

One of the most frequent problems in crystallization is poor quality of the crystals. In order to overcome this obstacle several methods have been utilized, including amino-acid substitutions of the target protein. Here, an example is presented of crystal-quality improvement by leucine-to-methionine substitutions. A variant protein with three amino-acid substitutions enabled improvement of the crystal quality of the histone chaperone SET/TAF-Ibeta/INHAT when combined with optimization of the cryoconditions. This procedure improved the resolution of the SET/TAF-Ibeta/INHAT crystals from around 5.5 to 2.3 A without changing the crystallization conditions.

Total Acid Value Titration of Hydrotreated Biomass Fast Pyrolysis Oil: Determination of Carboxylic Acids and Phenolics with Multiple End-Point Detection

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

Christensen, E.; Alleman, T. L.; McCormick, R. L.

Total acid value titration has long been used to estimate corrosive potential of petroleum crude oil and fuel oil products. The method commonly used for this measurement, ASTM D664, utilizes KOH in isopropanol as the titrant with potentiometric end point determination by pH sensing electrode and Ag/AgCl reference electrode with LiCl electrolyte. A natural application of the D664 method is titration of pyrolysis-derived bio-oil, which is a candidate for refinery upgrading to produce drop in fuels. Determining the total acid value of pyrolysis derived bio-oil has proven challenging and not necessarily amenable to the methodology employed for petroleum products duemore » to the different nature of acids present. We presented an acid value titration for bio-oil products in our previous publication which also utilizes potentiometry using tetrabutylammonium hydroxide in place of KOH as the titrant and tetraethylammonium bromide in place of LiCl as the reference electrolyte to improve the detection of these types of acids. This method was shown to detect numerous end points in samples of bio-oil that were not detected by D664. These end points were attributed to carboxylic acids and phenolics based on the results of HPLC and GC-MS studies. Additional work has led to refinement of the method and it has been established that both carboxylic acids and phenolics can be determined accurately. Use of pH buffer calibration to determine half-neutralization potentials of acids in conjunction with the analysis of model compounds has allowed us to conclude that this titration method is suitable for the determination of total acid value of pyrolysis oil and can be used to differentiate and quantify weak acid species. The measurement of phenolics in bio-oil is subject to a relatively high limit of detection, which may limit the utility of titrimetric methodology for characterizing the acidic potential of pyrolysis oil and products.« less

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

PubMed

Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

2014-05-20

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity and In Vitro and In Vivo Antiendotoxin Activity

PubMed Central

Kumar, Amit; Tripathi, Amit Kumar; Kathuria, Manoj; Shree, Sonal; Tripathi, Jitendra Kumar; Purshottam, R. K.; Ramachandran, Ravishankar; Mitra, Kalyan

2016-01-01

Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with

Nucleic Acids for Ultra-Sensitive Protein Detection

PubMed Central

Janssen, Kris P. F.; Knez, Karel; Spasic, Dragana; Lammertyn, Jeroen

2013-01-01

Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of “personalized medicine”. Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart, mainly because traditional antibody-based technologies struggle to meet the stringent sensitivity and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given. PMID:23337338

Sugar Substitutes

MedlinePlus

... Substitutes Share Print Sugar substitutes are chemical or plant-based substances used to sweeten or enhance the ... made with saccharin. Stevia sweeteners Stevia is a plant-based sugar substitute that has no calories. The ...

Fast analysis of domoic acid using microchip electrophoresis with laser-induced fluorescence detection.

PubMed

Cheng, Yongqiang; Guo, Cuilian; Zhao, Bin; Yang, Li

2017-04-01

A fast and effective method was developed to detect domoic acid based upon microchip electrophoresis combined with laser-induced fluorescence detection. Through study of the gated injection process on the cross channel of the microchip, the low-voltage mode with relatively longer sample loading time was adopted to reduce the sample discrimination and improve the signal sensitivity. Fluorescein isothiocyanate was used as the derivatizing reagent for domoic acid. Under the optimized conditions, domoic acid was completely separated in 60 s with separation efficiency of 1.35 × 10 5  m -1 . The calibration curve was obtained in the range of 1.0 × 10 -9 to 1.0 × 10 -7  mol/L, and the detection limit reached 2.8 × 10 -10  mol/L. This developed method was successfully applied to analyze domoic acid in real samples. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

PubMed

Markert, M L; Norby-Slycord, C; Ward, F E

1989-09-01

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.

X-ray photoelectron spectroscopy study of para-substituted benzoic acids chemisorbed to aluminum oxide thin films

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

Kreil, Justin; Ellingsworth, Edward; Szulczewski, Greg

A series of para-substituted, halogenated (F, Cl, Br, and I) benzoic acid monolayers were prepared on the native oxide of aluminum surfaces by solution self-assembly and spin-coating techniques. The monolayers were characterized by x-ray photoelectron spectroscopy (XPS) and water contact angles. Several general trends are apparent. First, the polarity of the solvent is critical to monolayer formation. Protic polar solvents produced low coverage monolayers; in contrast, nonpolar solvents produced higher coverage monolayers. Second, solution deposition yields a higher surface coverage than spin coating. Third, the thickness of the monolayers determined from XPS suggests the plane of the aromatic ring ismore » perpendicular to the surface with the carboxylate functional group most likely binding in a bidentate chelating geometry. Fourth, the saturation coverage (∼2.7 × 10{sup 14} molecules cm{sup −2}) is independent of the para-substituent.« less

Amino acid substitutions and intron polymorphism of acetylcholinesterase1 associated with mevinphos resistance in diamondback moth, Plutella xylostella (L.).

PubMed

Yeh, Shih-Chia; Lin, Chia-Li; Chang, Cheng; Feng, Hai-Tung; Dai, Shu-Mei

2014-06-01

The diamondback moth, Plutella xylostella L., is the most destructive insect pest of Brassica crops in the world. It has developed resistance rapidly to almost every insecticide used for its control. Mevinphos, a fast degrading and slow resistance evocating organophosphorus insecticide, has been recommended for controlling P. xylostella in Taiwan for more than 40years. SHM strain of P. xylostella, with ca. 22-fold resistance to this chemical, has been established from a field SH strain by selecting with mevinphos since 1997. Three mutations, i.e., G892T, G971C, and T1156T/G leading to A298S, G324A, and F386F/V amino acid substitutions in acetylcholinesterase1 (AChE1), were identified in these two strains; along with three haplotype pairs and a polymorphic intron in AChE1 gene (ace1). Two genetically pure lines, i.e., an SHggt wild type with intron AS and an SHMTCN mutant carrying G892T, G971C, T1156T/G mutations and intron AR in ace1, were established by single pair mating and haplotype determination. The F1 of SHMTCN strain had 52-fold resistance to mevinphos in comparison with the F1 of SHggt strain. In addition, AChE1 of this SHMTCN population, which exhibited lower maximum velocity (Vmax) and affinity (Km), was less susceptible to the inhibition of mevinphos, with an I50 32-fold higher than that of the SHggt F1 population. These results imply that amino acid substitutions in AChE1 of SHMTCN strain are associated with mevinphos resistance in this insect pest, and this finding is important for insecticide resistance management of P. xylostella in the field. Copyright © 2014 Elsevier Inc. All rights reserved.

Real-time detection of deoxyribonucleic acid bases via their negative differential conductance signature.

PubMed

Dragoman, D; Dragoman, M

2009-08-01

In this Brief Report, we present a method for the real-time detection of the bases of the deoxyribonucleic acid using their signatures in negative differential conductance measurements. The present methods of electronic detection of deoxyribonucleic acid bases are based on a statistical analysis because the electrical currents of the four bases are weak and do not differ significantly from one base to another. In contrast, we analyze a device that combines the accumulated knowledge in nanopore and scanning tunneling detection and which is able to provide very distinctive electronic signatures for the four bases.

A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides.

PubMed

Lavrov, K V; Zalunin, I A; Kotlova, E K; Yanenko, A S

2010-08-01

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

Detection of muramic acid in a carbohydrate fraction of human spleen.

PubMed Central

Hoijer, M A; Melief, M J; van Helden-Meeuwsen, C G; Eulderink, F; Hazenberg, M P

1995-01-01

In previous studies, we showed that peptidoglycan polysaccharides from anaerobic bacteria normally present in the human gut induced severe chronic joint inflammation in rats. Our hypothesis is that peptidoglycan from the gut flora is involved in perpetuation of idiopathic inflammation. However, in the literature, the presence of peptidoglycan or subunits like muramyl peptides in blood or tissues is still a matter of debate. We were able to stain red pulp macrophages in all six available human spleens by immunohistochemical techniques using a monoclonal antibody against gut flora-derived antigens. Therefore, these human spleens were extracted, and after removal of most of the protein, the carbohydrate fraction was investigated for the presence of muramic acid, an amino sugar characteristic for peptidoglycan. Using three different methods for detection of muramic acid, we found a mean of 3.3 mumol of muramic acid with high-pressure liquid chromatography, 1.9 mumol with a colorimetric method for detection of lactate, and 0.8 mumol with an enzymatic method for detection of D-lactate per spleen (D-lactate is a specific group of the muramic acid molecule). It is concluded that peptidoglycan is present in human spleen not as small muramyl peptides as were previously searched for by other investigators but as larger macromolecules probably stored in spleen macrophages. PMID:7729869

Visual detection of nucleic acids based on Mie scattering and the magnetophoretic effect.

PubMed

Zhao, Zichen; Chen, Shan; Ho, John Kin Lim; Chieng, Ching-Chang; Chen, Ting-Hsuan

2015-12-07

Visual detection of nucleic acid biomarkers is a simple and convenient approach to point-of-care applications. However, issues of sensitivity and the handling of complex bio-fluids have posed challenges. Here we report on a visual method detecting nucleic acids using Mie scattering of polystyrene microparticles and the magnetophoretic effect. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) were surface-functionalised with oligonucleotide probes, which can hybridise with target oligonucleotides in juxtaposition and lead to the formation of MMPs-targets-PMPs sandwich structures. Using an externally applied magnetic field, the magnetophoretic effect attracts the sandwich structure to the sidewall, which reduces the suspended PMPs and leads to a change in the light transmission via the Mie scattering. Based on the high extinction coefficient of the Mie scattering (∼3 orders of magnitude greater than that of the commonly used gold nanoparticles), our results showed the limit of detection to be 4 pM using a UV-Vis spectrometer or 10 pM by direct visual inspection. Meanwhile, we also demonstrated that this method is compatible with multiplex assays and detection in complex bio-fluids, such as whole blood or a pool of nucleic acids, without purification in advance. With a simplified operation procedure, low instrumentation requirement, high sensitivity and compatibility with complex bio-fluids, this method provides an ideal solution for visual detection of nucleic acids in resource-limited settings.

Transition metal-substituted cobalt ferrite nanoparticles for biomedical applications.

PubMed

Sanpo, Noppakun; Berndt, Christopher C; Wen, Cuie; Wang, James

2013-03-01

Transition metals of copper, zinc, chromium and nickel were substituted into cobalt ferrite nanoparticles via a sol-gel route using citric acid as a chelating agent. The microstructure and elemental composition were characterized using scanning electron microscopy combined with energy-dispersive X-ray spectroscopy. Phase analysis of transition metal-substituted cobalt ferrite nanoparticles was performed via X-ray diffraction. Surface wettability was measured using the water contact angle technique. The surface roughness of all nanoparticles was measured using profilometry. Moreover, thermogravimetric analysis and differential scanning calorimetry were performed to determine the temperature at which the decomposition and oxidation of the chelating agents took place. Results indicated that the substitution of transition metals influences strongly the microstructure, crystal structure and antibacterial property of the cobalt ferrite nanoparticles. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Carbocation Rearrangement in An Electrophilic Aromatic Substitution Discovery Laboratory

ERIC Educational Resources Information Center

Polito, Victoria; Hamann, Christian S.; Rhile, Ian J.

2010-01-01

In this discovery laboratory, students performed electrophilic aromatic substitution reactions between 1,4-dimethoxybenzene and either 2-methyl-2-butanol or 3-methyl-2-butanol with sulfuric acid as a catalyst. The carbocation from 3-methyl-2-butanol undergoes a hydride shift, and hence, both reactions afford…

Frequency of Interferon-Resistance Conferring Substitutions in Amino Acid Positions 70 and 91 of Core Protein of the Russian HCV 1b Isolates Analyzed in the T-Cell Epitopic Context

PubMed Central

Soboleva, N. V.; Karlsen, A. A.; Isaeva, O. V.; Isaguliants, M. G.; Mikhailov, M. I.

2018-01-01

Amino acid substitutions R70Q/H and L91M in HCV subtype 1b core protein can affect the response to interferon and are associated with the development of hepatocellular carcinoma. We found that the rate of R70Q/H in HCV 1b from Russia was 31.2%, similar to that in HCV strains from Asia (34.0%), higher than that in the European (18.0%, p = 0.0010), but lower than that in the US HCV 1b strains (62.8%, p < 0.0001). Substitution L91M was found in 80.4% of the Russian HCV 1b isolates, higher than in Asian isolates (43.8%, p < 0.0001). Thus, a significant proportion of Russian HCV 1b isolates carry the unfavorable R70Q/H and/or L91M substitution. In silico analysis of the epitopic structure of the regions of substitutions revealed that both harbor clusters of T-cell epitopes. Peptides encompassing these regions were predicted to bind to a panel of HLA class I molecules, with substitutions impairing peptide recognition by HLA I molecules of the alleles prevalent in Russia. This indicates that HCV 1b with R70Q/H and L91M substitutions may have evolved as the immune escape variants. Impairment of T-cell recognition may play a part in the negative effect of these substitutions on the response to IFN treatment. PMID:29577052

[Eucaloric substitution of medium chain triglycerides for dietary long chain fatty acids improves body composition and lipid profile in a patient with human immunodeficiency virus lipodystrophy].

PubMed

Vázquez, C; Reyes, R; Alcaraz, F; Balsa, J A; Botella-Carretero, J I

2006-01-01

Lipodystrophy is a frequent disorder among patients with human immunodeficiency virus (HIV) infection, characterized by a loss of adipose tissue from the extremities, gluteal region and face, with excess fat in the neck and abdominal region. Metabolic abnormalities such as hyperlipidaemia and diabetes mellitus frequently coexist, posing these patients to an increased cardiovascular risk. Drug therapy may improve some of these metabolic disturbances, but to date there are no treatments for lipodystrophy with proven benefit. A 42-year-old man with HIV lipodystrophy was started on a standard low caloric diet with <30% of total fat and <10% of saturated fat, together with rosiglitazone 8 mg daily. After five months of treatment, given that lipodystrophic features and dyslipidaemia were still present in our patient, we tried to further improve therapeutic results by eucaloric substitution of medium chain triglycerides for dietary long chain fatty acids. Three months later, a dramatic change in body composition was shown with an increase in lean mass and a decrease in fat mass, together with an improvement in lipid profile. Eucaloric substitution of medium chain triglycerides for dietary long chain fatty acids may produce therapeutic benefits in HIV lipodystrophy.

Affinity sensor using 3-aminophenylboronic acid for bacteria detection.

PubMed

Wannapob, Rodtichoti; Kanatharana, Proespichaya; Limbut, Warakorn; Numnuam, Apon; Asawatreratanakul, Punnee; Thammakhet, Chongdee; Thavarungkul, Panote

2010-10-15

Boronic acid that can reversibly bind to diols was used to detect bacteria through its affinity binding reaction with diol-groups on bacterial cell walls. 3-aminophenylboronic acid (3-APBA) was immobilized on a gold electrode via a self-assembled monolayer. The change in capacitance of the sensing surface caused by the binding between 3-APBA and bacteria in a flow system was detected by a potentiostatic step method. Under optimal conditions the linear range of 1.5×10(2)-1.5×10(6) CFU ml(-1) and the detection limit of 1.0×10(2) CFU ml(-1) was obtained. The sensing surface can be regenerated and reused up to 58 times. The method was used for the analysis of bacteria in several types of water, i.e., bottled, well, tap, reservoir and wastewater. Compared with the standard plate count method, the results were within one standard deviation of each other. The proposed method can save both time and cost of analysis. The electrode modified with 3-APBA would also be applicable to the detection of other cis-diol-containing analytes. The concept could be extended to other chemoselective ligands, offering less expensive and more robust affinity sensors for a wide range of compounds. Copyright © 2010 Elsevier B.V. All rights reserved.

Design, synthesis and biological evaluation of di-substituted cinnamic hydroxamic acids bearing urea/thiourea unit as potent histone deacetylase inhibitors.

PubMed

Ning, Chengqing; Bi, Yanjing; He, Yujun; Huang, WenYuan; Liu, Lifei; Li, Yi; Zhang, Sihan; Liu, Xiaoyu; Yu, Niefang

2013-12-01

A novel class of di-substituted cinnamic hydroxamic acid derivatives containing urea or thiourea unit was designed, synthesized and evaluated as HDAC inhibitors. All tested compounds demonstrated significant HDAC inhibitory activities and anti-proliferative effects against diverse human tumor cell lines. Among them, 7l exhibited most potent pan-HDAC inhibitory activity, with an IC50 value of 130 nM. It also showed strong cellular inhibition against diverse cell lines including HCT-116, MCF-7, MDB-MB-435 and NCI-460, with GI50 values of 0.35, 0.22, 0.51 and 0.48 μM, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

Influence of Avocado Purée as a Fat Replacer on Nutritional, Fatty Acid, and Organoleptic Properties of Low-Fat Muffins.

PubMed

Othman, Nurul Ain; Abdul Manaf, Marina; Harith, Sakinah; Wan Ishak, Wan Rosli

2018-04-13

The feasibility of developing reduced-fat muffins with avocado is investigated by preparing muffins with 25%, 50%, 75%, and 100% avocado purée as a fat (butter) replacer. The resulting products were compared to the control muffin, which was made with 100% butter. Muffins were analyzed for nutritional content, fatty acid profiles, and sensory acceptability. Muffins incorporated with avocado purée revealed a significant increase (p < 0.05) with respect to moisture, ash, and carbohydrate in comparison with the control sample. However, no significant changes (p > 0.05) were detected in all muffin formulations for protein and dietary fiber content. Both fat content and caloric value of muffins incorporated with avocado purée were significantly decreased (p < 0.05). The fatty acid profile showed that there was an increment in the monounsaturated fatty acids (MUFA) content by 16.51% at full-fat substitution. The sensory evaluation test demonstrated that muffins had acceptability at up to 50% substitution. Fat substitution at higher than 50% lead to undesirable flavor and aftertaste, which was significant (p < 0.05) to the panelists. The findings indicated the feasibility of avocado purée in fat-reduced muffin preparation with an optimal level of 50% avocado purée substitution.

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods.

PubMed

Westergaard Mulberg, Mads; Taskova, Maria; Thomsen, Rasmus P; Okholm, Anders H; Kjems, Jørgen; Astakhova, Kira

2017-08-17

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification-free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra-bright macrofluorophores of 9-84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target-specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kinetic and thermodynamic acidities of substituted 1-benzyl-1-methoxy-2-nitroethylenes. Strong reduction of the transition state imbalance compared to other nitroalkanes.

PubMed

Bernasconi, Claude F; Ali, Mahammad; Gunter, Janette C

2003-01-08

Acidity constants of six substituted 1-benzyl-1-methoxy-2-nitroethylenes (2-Z with Z = m-NO(2), m-CF(3), m-Cl, H, p-Me, p-MeO) and their respective nitronic acids were determined in 50% DMSO-50% water (v/v) at 20 degrees C. Kinetic data were obtained on the reversible deprotonation of all six 2-Z by OH(-) and piperidine and on the reversible deprotonation of 2-NO(2)() by piperazine, 1-(2-hydroxyethyl)piperazine, and morpholine in the same solvent. These data allowed a determination of the Brønsted coefficients alpha (dependence on acidity of 2-Z) and beta (dependence on amine basicity). The fact that alpha > beta indicates the presence of a transition state imbalance which, however, is much smaller than that for the deprotonation of arylnitromethanes. The reasons for this reduction in the imbalance and their relevance to a recent study of the deprotonation of Fischer carbene complexes are discussed.

Apparatus for point-of-care detection of nucleic acid in a sample

DOEpatents

Bearinger, Jane P.; Dugan, Lawrence C.

2016-04-19

Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

Methods for point-of-care detection of nucleic acid in a sample

DOEpatents

Bearinger, Jane P.; Dugan, Lawrence C.

2015-12-29

Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles.

PubMed

Li, Yuan; Tian, Rui; Zheng, Xingwang; Huang, Rongfu

2016-08-31

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and evaluation of a microdevice for amino acid biomarker detection and analysis on Mars

PubMed Central

Skelley, Alison M.; Scherer, James R.; Aubrey, Andrew D.; Grover, William H.; Ivester, Robin H. C.; Ehrenfreund, Pascale; Grunthaner, Frank J.; Bada, Jeffrey L.; Mathies, Richard A.

2005-01-01

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10–600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment. PMID:15657130

Development and evaluation of a microdevice for amino acid biomarker detection and analysis on Mars.

PubMed

Skelley, Alison M; Scherer, James R; Aubrey, Andrew D; Grover, William H; Ivester, Robin H C; Ehrenfreund, Pascale; Grunthaner, Frank J; Bada, Jeffrey L; Mathies, Richard A

2005-01-25

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10-600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment.

Neuronal detection thresholds during vestibular compensation: contributions of response variability and sensory substitution.

PubMed

Jamali, Mohsen; Mitchell, Diana E; Dale, Alexis; Carriot, Jerome; Sadeghi, Soroush G; Cullen, Kathleen E

2014-04-01

The vestibular system is responsible for processing self-motion, allowing normal subjects to discriminate the direction of rotational movements as slow as 1-2 deg s(-1). After unilateral vestibular injury patients' direction-discrimination thresholds worsen to ∼20 deg s(-1), and despite some improvement thresholds remain substantially elevated following compensation. To date, however, the underlying neural mechanisms of this recovery have not been addressed. Here, we recorded from first-order central neurons in the macaque monkey that provide vestibular information to higher brain areas for self-motion perception. Immediately following unilateral labyrinthectomy, neuronal detection thresholds increased by more than two-fold (from 14 to 30 deg s(-1)). While thresholds showed slight improvement by week 3 (25 deg s(-1)), they never recovered to control values - a trend mirroring the time course of perceptual thresholds in patients. We further discovered that changes in neuronal response variability paralleled changes in sensitivity for vestibular stimulation during compensation, thereby causing detection thresholds to remain elevated over time. However, we found that in a subset of neurons, the emergence of neck proprioceptive responses combined with residual vestibular modulation during head-on-body motion led to better neuronal detection thresholds. Taken together, our results emphasize that increases in response variability to vestibular inputs ultimately constrain neural thresholds and provide evidence that sensory substitution with extravestibular (i.e. proprioceptive) inputs at the first central stage of vestibular processing is a neural substrate for improvements in self-motion perception following vestibular loss. Thus, our results provide a neural correlate for the patient benefits provided by rehabilitative strategies that take advantage of the convergence of these multisensory cues.

Neuronal detection thresholds during vestibular compensation: contributions of response variability and sensory substitution

PubMed Central

Jamali, Mohsen; Mitchell, Diana E; Dale, Alexis; Carriot, Jerome; Sadeghi, Soroush G; Cullen, Kathleen E

2014-01-01

The vestibular system is responsible for processing self-motion, allowing normal subjects to discriminate the direction of rotational movements as slow as 1–2 deg s−1. After unilateral vestibular injury patients’ direction–discrimination thresholds worsen to ∼20 deg s−1, and despite some improvement thresholds remain substantially elevated following compensation. To date, however, the underlying neural mechanisms of this recovery have not been addressed. Here, we recorded from first-order central neurons in the macaque monkey that provide vestibular information to higher brain areas for self-motion perception. Immediately following unilateral labyrinthectomy, neuronal detection thresholds increased by more than two-fold (from 14 to 30 deg s−1). While thresholds showed slight improvement by week 3 (25 deg s−1), they never recovered to control values – a trend mirroring the time course of perceptual thresholds in patients. We further discovered that changes in neuronal response variability paralleled changes in sensitivity for vestibular stimulation during compensation, thereby causing detection thresholds to remain elevated over time. However, we found that in a subset of neurons, the emergence of neck proprioceptive responses combined with residual vestibular modulation during head-on-body motion led to better neuronal detection thresholds. Taken together, our results emphasize that increases in response variability to vestibular inputs ultimately constrain neural thresholds and provide evidence that sensory substitution with extravestibular (i.e. proprioceptive) inputs at the first central stage of vestibular processing is a neural substrate for improvements in self-motion perception following vestibular loss. Thus, our results provide a neural correlate for the patient benefits provided by rehabilitative strategies that take advantage of the convergence of these multisensory cues. PMID:24366259

ANAEROBIC BIODEGRADATION OF NITROGEN-SUBSTITUTED AND SULFONATED BENZENE AQUIFER CONTAMINANTS (JOURNAL)

EPA Science Inventory

A literature survey of ground water contaminants indicated that aquifers are repositories for hazardous wastes, including N- and 5-substituted benzene derivatives. We therefore examined the susceptibility of several anilines, benzamides, benenesulfonic acids and benenesulfonamide...

WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

PubMed Central

Liu, Qian; Chen, Haoqian; Ojode, Teresa; Gao, Xiangxi; Anaya-O'Brien, Sandra; Turner, Nicholas A.; Ulrick, Jean; DeCastro, Rosamma; Kelly, Corin; Cardones, Adela R.; Gold, Stuart H.; Hwang, Eugene I.; Wechsler, Daniel S.; Malech, Harry L.; Murphy, Philip M.

2012-01-01

WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4R334X, the most common truncation mutation in WHIM syndrome, CXCR4E343K mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4E343K had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling. PMID:22596258

Acetic Acid Detection Threshold in Synthetic Wine Samples of a Portable Electronic Nose

PubMed Central

Macías, Miguel Macías; Manso, Antonio García; Orellana, Carlos Javier García; Velasco, Horacio Manuel González; Caballero, Ramón Gallardo; Chamizo, Juan Carlos Peguero

2013-01-01

Wine quality is related to its intrinsic visual, taste, or aroma characteristics and is reflected in the price paid for that wine. One of the most important wine faults is the excessive concentration of acetic acid which can cause a wine to take on vinegar aromas and reduce its varietal character. Thereby it is very important for the wine industry to have methods, like electronic noses, for real-time monitoring the excessive concentration of acetic acid in wines. However, aroma characterization of alcoholic beverages with sensor array electronic noses is a difficult challenge due to the masking effect of ethanol. In this work, in order to detect the presence of acetic acid in synthetic wine samples (aqueous ethanol solution at 10% v/v) we use a detection unit which consists of a commercial electronic nose and a HSS32 auto sampler, in combination with a neural network classifier (MLP). To find the characteristic vector representative of the sample that we want to classify, first we select the sensors, and the section of the sensors response curves, where the probability of detecting the presence of acetic acid will be higher, and then we apply Principal Component Analysis (PCA) such that each sensor response curve is represented by the coefficients of its first principal components. Results show that the PEN3 electronic nose is able to detect and discriminate wine samples doped with acetic acid in concentrations equal or greater than 2 g/L. PMID:23262483

A cell-free stock of simian-human immunodeficiency virus that causes AIDS in pig-tailed macaques has a limited number of amino acid substitutions in both SIVmac and HIV-1 regions of the genome and has offered cytotropism.

PubMed

Stephens, E B; Mukherjee, S; Sahni, M; Zhuge, W; Raghavan, R; Singh, D K; Leung, K; Atkinson, B; Li, Z; Joag, S V; Liu, Z Q; Narayan, O

1997-05-12

We have examined both the sequence changes in the LTR, gag, vif, vpr, vpx, tat, rev, vpu, env, and nef genes and the cell tropism of a cell-free stock of chimeric simian-human immunodeficiency virus (SHIV) isolated from the cerebrospinal fluid of a pig-tailed macaque (PNb) that developed AIDS. This virus (SHIVKU-1) is highly pathogenic when inoculated into other macaques. DNA sequence analysis of PCR-amplified products revealed a total of 5 nucleotide changes in the LTR while vif had 2 consensus amino acid changes. The gag, vif, and vpx had no consensus amino acid substitutions, whereas vpr had 1 consensus substitution. The tat and rev genes of the HXB2 region of SHIVKU-1 had 2 and 1 consensus amino acid changes, respectively. The vpu gene of the HXB2 region of SHIV, which originally had an ACG at the beginning of the gene, reverted to an initiation ATG codon and in addition contained a consensus amino acid substitution at position 69 of this protein. As expected, the majority of the nucleotide substitutions were found in the env and nef genes. Thirteen and 5 amino acid changes were predicted for the corresponding Env and Nef proteins, respectively. In addition, one-third of the env gene clones isolated from the SHIVKU-1 stock had a 5-amino-acid deletion in the V4 region. Using three independent assays, we determined that the changes in the SHIVKU-1 were associated with an increase in the efficiency of replication in macrophages. The strikingly few consensus changes in the virus suggest that conversion of this virus to one capable of causing AIDS in pig-tailed macaques was associated with relatively few changes in the viral envelope and/or accessory genes. These results will provide the basis for the development of a pathogenic, molecular clone of SHIV capable of causing AIDS in pig-tailed macaques.

Isomers/enantiomers of perfluorocarboxylic acids: Method development and detection in environmental samples

EPA Science Inventory

Perfluoroalkyl substances are globally distributed in both urban and remote settings, and routinely are detected in wildlife, humans, and the environment. One of the most prominent and routinely detected perfluoroalkyl substances is perfluorooctanoic acid (PFOA), which has been s...

40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

Code of Federal Regulations, 2013 CFR

2013-07-01

... solely for research and development, have the same meaning as in § 720.3 of this chapter. (2... processor or distributor may not use the substance except in small quantities solely for research and... of a substance known to cause cancer in laboratory animals. The triethanolamine salt of a substituted...

40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

Code of Federal Regulations, 2014 CFR

2014-07-01

... solely for research and development, have the same meaning as in § 720.3 of this chapter. (2... processor or distributor may not use the substance except in small quantities solely for research and... of a substance known to cause cancer in laboratory animals. The triethanolamine salt of a substituted...

40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

Code of Federal Regulations, 2012 CFR

2012-07-01

... solely for research and development, have the same meaning as in § 720.3 of this chapter. (2... processor or distributor may not use the substance except in small quantities solely for research and... of a substance known to cause cancer in laboratory animals. The triethanolamine salt of a substituted...

Analysis of the breast milk of giant pandas (Ailuropoda melanoleuca) and the preparation of substitutes

PubMed Central

ZHANG, Zhihe; HOU, Rong; LAN, Jingchao; WANG, Hairui; KUROKAWA, Hiroyuki; TAKATSU, Zenta; KOBAYASHI, Toyokazu; KOIE, Hiroshi; KAMATA, Hiroshi; KANAYAMA, Kiichi; WATANABE, Toshi

2016-01-01

The first milk substitute for giant panda cubs was developed in 1988 based on limited data about giant panda breast milk and that of certain types of bear. Mixtures of other formulas have also been fed to cubs at some facilities. However, they are not of sufficient nutritional quality for promoting growth in panda cubs. Here, we report analysis of giant panda breast milk and propose new milk substitutes for cubs, which were developed based on the results of our analysis. The Chengdu Research Base of Giant Panda Breeding obtained breast milk samples from three giant pandas. Up to 30 ml of breast milk were collected from each mother by hand. Then, the milk samples were frozen and sent to Nihon University. The levels of protein, fat, carbohydrates, ash, moisture, vitamins, minerals, total amino acids, fatty acids, lactose and other carbohydrates in the milk were analyzed. The breast milk samples exhibited the following nutritional values: protein: 6.6–8.5%, fat: 6.9–16.4%, carbohydrates: 2.5–9.1%, ash: 0.9–1.0% and moisture: 67–83%. We designed two kinds of milk substitutes based on the data obtained and the nutritional requirements of dogs, cats and rodents. The nutritional composition of the milk substitutes for the first and second stages was as follows: protein: 38 and 26%, fat: 40 and 40%, carbohydrates: 13 and 25%, ash: 6 and 6% and moisture: 3 and 3%, respectively. In addition, the substitutes contained vitamins, minerals, taurine, docosahexaenoic acid, lactoferrin, nucleotides and other nutrients. PMID:26781707

Designing Inhibitors of Cytochrome c/Cardiolipin Peroxidase Complexes: Mitochondria-Targeted Imidazole-Substituted Fatty Acids

PubMed Central

Jiang, Jianfei; Bakan, Ahmet; Kapralov, Alexandr A.; Silva, K. Ishara; Huang, Zhentai; Amoscato, Andrew A.; Peterson, James; Garapati, Venkata Krishna; Saxena, Sunil; Bayir, Hülya; Atkinson, Jeffrey; Bahar, Ivet; Kagan, Valerian E.

2014-01-01

Mitochondria have emerged as the major regulatory platform responsible for coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (cyt) c. As this oxidation occurs within the peroxidase complex of cyt c with CL, the latter represents a promising target for the discovery and design of drugs with anti-apoptotic mechanism of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogues of stearic acid TPP-n-ISA with different positions of the attached imidazole group on the fatty acid (n=6, 8, 10, 13 and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance, and electron spin echo envelope modulation) we demonstrated that TPP-n-ISA indeed were able to potently suppress CL induced structural re-arrangements in cyt c paving the way to its peroxidase competence. TPP-n-ISA analogues preserved the low spin hexa-coordinated heme iron state in cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of cyt c/CL complexes with a significant anti-apoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all atom molecular dynamics simulations. Based on the experimental data and computations predictions, we identified TPP-6-ISA as a candidate drug with optimized anti-apoptotic potency. PMID:24631490

Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids.

PubMed

Jiang, Jianfei; Bakan, Ahmet; Kapralov, Alexandr A; Silva, K Ishara; Huang, Zhentai; Amoscato, Andrew A; Peterson, James; Garapati, Venkata Krishna; Saxena, Sunil; Bayir, Hülya; Atkinson, Jeffrey; Bahar, Ivet; Kagan, Valerian E

2014-06-01

Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency. Copyright © 2014 Elsevier Inc. All rights reserved.

Metal Binding by the D,DX35E Motif of Human Immunodeficiency Virus Type 1 Integrase: Selective Rescue of Cys Substitutions by Mn2+ In Vitro

PubMed Central

Gao, Kui; Wong, Steven; Bushman, Frederic

2004-01-01

The D,DX35E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg2+ or Mn2+), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg2+ and Mn2+, and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX35E motif. Mn2+ but not Mg2+ can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn2+ only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex. PMID:15194746

Probing structural features of Alzheimer's amyloid-β pores in bilayers using site-specific amino acid substitutions.

PubMed

Capone, Ricardo; Jang, Hyunbum; Kotler, Samuel A; Kagan, Bruce L; Nussinov, Ruth; Lal, Ratnesh

2012-01-24

A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes.

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: biochemical and spectroscopic studies.

PubMed Central

Yuan, T.; Vogel, H. J.

1999-01-01

Calmodulin (CaM) is a 148-residue regulatory calcium-binding protein that activates a wide range of target proteins and enzymes. Calcium-saturated CaM has a bilobal structure, and each domain has an exposed hydrophobic surface region where target proteins are bound. These two "active sites" of calmodulin are remarkably rich in Met residues. Here we have biosynthetically substituted (up to 90% incorporation) the unnatural amino acids ethionine (Eth) and norleucine (Nle) for the nine Met residues of CaM. The substituted proteins bind in a calcium-dependent manner to hydrophobic matrices and a synthetic peptide, encompassing the CaM-binding domain of myosin light-chain kinase (MLCK). Infrared and circular dichroism spectroscopy show that there are essentially no changes in the secondary structure of these proteins compared to wild-type CaM (WT-CaM). One- and two-dimensional NMR studies of the Eth-CaM and Nle-CaM proteins reveal that, while the core of the proteins is relatively unaffected by the substitutions, the two hydrophobic interaction surfaces adjust to accommodate the Eth and Nle residues. Enzyme activation studies with MLCK show that Eth-CaM and Nle-CaM activate the enzyme to 90% of its maximal activity, with little changes in dissociation constant. For calcineurin only 50% activation was obtained, and the K(D) for Nle-CaM also increased 3.5-fold compared with WT-CaM. These data show that the "active site" Met residues of CaM play a distinct role in the activation of different target enzymes, in agreement with site-directed mutagenesis studies of the Met residues of CaM. PMID:10210190

Mercury assisted fluorescent supramolecular assembly of hexaphenylbenzene derivative for femtogram detection of picric acid.

PubMed

Pramanik, Subhamay; Bhalla, Vandana; Kumar, Manoj

2013-09-02

Aggregates of hexaphenylbenzene derivatives 3, having pyrene groups form network of fluorescent nanofibres in presence of mercury ions. Further, fluorescent nanofibres of 3-Hg(2+) supramolecular ensemble exhibit sensitive and pronounced response towards the picric acid. In addition, the solution coated paper strips of 3-Hg(2+) supramolecular ensemble can detect picric acid in the range of 2.29 fg/cm(2), thus, providing a simple, portable and low cost method for detection of picric acid in solution and in contact mode. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of Xylan Side-Chain Substitutions on Xylan-Cellulose Interactions and Implications for Thermal Pretreatment of Cellulosic Biomass.

PubMed

Pereira, Caroline S; Silveira, Rodrigo L; Dupree, Paul; Skaf, Munir S

2017-04-10

Lignocellulosic biomass is mainly constituted by cellulose, hemicellulose, and lignin and represents an important resource for the sustainable production of biofuels and green chemistry materials. Xylans, a common hemicellulose, interact with cellulose and often exhibit various side chain substitutions including acetate, (4-O-methyl) glucuronic acid, and arabinose. Recent studies have shown that the distribution of xylan substitutions is not random, but follows patterns that are dependent on the plant taxonomic family and cell wall type. Here, we use molecular dynamics simulations to investigate the role of substitutions on xylan interactions with the hydrophilic cellulose face, using the recently discovered xylan decoration pattern of the conifer gymnosperms as a model. The results show that α-1,2-linked substitutions stabilize the binding of single xylan chains independently of the nature of the substitution and that Ca 2+ ions can mediate cross-links between glucuronic acid substitutions of two neighboring xylan chains, thus stabilizing binding. At high temperature, xylans move from the hydrophilic to the hydrophobic cellulose surface and are also stabilized by Ca 2+ cross-links. Our results help to explain the role of substitutions on xylan-cellulose interactions, and improve our understanding of the plant cell wall architecture and the fundamentals of biomass pretreatments.

Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids.

PubMed

Dutta, Subrata; Fülöp, Annabelle; Mokhir, Andriy

2013-09-18

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their

Convergent evolution of marine mammals is associated with distinct substitutions in common genes

PubMed Central

Zhou, Xuming; Seim, Inge; Gladyshev, Vadim N.

2015-01-01

Phenotypic convergence is thought to be driven by parallel substitutions coupled with natural selection at the sequence level. Multiple independent evolutionary transitions of mammals to an aquatic environment offer an opportunity to test this thesis. Here, whole genome alignment of coding sequences identified widespread parallel amino acid substitutions in marine mammals; however, the majority of these changes were not unique to these animals. Conversely, we report that candidate aquatic adaptation genes, identified by signatures of likelihood convergence and/or elevated ratio of nonsynonymous to synonymous nucleotide substitution rate, are characterized by very few parallel substitutions and exhibit distinct sequence changes in each group. Moreover, no significant positive correlation was found between likelihood convergence and positive selection in all three marine lineages. These results suggest that convergence in protein coding genes associated with aquatic lifestyle is mainly characterized by independent substitutions and relaxed negative selection. PMID:26549748

Atomic substitutions in synthetic apatite; Insights from solid-state NMR spectroscopy

NASA Astrophysics Data System (ADS)

Vaughn, John S.

Apatite, Ca5(PO4)3X (where X = F, Cl, or OH), is a unique mineral group capable of atomic substitutions for cations and anions of varied size and charge. Accommodation of differing substituents requires some kind of structural adaptation, e.g. new atomic positions, vacancies, or coupled substitutions. These structural adaptations often give rise to important physicochemical properties relevant to a range of scientific disciplines. Examples include volatile trapping during apatite crystallization, substitution for large radionuclides for long-term storage of nuclear fission waste, substitution for fluoride to improve acid resistivity in dental enamel composed dominantly of hydroxylapatite, and the development of novel biomaterials with enhanced biocompatibility. Despite the importance and ubiquity of atomic substitutions in apatite materials, many of the mechanisms by which these reactions occur are poorly understood. Presence of substituents at dilute concentration and occupancy of disordered atomic positions hinder detection by bulk characterization methods such as X-ray diffraction (XRD) and infrared (IR) spectroscopy. Solid-state nuclear magnetic resonance (NMR) spectroscopy is an isotope-specific structural characterization technique that does not require ordered atomic arrangements, and is therefore well suited to investigate atomic substitutions and structural adaptations in apatite. In the present work, solid-state NMR is utilized to investigate structural adaptations in three different types of apatite materials; a series of near-binary F, Cl apatite, carbonate-hydroxylapatite compositions prepared under various synthesis conditions, and a heat-treated hydroxylapatite enriched in 17O. The results indicate that hydroxyl groups in low-H, near binary F,Cl apatite facilitate solid-solution between F and Cl via column reversals, which result in average hexagonal symmetry despite very dilute OH concentration ( 2 mol percent). In addition, 19F NMR spectra indicate

A Single-Amino-Acid Substitution in a Polymerase Protein of an H5N1 Influenza Virus Is Associated with Systemic Infection and Impaired T-Cell Activation in Mice▿ †

PubMed Central

Fornek, Jamie L.; Gillim-Ross, Laura; Santos, Celia; Carter, Victoria; Ward, Jerrold M.; Cheng, Lily I.; Proll, Sean; Katze, Michael G.; Subbarao, Kanta

2009-01-01

The transmission of H5N1 influenza viruses from birds to humans poses a significant public health threat. A substitution of glutamic acid for lysine at position 627 of the PB2 protein of H5N1 viruses has been identified as a virulence determinant. We utilized the BALB/c mouse model of H5N1 infection to examine how this substitution affects virus-host interactions and leads to systemic infection. Mice infected with H5N1 viruses containing lysine at amino acid 627 in the PB2 protein exhibited an increased severity of lesions in the lung parenchyma and the spleen, increased apoptosis in the lungs, and a decrease in oxygen saturation. Gene expression profiling revealed that T-cell receptor activation was impaired at 2 days postinfection (dpi) in the lungs of mice infected with these viruses. The inflammatory response was highly activated in the lungs of mice infected with these viruses and was sustained at 4 dpi. In the spleen, immune-related processes including NK cell cytotoxicity and antigen presentation were highly activated by 2 dpi. These differences are not attributable solely to differences in viral replication in the lungs but to an inefficient immune response early in infection as well. The timing and magnitude of the immune response to highly pathogenic influenza viruses is critical in determining the outcome of infection. The disruption of these factors by a single-amino-acid substitution in a polymerase protein of an influenza virus is associated with severe disease and correlates with the spread of the virus to extrapulmonary sites. PMID:19692471

Determination of acrylamide and acrylic acid by isocratic liquid chromatography with pulsed electrochemical detection.

PubMed

Casella, Innocenzo G; Pierri, Marianna; Contursi, Michela

2006-02-24

The electrochemical behaviour of the polycrystalline platinum electrode towards the oxidation/reduction of short-chain unsaturated aliphatic molecules such as acrylamide and acrylic acid was investigated in acidic solutions. Analytes were separated by reverse phase liquid chromatographic and quantified using a pulsed amperometric detection. A new two-step waveform, is introduced for detection of acrylamide and acrylic acid. Detection limits (LOD) of 20 nM (1. 4 microg/kg) and 45 nM (3.2 microg/kg) were determined in water solutions containing acrylamide and acrylic acid, respectively. Compared to the classical three-step waveform, the proposed two-step waveform shows favourable analytical performance in terms of LOD, linear range, precision and improved long-term reproducibility. The proposed analytical method combined with clean-up procedure accomplished by Carrez clearing reagent and subsequent extraction with a strong cation exchanger cartridges (SPE), was successfully used for the quantification of low concentrations of acrylamide in foodstuffs such as coffee and potato fries.

Effects of two novel amino acid substitutions on the penicillin binding properties of the PBP5 C‑terminal from Enterococcus faecium.

PubMed

Zhou, Chengjiang; Niu, Haiying; Yu, Hui; Zhou, Lishe; Wang, Zhanli

2015-10-01

The low‑affinity penicillin‑binding protein (PBP)5 is responsible for resistance to β‑lactam antibiotics in Enterococcus faecium. (E. faecium). In order to evaluate more fully the potential of this species for the development of resistance to β-lactam antibiotics, the present study aimed to examine the extent of penicillin-binding protein (PBP) variations in a collection of clinical E. faecium isolates. In the present study, the C‑terminal domain of PBP5 (PBP5‑CD) of 13 penicillin‑resistant clinical isolates of E. faecium were sequenced and the correlation between penicillin resistance and particular amino acid changes were analyzed. The present study identified for the first time, to the best of our knowledge, two novel substitutions (Tyr460Phe and Ala462Thr or Val462Thr) of E. faecium PBP5‑CD. The covalent interaction between penicillin and PBP5‑CD was also investigated using homology modeling and molecular docking methods. The theoretical calculation revealed that Phe460 and Thr462 were involved in penicillin binding, suggesting that substitutions at these positions exert effects on the affinity for penicillin, and this increased affinity translates into lower resistance in vitro.

Peroxyoxalate chemiluminescence detection of condensates of malondialdehyde with thiobarbituric acids using a flow system.

PubMed

Nakashima, K; Nagata, M; Takahashi, M; Akiyama, S

1992-01-01

The peroxyoxalate chemiluminescence(CL) detection method for the evaluation of the CL intensity of malondialdehyde(MDA) condensates with seven 2-thiobarbituric acid derivatives is described. The method consists of a flow injection technique together with a CL detection system using bis(2,4,6-trichlorophenyl) oxalate(TCPO) and hydrogen peroxide as chemiluminogenic reagents. Linear correlations between CL intensity and concentration are obtained for pmol levels of condensates. Among the condensates, 1,3-diethyl-2-thiobarbituric acid(DETBA)-MDA shows the largest CL intensity. High performance liquid chromatography (HPLC)/CL detection of DETBA-MDA and 1,3-diphenyl-2-thiobarbituric acid(DPTBA)-MDA using a mixture of TCPO and hydrogen peroxide in acetonitrile as a postcolumn reagent solution is also described. The detection limits for DETBA-MDA and DPTBA-MDA are 20 and 200 fmol, respectively, per 20 microL injection at a signal-to-noise ratio of 2. This HPLC/CL detection system was applied to the determination of MDA in rat brains by using DETBA as a fluorescent derivatizing reagent.

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9.

PubMed

Willson, T A; Nagley, P

1987-09-01

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions.

PubMed

Nishizawa, M; Nishizawa, K

2000-10-01

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the 'between gene' GC content heterogeneity, which is linked to 'isochores', is a principal factor associated with the bias in substitution patterns in human, 'within gene' heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed.

Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions

PubMed Central

Nishizawa, Manami; Nishizawa, Kazuhisa

2000-01-01

The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the ‘between gene’ GC content heterogeneity, which is linked to ‘isochores’, is a principal factor associated with the bias in substitution patterns in human, ‘within gene’ heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed. PMID:11000273

Implantation of silicon dioxide-based nanocrystalline hydroxyapatite and pure phase beta-tricalciumphosphate bone substitute granules in caprine muscle tissue does not induce new bone formation.

PubMed

Ghanaati, Shahram; Udeabor, Samuel E; Barbeck, Mike; Willershausen, Ines; Kuenzel, Oliver; Sader, Robert A; Kirkpatrick, C James

2013-01-04

Osteoinductive bone substitutes are defined by their ability to induce new bone formation even at heterotopic implantation sites. The present study was designed to analyze the potential osteoinductivity of two different bone substitute materials in caprine muscle tissue. One gram each of either a porous beta-tricalcium phosphate (β-TCP) or an hydroxyapatite/silicon dioxide (HA/SiO2)-based nanocrystalline bone substitute material was implanted in several muscle pouches of goats. The biomaterials were explanted at 29, 91 and 181 days after implantation. Conventional histology and special histochemical stains were performed to detect osteoblast precursor cells as well as mineralized and unmineralized bone matrix. Both materials underwent cellular degradation in which tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and TRAP-negative multinucleated giant cells were involved. The ß-TCP was completely resorbed within the observation period, whereas some granules of the HA-groups were still detectable after 180 days. Neither osteoblasts, osteoblast precursor cells nor extracellular bone matrix were found within the implantation bed of any of the analyzed biomaterials at any of the observed time points. This study showed that ß-TCP underwent a faster degradation than the HA-based material. The lack of osteoinductivity for both materials might be due to their granular shape, as osteoinductivity in goat muscle has been mainly attributed to cylindrical or disc-shaped bone substitute materials. This hypothesis however requires further investigation to systematically analyze various materials with comparable characteristics in the same experimental setting.

Influence of Al substitution on magnetism and adsorption properties of hematite

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

Cao, Shanshan; Kang, Feifei; Yang, Xin

2015-08-15

A series of Al-substituted hematite was prepared. The structures and properties of as-prepared samples were characterized by various techniques. The magnetic property of the samples was determined and the adsorption of three dyes Acid Blue 74, Methylene Blue and Phenol Red onto the samples was investigated. The results showed that Al incorporation into the crystal structure of hematite occurs via isomorphous ionic substitution of Al for Fe. With increasing Al content, the particle size of samples decreases, the magnetization increases and the remanent magnetization remains unchanged. The coercivity of the samples increases with Al substitution up to n{sub Al}/n{sub Fe}more » 0.03, and then decreases as Al content further increases. Compared with Al-free hematite, Al-substituted samples exhibit better adsorption ability to all of the three dyes. The adsorption rates of the three dyes on the surface of Al substituted samples depend on the structure of dye, pH and Al content in hematite. - Graphical abstract: Effect of Al on the structure, magnetic properties and adsorption performance of hematite was investigated. - Highlights: • A series of Al-substituted α-Fe{sub 2}O{sub 3} was prepared. • Effect of Al content on the crystal structure and magnetic property of hematite was investigated. • Al-substituted hematite exhibits better adsorption ability than hematite.« less

Adaptation of tick-borne encephalitis virus from human brain to different cell cultures induces multiple genomic substitutions.

PubMed

Ponomareva, Eugenia P; Ternovoi, Vladimir A; Mikryukova, Tamara P; Protopopova, Elena V; Gladysheva, Anastasia V; Shvalov, Alexander N; Konovalova, Svetlana N; Chausov, Eugene V; Loktev, Valery B

2017-10-01

The C11-13 strain from the Siberian subtype of tick-borne encephalitis virus (TBEV) was isolated from human brain using pig embryo kidney (PEK), 293, and Neuro-2a cells. Analysis of the complete viral genome of the C11-13 variants during six passages in these cells revealed that the cell-adapted C11-13 variants had multiple amino acid substitutions as compared to TBEV from human brain. Seven out of eight amino acids substitutions in the high-replicating C11-13(PEK) variant mapped to non-structural proteins; 13 out of 14 substitutions in the well-replicating C11-13(293) variant, and all four substitutions in the low-replicating C11-13(Neuro-2a) variant were also localized in non-structural proteins, predominantly in the NS2a (2), NS3 (6) and NS5 (3) proteins. The substitutions NS2a 1067 (Asn → Asp), NS2a 1168 (Leu → Val) in the N-terminus of NS2a and NS3 1745 (His → Gln) in the helicase domain of NS3 were found in all selected variants. We postulate that multiple substitutions in the NS2a, NS3 and NS5 genes play a key role in adaptation of TBEV to different cells.

Fluorescent hybridization probes for nucleic acid detection.

PubMed

Guo, Jia; Ju, Jingyue; Turro, Nicholas J

2012-04-01

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

A graphene-based electrochemical competitive immunosensor for the sensitive detection of okadaic acid in shellfish

NASA Astrophysics Data System (ADS)

Eissa, Shimaa; Zourob, Mohammed

2012-11-01

A novel graphene-based voltammetric immunosensor for sensitive detection of okadaic acid (OA) was developed. A simple and efficient electrografting method was utilized to functionalize graphene-modified screen-printed carbon electrodes (GSPE) by the electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt in acidic aqueous solution. Next, the okadaic acid antibody was covalently immobilized on the carboxyphenyl modified graphene electrodes via carbodiimide chemistry. Square wave voltammetry (SWV) was used to investigate the stepwise assembly of the immunosensor. A competitive assay between OA and a fixed concentration of okadaic acid-ovalbumin conjugate (OA-OVA) for the immobilized antibodies was employed for the detection of okadaic acid. The decrease of the [Fe(CN)6]3-/4- reduction peak current in the square wave voltammetry for various concentrations of okadaic acid was used for establishing the calibration curve. A linear relationship between the SWV peak current difference and OA concentration was obtained up to ~5000 ng L-1. The developed immunosensor allowed a detection limit of 19 ng L-1 of OA in PBS buffer. The matrix effect studied with spiked shellfish tissue extracts showed a good percentage of recovery and the method was also validated with certified reference mussel samples.A novel graphene-based voltammetric immunosensor for sensitive detection of okadaic acid (OA) was developed. A simple and efficient electrografting method was utilized to functionalize graphene-modified screen-printed carbon electrodes (GSPE) by the electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt in acidic aqueous solution. Next, the okadaic acid antibody was covalently immobilized on the carboxyphenyl modified graphene electrodes via carbodiimide chemistry. Square wave voltammetry (SWV) was used to investigate the stepwise assembly of the immunosensor. A competitive assay between OA and a fixed concentration of okadaic acid

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2016-08-15

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. © 2016. Published by The Company of Biologists Ltd.

A single amino acid substitution in the Bombyx-specific mucin-like membrane protein causes resistance to Bombyx mori densovirus.

PubMed

Ito, Katsuhiko; Kidokoro, Kurako; Katsuma, Susumu; Sezutsu, Hideki; Uchino, Keiro; Kobayashi, Isao; Tamura, Toshiki; Yamamoto, Kimiko; Mita, Kazuei; Shimada, Toru; Kadono-Okuda, Keiko

2018-05-09

Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain. The NSD-1 protein was specifically expressed in the larval midgut epithelium, the known infection site of BmDV. Sequence analysis of the nsd-1 gene from 13 resistant and 12 susceptible strains suggested that a specific arginine residue in the extracellular tail of the NSD-1 protein was common among susceptible strains. Germline transformation of the susceptible-type nsd-1 (with a single nucleotide substitution) conferred partial susceptibility to resistant larvae, indicating that the +  nsd-1 gene is required for the susceptibility of B. mori larvae to BmDV and the susceptibility is solely a result of the substitution of a single amino acid with arginine. Taken together, our results provide striking evidence that a novel membrane-bound mucin-like protein functions as a cell-surface receptor for a densovirus.

Design, synthesis, and evaluation of a novel series of alpha-substituted phenylpropanoic acid derivatives as human peroxisome proliferator-activated receptor (PPAR) alpha/delta dual agonists for the treatment of metabolic syndrome.

PubMed

Kasuga, Jun-ichi; Yamasaki, Daisuke; Araya, Yoko; Nakagawa, Aya; Makishima, Makoto; Doi, Takefumi; Hashimoto, Yuichi; Miyachi, Hiroyuki

2006-12-15

A series of alpha-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated receptors alpha and delta (PPARalpha/delta). Structure-activity relationship studies indicated that the shape of the linking group and the shape of the substituent at the distal benzene ring play key roles in determining the potency and the selectivity of PPAR subtype transactivation. Structure-activity relationships among the amide series (10) and the reversed amide series (13) are similar, but not identical, especially in the case of the compounds bearing a bulky hydrophobic substituent at the distal benzene ring, indicating that the hydrophobic tail part of the molecules in these two series binds at somewhat different positions in the large binding pocket of PPAR. alpha-Alkyl-substituted phenylpropanoic acids of (S)-configuration were identified as potent human PPARalpha/delta dual agonists. Representative compounds exhibited marked nuclear receptor selectivity for PPARalpha and PPARdelta. Subtype-selective PPAR activation was also examined by analysis of the mRNA expression of PPAR-regulated genes.

Amino acid substitutions in the heptad repeat A and C regions of the F protein responsible for neurovirulence of measles virus Osaka-1 strain from a patient with subacute sclerosing panencephalitis.

PubMed

Ayata, Minoru; Tanaka, Miyuu; Kameoka, Kazuo; Kuwamura, Mitsuru; Takeuchi, Kaoru; Takeda, Makoto; Kanou, Kazuhiko; Ogura, Hisashi

2016-01-01

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE). We previously reported that the F gene of the SSPE Osaka-2 strain is the major determinant of MV neurovirulence. Because the sites and extents of mutations differ among SSPE strains, it is necessary to determine the mutations responsible for the SSPE-specific phenotypes of individual viral strain. In this study, recombinant viruses containing the envelope-associated genes from the SSPE Osaka-1 strain were generated in the IC323 wild-type MV background. Hamsters inoculated with MV containing the H gene of the Osaka-1 strain displayed hyperactivity and seizures, but usually recovered and survived. Hamsters inoculated with MV containing the F gene of the Osaka-1 strain displayed severe neurologic signs and died. Amino acid substitutions in the heptad repeat A and C regions of the F protein, including a methionine-to-valine substitution at amino acid 94, play major roles in neurovirulence. Copyright © 2015 Elsevier Inc. All rights reserved.

Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag.

PubMed

Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

2006-12-01

The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

Rapid Detection of Positive Selection in Genes and Genomes Through Variation Clusters

PubMed Central

Wagner, Andreas

2007-01-01

Positive selection in genes and genomes can point to the evolutionary basis for differences among species and among races within a species. The detection of positive selection can also help identify functionally important protein regions and thus guide protein engineering. Many existing tests for positive selection are excessively conservative, vulnerable to artifacts caused by demographic population history, or computationally very intensive. I here propose a simple and rapid test that is complementary to existing tests and that overcomes some of these problems. It relies on the null hypothesis that neutrally evolving DNA regions should show a Poisson distribution of nucleotide substitutions. The test detects significant deviations from this expectation in the form of variation clusters, highly localized groups of amino acid changes in a coding region. In applying this test to several thousand human–chimpanzee gene orthologs, I show that such variation clusters are not generally caused by relaxed selection. They occur in well-defined domains of a protein's tertiary structure and show a large excess of amino acid replacement over silent substitutions. I also identify multiple new human–chimpanzee orthologs subject to positive selection, among them genes that are involved in reproductive functions, immune defense, and the nervous system. PMID:17603100

Green synthesis of carbon dots from pork and application as nanosensors for uric acid detection

NASA Astrophysics Data System (ADS)

Zhao, Chunxi; Jiao, Yang; Hu, Feng; Yang, Yaling

2018-02-01

In this work, a green, simple, economical method was developed in the synthesis of fluorescent carbon dots using pork as carbon source. The as-prepared carbon dots exhibit exceptional advantages including high fluorescent quantum yield (17.3%) and satisfactory chemical stability. The fluorescence of carbon dots based nanosensor can be selectively and efficiently quenched by uric acid. This phenomenon was used to develop a fluorescent method for facile detection of uric acid within a linear range of 0.1-100 μM and 100-500 μM, with a detection limit of 0.05 μM (S/N = 3). Finally, the proposed method was successfully applied in the determination of uric acid in human serum and urine samples with satisfactory recoveries, which suggested that the new nanosensors have great prospect toward the detection of uric acid in human fluids.

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

PubMed

Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

2017-12-01

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

A new detection scheme for ultrafast 2D J-resolved spectroscopy

NASA Astrophysics Data System (ADS)

Giraudeau, Patrick; Akoka, Serge

2007-06-01

Recent ultrafast techniques enable 2D NMR spectra to be obtained in a single scan. A modification of the detection scheme involved in this technique is proposed, permitting the achievement of 2D 1H J-resolved spectra in 500 ms. The detection gradient echoes are substituted by spin echoes to obtain spectra where the coupling constants are encoded along the direct ν2 domain. The use of this new J-resolved detection block after continuous phase-encoding excitation schemes is discussed in terms of resolution and sensitivity. J-resolved spectra obtained on cinnamic acid and 3-ethyl bromopropionate are presented, revealing the expected 2D J-patterns with coupling constants as small as 2 Hz.

40 CFR 721.10214 - Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle...

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Poly(oxyalkylenediyl),.alpha... Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle (generic... identified generically as poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted...

40 CFR 721.10214 - Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Poly(oxyalkylenediyl),.alpha... Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle (generic... identified generically as poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted...

40 CFR 721.10214 - Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle...

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Poly(oxyalkylenediyl),.alpha... Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle (generic... identified generically as poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted...

40 CFR 721.10214 - Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle...

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Poly(oxyalkylenediyl),.alpha... Poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted carbomonocycle (generic... identified generically as poly(oxyalkylenediyl),.alpha.-substituted carbomonocycle-.omega.-substituted...

Sensitive detection of dopamine via leucodopaminechrome on polyacrylic acid-coated ceria nanorods

NASA Astrophysics Data System (ADS)

Sheng, Weiqin; Zheng, Liang; Liu, Yan; Zhao, Xueqin; Weng, Jian; Zhang, Yang

2017-09-01

The major hurdle in detection of dopamine (DA) by electro-analysis is the presence of physiological interferents with a similar oxidation potential of DA. The conventional method is to enlarge the difference of their oxidation potentials. Here, we report an unconventional method to detect DA via leucodopaminechrome on CeO2 nanorods. Leucodopaminechrome is produced from the cyclization of dopamine-quinone, a product of two-electron oxidation of DA. Thus, its concentration is proportional to the DA concentration. Determining DA is demonstrated by measuring the reduction current of leucodopaminechrome on CeO2 nanorods. CeO2 nanorods demonstrate high electrocatalytic activity for reduction of leucodopaminechrome with a low potential at -0.27 V. The low detection potential of leucodopaminechrome can avoid the interference from ascorbic acid (AA) and uric acid (UA). Therefore, detecting DA via leucodopaminechrome is an effective method to avoid interference from AA and UA, and the suggested biosensor also displays good reproducibility and stability.

SubVis: an interactive R package for exploring the effects of multiple substitution matrices on pairwise sequence alignment

PubMed Central

Coan, Heather B.; Youker, Robert T.

2017-01-01

Understanding how proteins mutate is critical to solving a host of biological problems. Mutations occur when an amino acid is substituted for another in a protein sequence. The set of likelihoods for amino acid substitutions is stored in a matrix and input to alignment algorithms. The quality of the resulting alignment is used to assess the similarity of two or more sequences and can vary according to assumptions modeled by the substitution matrix. Substitution strategies with minor parameter variations are often grouped together in families. For example, the BLOSUM and PAM matrix families are commonly used because they provide a standard, predefined way of modeling substitutions. However, researchers often do not know if a given matrix family or any individual matrix within a family is the most suitable. Furthermore, predefined matrix families may inaccurately reflect a particular hypothesis that a researcher wishes to model or otherwise result in unsatisfactory alignments. In these cases, the ability to compare the effects of one or more custom matrices may be needed. This laborious process is often performed manually because the ability to simultaneously load multiple matrices and then compare their effects on alignments is not readily available in current software tools. This paper presents SubVis, an interactive R package for loading and applying multiple substitution matrices to pairwise alignments. Users can simultaneously explore alignments resulting from multiple predefined and custom substitution matrices. SubVis utilizes several of the alignment functions found in R, a common language among protein scientists. Functions are tied together with the Shiny platform which allows the modification of input parameters. Information regarding alignment quality and individual amino acid substitutions is displayed with the JavaScript language which provides interactive visualizations for revealing both high-level and low-level alignment information. PMID:28674656

Application of HPLC with ELSD Detection for the Assessment of Azelaic Acid Impurities in Liposomal Formulation

PubMed Central

Han, Stanislaw; Karlowicz-Bodalska, Katarzyna; Ozimek, Lukasz

2013-01-01

In the course of research and development of a new pharmaceutical formulation of azelaic acid in the liposomal form, we developed a rapid and accurate method for the detection of impurities using high-performance liquid chromatography. A chromatographic column from Merck (Purospher Star RP C18, 250–4 mm (5 μm) was used in the assay, and the mobile phase gradient consisted of three phases: A—methanol : water (5 : 95) + 1.5% (v/v) acetic acid; B—water : methanol (5 : 95) + 1.5% (v/v) acetic acid; and C—chloroform. Detection of the impurities and the active substance was performed by an evaporative light-scattering detector. The method was validated for selectivity, system precision, method precision, limit of detection, and response rates. The proposed method can be used to detect impurities in the liposomal formulation of azelaic acid. The method enables separation of azelaic acid from the identified and unidentified impurities and from the excipients used in the drug form. PMID:24228008

Application of HPLC with ELSD detection for the assessment of azelaic acid impurities in liposomal formulation.

PubMed

Han, Stanislaw; Karlowicz-Bodalska, Katarzyna; Szura, Dorota; Ozimek, Lukasz; Musial, Witold

2013-01-01

In the course of research and development of a new pharmaceutical formulation of azelaic acid in the liposomal form, we developed a rapid and accurate method for the detection of impurities using high-performance liquid chromatography. A chromatographic column from Merck (Purospher Star RP C18, 250-4 mm (5 μm) was used in the assay, and the mobile phase gradient consisted of three phases: A--methanol : water (5 : 95) + 1.5% (v/v) acetic acid; B--water : methanol (5 : 95) + 1.5% (v/v) acetic acid; and C--chloroform. Detection of the impurities and the active substance was performed by an evaporative light-scattering detector. The method was validated for selectivity, system precision, method precision, limit of detection, and response rates. The proposed method can be used to detect impurities in the liposomal formulation of azelaic acid. The method enables separation of azelaic acid from the identified and unidentified impurities and from the excipients used in the drug form.

Simple, heart-smart substitutions

MedlinePlus

Coronary artery disease - heart smart substitutions; Atherosclerosis - heart smart substitutions; Cholesterol - heart smart substitutions; Coronary heart disease - heart smart substitutions; Healthy diet - heart ...

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Self-assembled pentacenequinone derivative for trace detection of picric acid.

PubMed

Bhalla, Vandana; Gupta, Ankush; Kumar, Manoj; Rao, D S Shankar; Prasad, S Krishna

2013-02-01

Pentacenequinone derivative 3 forms luminescent supramolecular aggregates both in bulk as well as in solution phase. In bulk phase at high temperature, long-range stacking of columns leads to formation of stable and ordered columnar mesophase. Further, derivative 3 works as sensitive chemosensor for picric acid (PA) and gel-coated paper strips detect PA at nanomolar level and provide a simple, portable, and low-cost method for detection of PA in aqueous solution, vapor phase, and in contact mode.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

An advanced LC-MS (Q-TOF) technique for the detection of amino acids in atmospheric aerosols

EPA Science Inventory

Methodology for detection of native (underivitized) amino acids in atmospheric aerosols has been developed. This article describes the use of LC-MS (Q-TOF) and microwave-assisted gas phase hydrolysis for detection of free and combined amino acids in aerosols collected in a Southe...

Synthesis of substituted tetrahydroisoquinolines by lithiation then electrophilic quench.

PubMed

Talk, Ruaa A; Duperray, Alexia; Li, Xiabing; Coldham, Iain

2016-06-07

Substituted N-tert-butoxycarbonyl (Boc)-1,2,3,4-tetrahydroisoquinolines were prepared and treated with n-butyllithium in THF at -50 °C to test the scope of the metallation and electrophilic quench. The lithiation was optimised by using in situ ReactIR spectroscopy and the rate of rotation of the carbamate was determined. The 1-lithiated intermediates could be trapped with a variety of electrophiles to give good yields of 1-substituted tetrahydroisoquinoline products. Treatment with acid or reduction with LiAlH4 allows conversion to the N-H or N-Me compound. The chemistry was applied to the efficient total syntheses of the alkaloids (±)-crispine A and (±)-dysoxyline.

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.

PubMed

Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

2010-08-01

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

Amino Acid Substitutions in Polymerase Basic Protein 2 Gene Contribute to the Pathogenicity of the Novel A/H7N9 Influenza Virus in Mammalian Hosts

PubMed Central

Mok, Chris Ka Pun; Lee, Horace Hok Yeung; Lestra, Maxime; Nicholls, John Malcolm; Chan, Michael Chi Wai; Sia, Sin Fun; Zhu, Huachen; Poon, Leo Lit Man; Guan, Yi

2014-01-01

ABSTRACT A novel avian-origin influenza A/H7N9 virus emerged in 2013 to cause more than 130 cases of zoonotic human disease, with an overall case fatality rate of around 30% in cases detected. It has been shown that an E-to-K amino acid change at residue 627 of polymerase basic protein 2 (PB2) occurred frequently in the H7N9 isolates obtained from humans but not in viruses isolated from poultry. Although this mutation has been reported to confer increased mammalian pathogenicity in other avian influenza subtypes, it has not been experimentally investigated in the H7N9 virus. In this study, we determined the contribution of PB2-E627K in H7N9 virus to its pathogenicity in mammalian hosts. In addition, the compensatory role of the PB2 mutations T271A, Q591K, and D701N in H7N9 virus was investigated. We characterized the activity of polymerase complexes with these PB2 mutations and found that they enhance the polymerase activity in human 293T cells. The rescued mutants enhanced growth in mammalian cells in vitro. Mice infected with the H7N9 mutant containing the avian signature protein PB2-627E showed a marked decrease in disease severity (weight loss) and pathology compared to mice infected with the wild-type strain (PB2-627K) or other PB2 mutants. Also, mutants with PB2-627E showed lower virus replication and proinflammatory cytokine responses in the lungs of the virus-infected mice, which may contribute to pathogenicity. Our results suggest that these amino acid substitutions contribute to mouse pathogenicity and mammalian adaptation. IMPORTANCE A novel avian H7N9 influenza A virus emerged in east China in 2013 to cause zoonotic human disease associated with significant mortality. It is important to understand the viral genetic markers of mammalian adaptation and disease severity in this H7N9 virus. Since many human (but not avian) H7N9 virus isolates have an amino acid substitution at position E627K in the polymerase basic protein 2 (PB2) gene, we investigated the

Poly(ionic liquid) based chemosensors for detection of basic amino acids in aqueous medium

NASA Astrophysics Data System (ADS)

Li, Xinjuan; Wang, Kai; Ma, Nana; Jia, Xianbin

2017-09-01

Naked-eye detection of amino acids in water is of great significance in the field of bio-analytical applications. Herein, polymerized ionic liquids (PILs) with controlled chain length structures were synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization and post-quaternization approach. The amino acids recognition performance of PILs with different alkyl chain lengths and molecular weights was evaluated by naked-eye color change and ultraviolet-visible (UV-vis) spectral studies. These PILs were successfully used for highly sensitive and selective detection of Arg, Lys and His in water. The recognition performance was improved effectively with increased molecular weight of PILs. The biosensitivity of the PILs in water was strongly dependent on their aggregation effect and polarization effect. Highly sensitive and selective detection of amino acids was successfully accomplished by introducing positively charged pyridinium moieties and controlled RAFT radical polymerization.

Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection.

PubMed

Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T; Carr, Christopher E

2017-08-01

Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments-Nucleic acids-Mars-Panspermia. Astrobiology 17, 747-760.

Application of carbon and hydrogen stable isotope analyses to detect exogenous citric acid in Japanese apricot liqueur.

PubMed

Akamatsu, Fumikazu; Oe, Takaaki; Hashiguchi, Tomokazu; Hisatsune, Yuri; Kawao, Takafumi; Fujii, Tsutomu

2017-08-01

Japanese apricot liqueur manufacturers are required to control the quality and authenticity of their liqueur products. Citric acid made from corn is the main acidulant used in commercial liqueurs. In this study, we conducted spiking experiments and carbon and hydrogen stable isotope analyses to detect exogenous citric acid used as an acidulant in Japanese apricot liqueurs. Our results showed that the δ 13 C values detected exogenous citric acid originating from C 4 plants but not from C 3 plants. The δ 2 H values of citric acid decreased as the amount of citric acid added increased, whether the citric acid originated from C 3 or C 4 plants. Commercial liqueurs with declared added acidulant provided higher δ 13 C values and lower δ 2 H values than did authentic liqueurs and commercial liqueurs with no declared added acidulant. Carbon and hydrogen stable isotope analyses are suitable as routine methods for detecting exogenous citric acid in Japanese apricot liqueur. Copyright © 2017 Elsevier Ltd. All rights reserved.

Arsenic Scavenging by Al-Substituted Ferrihydrites in a Circumneutral pH River Impacted by the Acid Mine Drainage of Carnoulès, Gard, France

NASA Astrophysics Data System (ADS)

ADRA, A.; Morin, G.; ona-Nguema, G.; Maillot, F.; Casiot, C.; Bruneel, O.

2013-12-01

Ferrihydrite (Fh) is a nanocrystalline ferric oxyhydroxide involved in the retention of pollutants in natural systems and in water-treatment processes. The status and properties of major chemical impurities in natural Fh is however still scarcely documented. Here we investigated the structure and reactivity of aluminum-rich Fh from river-bed sediments collected in a circumneutral river (pH 6-7) impacted by an arsenic-rich acid mine drainage (AMD). Extended X-ray absorption fine structure (EXAFS) spectroscopy at the Fe K-edge shows that Fh is the predominant mineral phase forming after neutralization of the AMD, in association with minor amount of schwertmannite transported from the AMD. EXAFS analysis indicates that Al(III) substitutes for Fe(III) ions into the Fh structure in the natural sediment samples, with local aluminum concentration within the 20-37×7 mol%Al range, in agreement with bulk chemical compositions. Synthetic aluminous Fh analogues prepared in the present study are found to be less Al-substituted (14-18×4 mol%Al). Finally, EXAFS analysis at the arsenic K-edge indicates that As(V) form similar inner-sphere surface complexes on the natural and synthetic Al-substituted Fh studied. Our results provide direct evidences for the scavenging of arsenic by natural Al- Fh, with possible implications for other pollutants in natural or engineered systems.

A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection.

PubMed

Tang, Ruihua; Yang, Hui; Gong, Yan; You, MinLi; Liu, Zhi; Choi, Jane Ru; Wen, Ting; Qu, Zhiguo; Mei, Qibing; Xu, Feng

2017-03-29

Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. At present, NAT mainly involves nucleic acid extraction, amplification and detection steps that heavily rely on large equipment and skilled workers, making the test expensive, time-consuming, and thus less suitable for point-of-care (POC) applications. With advances in paper-based microfluidic technologies, various integrated paper-based devices have recently been developed for NAT, which however require off-chip reagent storage, complex operation steps and equipment-dependent nucleic acid amplification, restricting their use for POC testing. To overcome these challenges, we demonstrate a fully disposable and integrated paper-based sample-in-answer-out device for NAT by integrating nucleic acid extraction, helicase-dependent isothermal amplification and lateral flow assay detection into one paper device. This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps, which could be performed by untrained users in remote settings. The proposed device consists of a sponge-based reservoir and a paper-based valve for nucleic acid extraction, an integrated battery, a PTC ultrathin heater, temperature control switch and on-chip dried enzyme mix storage for isothermal amplification, and a lateral flow test strip for naked-eye detection. It can sensitively detect Salmonella typhimurium, as a model target, with a detection limit of as low as 10 2 CFU ml -1 in wastewater and egg, and 10 3 CFU ml -1 in milk and juice in about an hour. This fully disposable and integrated paper-based device has great potential for future POC applications in resource-limited settings.

Direct detection by real-time PCR of ftsI gene mutations affecting MICs of β-lactam agents for Haemophilus influenzae isolates from meningitis.

PubMed

Kishii, Kozue; Morozumi, Miyuki; Chiba, Naoko; Ono, Akiko; Ubukata, Kimiko

2011-10-01

One resistance mechanism of Haemophilus influenzae to ampicillin involves decreased affinity of penicillin-binding protein (PBP) 3 for β-lactam antibiotics reflecting amino acid substitutions in PBP3 encoded by the ftsI gene. Three amino acid substitutions, Ser385Thr, Arg517His, and Asn526Lys, are especially responsible for β-lactam resistance. We constructed a new real-time polymerase chain reaction (PCR) to directly detect these substitutions in addition to 16S ribosomal RNA (rRNA), cap, and bla(TEM) genes. Our real-time PCR was evaluated using 206 clinical H. influenzae strains isolated from pediatric patients with meningitis. Relative sensitivities and specificities of real-time PCR were 90.5-100% and 96.3-100% for all resistance classes compared with our previously reported conventional PCR. In addition, real-time PCR shortened time required from 3 h by conventional PCR to 1.5 h. When correlations between combinations of amino acid substitutions in the ftsI gene detected by real-time PCR and minimum inhibitory concentrations (MICs) of β-lactam antibiotics were evaluated, MIC(90)s of ampicillin for β-lactamase-nonproducing ampicillin-intermediate-resistant strains with Asn526Lys, β-lactamase-nonproducing, ampicillin-resistant strains with Ser385Thr, and β-lactamase-nonproducing ampicillin-resistant strains with both Asn526Lys and Ser385Thr, respectively, were two, four, and eight times higher than those for sensitive strains. Similarly, MIC(90)s of cephalosporins for these strains, respectively, were two, 16-32, and 16-32 times higher than those for sensitive strains. Thus, real-time PCR can guide antibiotic use.

The Quality Improvement of Emulsion-type Pork Sausages Formulated by Substituting Pork Back fat with Rice Bran Oil

PubMed Central

Yum, Hyeon-Woong; Seo, Jin-Kyu; Jeong, Jin-Yeon; Kim, Gap-Don; Rahman, M. Shafiur; Yang, Han-Sul

2018-01-01

The effects of pork back fat (PBF) substitution with various concentrations of rice bran oil (RBO) (50%, 45%, 40% and 35%) on the physicochemical characteristics and sensory attributes of emulsion-type pork sausages were studied. The modified pork sausages were compared with control sausages produced using PBF only. The sausages with RBO had significantly lower (p<0.05) moisture content than the control sausages. Sausages made from PBF substituted with 40% RBO showed the lowest cooking loss. Substitution of PBF with RBO had no significant effect on the emulsion stability of pork sausages. All sausages with RBO showed significantly lower (p<0.05) hardness values than control sausages. Sausages with RBO also had significantly higher values (p<0.05) of unsaturated fatty acid and polyunsaturated to saturated fatty acid contents than the controls. RBO substitution had no effect on the flavor intensity of sausages, but it improved the tenderness and produced a softer texture. PMID:29725230

Amino acid substitutions in the hepatitis C virus core region predict hepatocarcinogenesis following eradication of HCV RNA by all-oral direct-acting antiviral regimens.

PubMed

Ogata, Fumihiro; Akuta, Norio; Kobayashi, Masahiro; Fujiyama, Shunichiro; Kawamura, Yusuke; Sezaki, Hitomi; Hosaka, Tetsuya; Kobayashi, Mariko; Saitoh, Satoshi; Suzuki, Yoshiyuki; Suzuki, Fumitaka; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

2018-06-01

Impact of substitution of aa70 in the core region (Core aa70) in HCV genotype 1b (HCV-1b) on hepatocarcinogenesis following eradication of HCV RNA by direct-acting antiviral therapy is not clear. In a retrospective study, 533 patients with HCV-related chronic liver disease, with sustained virological response defined as negative HCV RNA at 12 weeks after cessation of direct-acting antiviral therapy, were examined to evaluate the relationship between Core aa70 substitution and hepatocarcinogenesis. Twelve patients developed hepatocellular carcinoma during the follow-up period. The cumulative hepatocarcinogenesis rates were 1.7% and 2.4% at the end of 1 and 2 years, respectively. Overall, multivariate analysis identified HCV subgroup (HCV-1b with Gln70(His70); P = 0.003) and age (>65 years; P = 0.049), as pretreatment predictors of hepatocarcinogenesis. In HCV-1b patients, multivariate analysis identified post-treatment Wisteria floribunda agglutinin positive Mac-2 binding protein (>1.8 COI; P = 0.042) and HCV subgroup (HCV-1b with Gln70(His70); P = 0.071), as predictors of hepatocarcinogenesis, including post-treatment parameter. In conclusion, Core aa70 substitution in HCV-1b at the start of direct-acting antiviral therapy is an important predictor of hepatocarcinogenesis following eradication of HCV RNA. This study emphasizes the importance of detection of Core aa70 substitution before initiating antiviral therapy. © 2018 Wiley Periodicals, Inc.

Triphenylbenzene Sensor for Selective Detection of Picric Acid.

PubMed

Nagendran, S; Vishnoi, Pratap; Murugavel, Ramaswamy

2017-07-01

A C 3 -symmetric triphenylbenzene based photoluminescent compound, 1,3,5-tris(4'-(N-methylamino)phenyl) benzene ([NHMe] 3 TAPB), has been synthesized by mono-N-methylation of 1,3,5-tris(4'-aminophenyl) benzene (TAPB) and structurally characterized. [NHMe] 3 TAPB acts as a selective fluorescent sensor for picric acid (PA) with a detection limit as low as 2.25 ppm at a signal to noise ratio of 3. Other related analytes (i.e. TNT, DNT and DNB) show very little effect on the fluorescence intensity of [NHMe] 3 TAPB. The selectivity is triggered by proton transfer from picric acid to the fluorophore and ground-state complex formation between the protonated fluorophore and picrate anion through hydrogen bonding interactions. The fluorescence lifetime measurements reveal static nature of fluorescence quenching.

Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

PubMed

Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

2014-08-07

Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

Ion-exclusion chromatography with conductimetric detection of aliphatic carboxylic acids on a weakly acidic cation-exchange resin by elution with benzoic acid-beta-cyclodextrin.

PubMed

Tanaka, Kazuhiko; Mori, Masanobu; Xu, Qun; Helaleh, Murad I H; Ikedo, Mikaru; Taoda, Hiroshi; Hu, Wenzhi; Hasebe, Kiyoshi; Fritz, James S; Haddad, Paul R

2003-05-16

In this study, an aqueous solution consisting of benzoic acid with low background conductivity and beta-cyclodextrin (beta-CD) of hydrophilic nature and the inclusion effect to benzoic acid were used as eluent for the ion-exclusion chromatographic separation of aliphatic carboxylic acids with different pKa values and hydrophobicity on a polymethacrylate-based weakly acidic cation-exchange resin in the H+ form. With increasing concentration of beta-cyclodextrin in the eluent, the retention times of the carboxylic acids decreased due to the increased hydrophilicity of the polymethacrylate-based cation-exchange resin surface from the adsorption of OH groups of beta-cyclodextrin. Moreover, the eluent background conductivity decreased with increasing concentration of beta-cyclodextrin in 1 mM benzoic acid, which could result in higher sensitivity for conductimetric detection. The ion-exclusion chromatographic separation of carboxylic acids with high resolution and sensitivity was accomplished successfully by elution with a 1 mM benzoic acid-10 mM cyclodextrin solution without chemical suppression.

Determination of polyadipates migrating from lid gaskets of glass jars. Hydrolysis to adipic acid and measurement by LC-MS/MS.

PubMed

Driffield, M; Bradley, E L; Harmer, N; Castle, L; Klump, S; Mottier, P

2010-10-01

Polyadipate plasticizers can be present in the polyvinylchloride (PVC) gaskets used to seal the lids of glass jars. As the gaskets can come into direct contact with the foodstuffs inside the jar, the potential exists for polyadipate migration into the food. The procedure and performance characteristics of a test method for the analysis of polyadipates in food simulants (3% aqueous acetic acid and 10% aqueous ethanol) and the volatile test media used in substitute fat tests (isooctane and 95% aqueous ethanol) are described. The PVC gaskets were exposed to the food simulants or their substitutes under standard test conditions. Studies were initially carried out using direct measurement of the polyadipate oligomers by liquid chromatography with time-of-flight mass spectrometric detection (LC-TOF-MS) but this was not practical due to the number of peaks detected. Instead, the migrating polyadipates were hydrolysed to adipic acid and measured by liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The amount of polyadipate that this measurement of adipic acid represents was then calculated. Method performance was assessed by analysis of gaskets from two types of jar lids by single-laboratory validation. Linearity, sensitivity, repeatability, intermediate reproducibility and recovery were determined to be suitable for checking compliance with the 30 mg/kg specific migration limits for polyesters of 1,2-propane diol and/or 1,3- and/or 1,4-butanediol and/or polypropylene-glycol with adipic acid, which may be end-capped with acetic acid or fatty acids C(12)-C(18) or n-octanol and/or n-decanol. The method was found to be much quicker than previous methods involving extraction, clean-up, hydrolysis, esterification, derivatisation and GC measurement, consequently saving time and money.

Micro-Detection System for Determination of the Biotic or Abiotic Origin of Amino Acids

NASA Technical Reports Server (NTRS)

Bada, Jeffrey L.; Betts, Bruce (Technical Monitor)

2002-01-01

The research involved the development of a breadboard version of a spacecraft based system for the detection of amino acid chirality (handedness) on solar system bodies. The design concept has three distinct components: a sublimation chamber for the release of amino acids from an acquired sample; a microchip based capillary electrophoresis (CE) chip for the separation of amino acids and their enantiomers; and a fluorescent based detection system. In addition, we have investigated the use of a microfluidics system for the extraction of amino acids in samples in which sublimation has proven to be problematic. This is a joint project carried out at the Scripps Institution of Oceanography (SIO), University of California at San Diego; the Jet Propulsion Laboratory (JPL), Pasadena; and the Department of Chemistry, University of California, Berkeley.

How Do Substitute Teachers Substitute? An Empirical Study of Substitute-Teacher Labor Supply

ERIC Educational Resources Information Center

Gershenson, Seth

2012-01-01

This paper examines the daily labor supply of a potentially important, but often overlooked, source of instruction in U.S. public schools: substitute teachers. I estimate a sequential binary-choice model of substitute teachers' job-offer acceptance decisions using data on job offers made by a randomized automated calling system. Importantly, this…

Probing Structural Features of Alzheimer’s Amyloid-β Pores in Bilayers Using Site-Specific Amino Acid Substitutions

PubMed Central

2012-01-01

A current hypothesis for the pathology of Alzheimer’s disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ1–42 peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes. PMID:22242635

Structure-property study of the Raman spectroscopy detection of fusaric acid and analogs

USDA-ARS?s Scientific Manuscript database

Food security can benefit from the development of selective methods to detect toxins. Fusaric acid is a mycotoxin produced by certain fungi occasionally found in agricultural commodities. Raman spectroscopy allows selective detection of analytes associated with certain spectral characteristics relat...

Multi-chamber nucleic acid amplification and detection device

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

Dugan, Lawrence

A nucleic acid amplification and detection device includes an amplification cartridge with a plurality of reaction chambers for containing an amplification reagent and a visual detection reagent, and a plurality of optically transparent view ports for viewing inside the reaction chambers. The cartridge also includes a sample receiving port which is adapted to receive a fluid sample and fluidically connected to distribute the fluid sample to the reaction chamber, and in one embodiment, a plunger is carried by the cartridge for occluding fluidic communication to the reaction chambers. The device also includes a heating apparatus having a heating element whichmore » is activated by controller to generate heat when a trigger event is detected. The heating apparatus includes a cartridge-mounting section which positioned a cartridge in thermal communication with the heating element so that visual changes to the contents of the reaction chambers are viewable through the view ports.« less

Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil.

PubMed

Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

2017-04-15

The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil. Copyright © 2016 Elsevier Ltd. All rights reserved.

Natural single amino acid polymorphism (F19Y) in human galectin-8: detection of structural alterations and increased growth-regulatory activity on tumor cells.

PubMed

Ruiz, Federico M; Scholz, Barbara A; Buzamet, Eliza; Kopitz, Jürgen; André, Sabine; Menéndez, Margarita; Romero, Antonio; Solís, Dolores; Gabius, Hans-Joachim

2014-03-01

Natural amino acid substitution by single-site nucleotide polymorphism can become a valuable tool for structure-activity correlations, especially if evidence for association to disease parameters exists. Focusing on the F19Y change in human galectin-8, connected clinically to rheumatoid arthritis, we here initiate the study of consequences of a single-site substitution in the carbohydrate recognition domain of this family of cellular effectors. We apply a strategically combined set of structural and cell biological techniques for comparing properties of the wild-type and variant proteins. The overall hydrodynamic behavior of the full-length protein and of the separate N-domain is not noticeably altered, but displacements in the F0 β-strand of the β-sandwich fold in the N-domain are induced, as evidenced by protein crystallography. Analysis of thermal stability by circular dichroism spectroscopy revealed perceptible differences for the full-length proteins, pointing to an impact of the substitution beyond the N-domain. In addition, small differences in thermodynamic parameters of carbohydrate binding are detected. On the level of two types of tumor cells, characteristics of binding appeared rather similar. In further comparison of the influence on proliferation, the variant proved to be more active as growth regulator in the six tested lines of neuroblastoma, erythroleukemia and colon adenocarcinoma. The seemingly subtle structural change identified here thus has functional implications in vitro, encouraging further analysis in autoimmune regulation and, in a broad context, in work with other natural single-site variants, using the documented combined strategy. The atomic coordinates and structure factors (codes 4BMB, 4BME) have been deposited in the Protein Data Bank. © 2014 FEBS.

Micro-Detection System for Determination of the Biotic or Abiotic Origin of Amino Acids

NASA Technical Reports Server (NTRS)

Bada, Jeffrey L.

2003-01-01

The research carried out under this PIDDP involves the development of a breadboard version of a spacecraft-based system for the detection of amino acid chirality (molecular handedness) on solar system bodies. Chirality provides an unambiguous way of distinguishing between abiotic and biotic origins since only one mirror-image form is used in the functional molecules of life. Recent advances in a variety of nano-fabrication technologies have resulted in concepts for enabling miniaturized chemical and biological analytical systems. These are complete application-specific systems that integrate fluid micro handling systems for extracting and reacting target molecules, micro-separation technologies for enhanced sensitivity and resolution, and advanced detection technologies. This effort makes use of a relatively new technology that shows demonstrated promise for spacecraft-based amino acid analysis: microchip-based capillary electrophoresis (muCE). The muCE system is capable of analyzing the type of amino acids present as well as the relative amounts of their mirror image forms. The system we developed will be able to chirally resolve all of the major amino acids found in extraterrestrial material (Gly, Ala, Val, Pro, Asp, Glu, a-aminoisobutyric acid, and isovaline) at sub-part-per-billion levels. The _CE analysis requires that the amino acids be extracted from the sample and derivatized for either optical or electrochemical detection. In our implementation, the amino acids are released from the sample by sublimation and prepared for muCE analysis using a microfluidic circuit. In addition, we have investigated the use of a microfluidic circuit for the release of amino acids from samples in which sublimation has proven to be problematic.

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons.

PubMed

Vollenhofer-Schrumpf, Sabine; Buresch, Ronald; Schinkinger, Manfred

2007-03-01

We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.

Simultaneous determination of low-molecular-weight organic acids and chlorinated acid herbicides in environmental water by a portable CE system with contactless conductivity detection.

PubMed

Xu, Yan; Wang, Weilong; Li, Sam Fong Yau

2007-05-01

This report describes a method to simultaneously determine 11 low-molecular-weight (LMW) organic acids and 16 chlorinated acid herbicides within a single run by a portable CE system with contactless conductivity detection (CCD) in a poly(vinyl alcohol) (PVA)-coated capillary. Under the optimized condition, the LODs of CE-CCD ranged from 0.056 to 0.270 ppm, which were better than for indirect UV (IUV) detection of the 11 LMW organic acids or UV detection of the 16 chlorinated acid herbicides. Combined with an on-line field-amplified sample stacking (FASS) procedure, sensitivity enhancement of 632- to 1078-fold was achieved, with satisfactory reproducibility (RSDs of migration times less than 2.2%, and RSDs of peak areas less than 5.1%). The FASS-CE-CCD method was successfully applied to determine the two groups of acidic pollutants in two kinds of environmental water samples. The portable CE-CCD system shows advantages such as simplicity, cost effectiveness, and miniaturization. Therefore, the method presented in this report has great potential for onsite analysis of various pollutants at the trace level.

Hydroxycinnamic acid bound arabinoxylans from millet brans-structural features and antioxidant activity.

PubMed

Bijalwan, Vandana; Ali, Usman; Kesarwani, Atul Kumar; Yadav, Kamalendra; Mazumder, Koushik

2016-07-01

Hydroxycinnamic acid bound arabinoxylans (HCA-AXs) were extracted from brans of five Indian millet varieties and response surface methodology was used to optimize the extraction conditions. The optimal condition to obtain highest yield of millet HCA-AXs was determined as follows: time 61min, temperature 66°C, ratio of solvent to sample 12ml/g. Linkage analysis indicated that hydroxycinnamic acid bound arabinoxylan from kodo millet (KM-HCA-AX) contained comparatively low branched arabinoxylan consisting of 14.6% mono-substituted, 1.2% di-substituted and 41.2% un-substituted Xylp residues. The HPLC analysis of millet HCA-AXs showed significant variation in the content of three major bound hydroxycinnamic acids (caffeic, p-coumaric and ferulic acid). The antioxidant activity of millet HCA-AXs were evaluated using three in vitro assay methods (DPPH, FRAP and β-carotene linoleate emulsion assays) which suggested both phenolic acid composition and structural characteristics of arabinoxylans could be correlated to their antioxidant potential, the detailed structural analysis revealed that low substituted KM-HCA-AX exhibited relatively higher antioxidant activity compared to other medium and highly substituted HCA-AXs from finger (FM), proso (PM), barnyard (BM) and foxtail (FOXM) millet. Copyright © 2016. Published by Elsevier B.V.

Carbon nanotube-based labels for highly sensitive colorimetric and aggregation-based visual detection of nucleic acids

NASA Astrophysics Data System (ADS)

Lee, Ai Cheng; Ye, Jian-Shan; Ngin Tan, Swee; Poenar, Daniel P.; Sheu, Fwu-Shan; Kiat Heng, Chew; Meng Lim, Tit

2007-11-01

A novel carbon nanotube (CNT) derived label capable of dramatic signal amplification of nucleic acid detection and direct visual detection of target hybridization has been developed. Highly sensitive colorimetric detection of human acute lymphocytic leukemia (ALL) related oncogene sequences amplified by the novel CNT-based label was demonstrated. Atomic force microscope (AFM) images confirmed that a monolayer of horseradish peroxidase and detection probe molecules was immobilized along the carboxylated CNT carrier. The resulting CNT labels significantly enhanced the nucleic acid assay sensitivity by at least 1000 times compared to that of conventional labels used in enzyme-linked oligosorbent assay (ELOSA). An excellent detection limit of 1 × 10-12 M (60 × 10-18 mol in 60 µl) and a four-order wide dynamic range of target concentration were achieved. Hybridizations using these labels were coupled to a concentration-dependent formation of visible dark aggregates. Targets can thus be detected simply with visual inspection, eliminating the need for expensive and sophisticated detection systems. The approach holds promise for ultrasensitive and low cost visual inspection and colorimetric nucleic acid detection in point-of-care and early disease diagnostic application.

Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection

PubMed Central

Mojarro, Angel; Ruvkun, Gary; Zuber, Maria T.

2017-01-01

Abstract Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry–dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a “universal” nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments—Nucleic acids—Mars—Panspermia. Astrobiology 17, 747–760. PMID:28704064

Implantation of silicon dioxide-based nanocrystalline hydroxyapatite and pure phase beta-tricalciumphosphate bone substitute granules in caprine muscle tissue does not induce new bone formation

PubMed Central

2013-01-01