Purification and characteristics of a novel bacteriocin produced by Enterococcus faecalis L11 isolated from Chinese traditional fermented cucumber.

PubMed

Gao, Yurong; Li, Benling; Li, Dapeng; Zhang, Liyuan

2016-05-01

To purify and characterize a novel bacteriocin with broad inhibitory spectrum produced by an isolate of Enterococcus faecalis from Chinese fermented cucumber. E. faecalis L11 produced a bacteriocin with antimicrobial activity against both Escherichia coli and Staphylococcus aureus. The amino acid sequence of the purified bacteriocin, enterocin L11, was assayed by Edman degradation method. It differs from other class II bacteriocins and exhibited a broad antimicrobial activity against not only Gram-positive bacteria, including Bacillus subtilis, S. aureus, Listeria monocytogenes, Sarcina flava, Lactobacillus acidophilus, L. plantarum, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, but also some Gram-negative bacteria including Salmonella typhimurium, E. coli and Shigella flexneri. Enterocin L11 retained 91 % of its activity after holding at 121 °C for 30 min. It was also resistant to acids and alkalis. Enterocin L11 is a novel broad-spectrum Class II bacteriocin produced by E. faecalis L11, and may have potential as a food biopreservative.

A proteomic analysis of leaf sheaths from rice.

PubMed

Shen, Shihua; Matsubae, Masami; Takao, Toshifumi; Tanaka, Naoki; Komatsu, Setsuko

2002-10-01

The proteins extracted from the leaf sheaths of rice seedlings were separated by 2-D PAGE, and analyzed by Edman sequencing and mass spectrometry, followed by database searching. Image analysis revealed 352 protein spots on 2-D PAGE after staining with Coomassie Brilliant Blue. The amino acid sequences of 44 of 84 proteins were determined; for 31 of these proteins, a clear function could be assigned, whereas for 12 proteins, no function could be assigned. Forty proteins did not yield amino acid sequence information, because they were N-terminally blocked, or the obtained sequences were too short and/or did not give unambiguous results. Fifty-nine proteins were analyzed by mass spectrometry; all of these proteins were identified by matching to the protein database. The amino acid sequences of 19 of 27 proteins analyzed by mass spectrometry were similar to the results of Edman sequencing. These results suggest that 2-D PAGE combined with Edman sequencing and mass spectrometry analysis can be effectively used to identify plant proteins.

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812.

PubMed

Chen, Yi-Sheng; Yu, Chi-Rong; Ji, Si-Hua; Liou, Min-Shiuan; Leong, Kun-Hon; Pan, Shwu-Fen; Wu, Hui-Chung; Lin, Yu-Hsuan; Yu, Bi; Yanagida, Fujitoshi

2013-09-01

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.

Biomonitoring of carcinogenic substances: enzymatic digestion of globin for detecting alkylated amino acids

NASA Astrophysics Data System (ADS)

Bader, Michael; Rauscher, Dankwart; Geibel, Kurt; Angerer, Juergen

1993-03-01

We report the application of proteases for the total hydrolysis of globin with subsequent determination of amino acids. Optimization of the proteolysis was made with respect to enzyme concentration, time of incubation and type of protease. Ethylene oxide modified globin was used to compare the results of the analysis of the N-terminal amino acid valine after enzymatic cleavage to those obtained from the widely used modified Edman procedure. It is shown that the cleavage is of good reproducibility and yields more alkylated amino acid than the Edman procedure.

New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

PubMed Central

Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

2015-01-01

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation. PMID:26404319

Primary structure of a guanyl-specific ribonuclease from the fungus Penicillium brevicompactum

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

Kulikov, V.A.; Shlyapnikov, S.V.; Yakovlev, G.I.

1986-01-01

By the automatic Edman degradation of the intact S-carboxymethylated protein and a mixture of the products of its proteolytic cleavage at Arg, Lys, and Glu residues, together with results on the kinetics of the proteolysis of the protein under the action of carboxypeptidase Y, the primary structure of the extracellular guanyl-specific RNase of the fungus Penicillium brevicompactum has been determined. The RNase contains 102 amino acid residues: 7 Asp, 7 Asn, 9 Thr, 11 Ser, 4 Glu, 1 Gln, 4 Pro, 10 Gly, 11 Ala, 4 Cys, 7 Val, 4 Ile, 3 Leu, 9 Tyr, 5 Phe, 2 Lys, 3more » His, 1 Arg (M/sub r/ 10,801). It has been established that four hemicystine residues of the P. compactum RNase form, in pairs, two disulfide bonds« less

Identification of a major continuous epitope of human alpha crystallin

NASA Technical Reports Server (NTRS)

Takemoto, L.; Emmons, T.; Spooner, B. S. (Principal Investigator)

1992-01-01

Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.

Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese▿

PubMed Central

Izquierdo, Esther; Wagner, Camille; Marchioni, Eric; Aoude-Werner, Dalal; Ennahar, Saïd

2009-01-01

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains. PMID:19411428

Enterocin 96, a novel class II bacteriocin produced by Enterococcus faecalis WHE 96, isolated from Munster cheese.

PubMed

Izquierdo, Esther; Wagner, Camille; Marchioni, Eric; Aoude-Werner, Dalal; Ennahar, Saïd

2009-07-01

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.

Biomimetic Synthesis of Macahydantoins A and B from Lepidium meyenii, and Structure Revision of Macahydantoin B as a Class of Thiohydantoin with a 4-Methyl-hexahydropyrrolo[1,2-c]imidazole Skeleton.

PubMed

Zhou, Min; Ma, Hang-Ying; Xing, Huan-Huan; Li, Ping; Li, Gan-Peng; Geng, Hui-Chun; Hu, Qiu-Fen; Yang, Guang-Yu

2017-09-15

Phytochemical investigation on Lepidium meyenii led to the discovery of macahydantoin C (3), a new thiohydantoin with a 1,3-diazabicyclo[3.3.1]nonane core, the spectral properties of which indicate a potential structural misassignment of its previously reported analogue, macahydantoin B (2a). To probe this hypothesis, a concise, scalable, and biomimetic synthesis of the originally proposed 2a and its revised structure (2b) was efficiently accomplished using the modified Edman degradation as the key step from commercially available materials in 65% (three steps) and 52% (three steps) overall yields, respectively. These synthetic endeavors undoubtedly reassigned the structure of macahydantoin B as an unreported type of thiohydantoin featuring a 4-methyl-hexahydropyrrolo[1,2-c]imidazole scaffold.

An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

PubMed Central

Rose, K; Kocher, H P; Blumberg, B M; Kolakofsky, D

1984-01-01

A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure. PMID:6421284

Protein Sequencing with Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Ziady, Assem G.; Kinter, Michael

The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

PubMed

Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad; Schaefer, Jacob

2017-03-14

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [ 19 F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides

NASA Astrophysics Data System (ADS)

Zhokhov, Sergey S.; Kovalyov, Sergey V.; Samgina, Tatiana Yu.; Lebedev, Albert T.

2017-08-01

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination.

Shotgun protein sequencing: assembly of peptide tandem mass spectra from mixtures of modified proteins.

PubMed

Bandeira, Nuno; Clauser, Karl R; Pevzner, Pavel A

2007-07-01

Despite significant advances in the identification of known proteins, the analysis of unknown proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for sequencing of unknown proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA sequencing strategies have not been extended to automated protein sequencing. We demonstrate, for the first time, the feasibility of automated shotgun protein sequencing of protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various proteins in western diamondback rattlesnake venom. We further argue that shotgun protein sequencing has the potential to overcome the limitations of current protein sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown proteins.

Isolation, characterization, and primary structure of rubredoxin from the photosynthetic bacterium, Heliobacillus mobilis

NASA Technical Reports Server (NTRS)

Lee, W. Y.; Brune, D. C.; LoBrutto, R.; Blankenship, R. E.

1995-01-01

Rubredoxin is a small nonheme iron protein that serves as an electron carrier in bacterial systems. Rubredoxin has now been isolated and characterized from the strictly anaerobic phototroph, Heliobacillus mobilis. THe molecular mass (5671.3 Da from the amino acid sequence) was confirmed and partial formylation of the N-terminal methionyl residue was established by matrix-assisted laser desorption mass spectroscopy. The complete 52-amino-acid sequence was determined by a combination of N-terminal sequencing by Edman degradation and C-terminal sequencing by a novel method using carboxypeptidase treatment in conjunction with amino acid analysis and laser desorption time of flight mass spectrometry. The molar absorption coefficient of Hc. mobilis rubredoxin at 490 nm is 6.9 mM-1 cm-1 and the midpoint redox potential at pH 8.0 is -46 mV. The EPR spectrum of the oxidized form shows resonances at g = 9.66 and 4.30 due to a high-spin ferric iron. The amino acid sequence is homologous to those of rubredoxins from other species, in particular, the gram-positive bacteria, and the phototrophic green sulfur bacteria, and the evolutionary implications of this are discussed.

Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli.

PubMed

Szewczak, Joanna; Bierczyńska-Krzysik, Anna; Piejko, Marcin; Mak, Paweł; Stadnik, Dorota

2015-07-01

Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro--one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro.

Personal Protective Measures Against Insects and Other Arthropods of Military Significance

DTIC Science & Technology

2009-10-01

responsibility, although it is also an important adjunct to unit- level and higher echelon preventive medicine countermeasures. Military personnel must be aware...fabric treatment level of 0.52% weight by weight of active ingredient. (d) Several steps are essential in properly using this treatment method...Control as a Force Multiplier. Defense 90. pp. 26-35. Elridge, B.F. and Edman, J.D. 2004. Medical Entomology: A Textbook on Public Health and

Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor.

PubMed Central

Daly, J W; Caceres, J; Moni, R W; Gusovsky, F; Moos, M; Seamon, K B; Milton, K; Myers, C W

1992-01-01

A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans. Images PMID:1438301

Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor.

PubMed

Daly, J W; Caceres, J; Moni, R W; Gusovsky, F; Moos, M; Seamon, K B; Milton, K; Myers, C W

1992-11-15

A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans.

Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

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

Semler, B.L.; Hanecak, R.; Dorner, L.F.

1983-01-01

The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage ofmore » the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.« less

Antifungal mechanism of a novel antifungal protein from pumpkin rinds against various fungal pathogens.

PubMed

Park, Seong-Cheol; Kim, Jin-Young; Lee, Jong-Kook; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

2009-10-14

A novel antifungal protein (Pr-2) was identified from pumpkin rinds using water-soluble extraction, ultrafiltration, cation exchange chromatography, and reverse-phase high-performance liquid chromatography. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry indicated that the protein had a molecular mass of 14865.57 Da. Automated Edman degradation showed that the N-terminal sequence of Pr-2 was QGIGVGDNDGKRGKR-. The Pr-2 protein strongly inhibited in vitro growth of Botrytis cinerea, Colletotrichum coccodes, Fusarium solani, Fusarium oxysporum, and Trichoderma harzianum at 10-20 microM. The results of confocal laser scanning microscopy and SYTOX Green uptake demonstrated that its effective region was the membrane of the fungal cell surface. In addition, this protein was found to be noncytotoxic and heat-stable. Taken together, the results of this study indicate that Pr-2 is a good candidate for use as a natural antifungal agent.

Characterization of the major cyanogen bromide fragment of alpha-A crystallin

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Alpha crystallin from the bovine lens has been digested with cyanogen bromide, and the major fragment (CB-1) has been purified using reverse phase HPLC. Characterization of this fragment by Edman degradation and antisera to synthetic peptides indicates that it originates from alpha-A crystallin, but lacks the N-terminal methionine and the last 35 amino acids from the C-terminus of the molecule. The purified CB-1 fragment binds as well as native alpha crystallin to lens membrane, but is unable to self-assemble into the correct size of high molecular weight oligomeric complexes characteristic of the intact alpha-A chain. Together, these results demonstrate that the alpha-A chain is comprised of at least two functional domains, one of which is involved in binding of alpha-A crystallin to lens membrane, and another which is necessary for correct self-assembly of the molecule into high molecular weight oligomers.

De novo protein sequencing by combining top-down and bottom-up tandem mass spectra.

PubMed

Liu, Xiaowen; Dekker, Lennard J M; Wu, Si; Vanduijn, Martijn M; Luider, Theo M; Tolić, Nikola; Kou, Qiang; Dvorkin, Mikhail; Alexandrova, Sonya; Vyatkina, Kira; Paša-Tolić, Ljiljana; Pevzner, Pavel A

2014-07-03

There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

Purification and Molecular Characterization of the Novel Highly Potent Bacteriocin TSU4 Produced by Lactobacillus animalis TSU4.

PubMed

Sahoo, Tapasa Kumar; Jena, Prasant Kumar; Patel, Amiya Kumar; Seshadri, Sriram

2015-09-01

Bacterial infections causing fish diseases and spoilage during fish food processing and storage are major concerns in aquaculture. Use of bacteriocins has recently been considered as an effective strategy for prevention of bacterial infections. A novel bacteriocin produced by Catla catla gut isolates, Lactobacillus animalis TSU4, designated as bacteriocin TSU4 was purified to homogeneity by a three-step protocol. The molecular mass of bacteriocin TSU4 was 4117 Da determined by Q-TOF LC/MS analysis. Its isoelectric point was ~9. Secondary conformation obtained by circular dichroism spectroscopy showed molecular conformation with significant proportions of the structure in α-helix (23.7 %) and β-sheets (17.1 %). N-terminal sequencing was carried out by the Edman degradation method; partial sequence identified was NH2-SMSGFSKPHD. Bacteriocin TSU4 exhibited a wide range of antimicrobial activity, pH and thermal stability. It showed a bacteriocidal mode of action against the indicator strain Aeromonas hydrophila MTCC 646. Bacteriocin TSU4 is the first reported bacteriocin produced by fish isolate Lactobacillus animalis. The characterization of bacteriocin TSU4 suggested that it is a novel bacteriocin with potential value against infections of bacteria such as A. hydrophila MTCC 646 and Pseudomonas aeruginosa MTCC 1688 and application to prevent spoilage during food preservation.

The tick plasma lectin, Dorin M, is a fibrinogen-related molecule.

PubMed

Rego, Ryan O M; Kovár, Vojtĕch; Kopácek, Petr; Weise, Christoph; Man, Petr; Sauman, Ivo; Grubhoffer, Libor

2006-04-01

A lectin, named Dorin M, previously isolated and characterized from the hemolymph plasma of the soft tick, Ornithodoros moubata, was cloned and sequenced. The immunofluorescence using confocal microscopy revealed that Dorin M is produced in the tick hemocytes. A tryptic cleavage of Dorin M was performed and the resulting peptide fragments were sequenced by Edman degradation and/or mass spectrometry. Two of three internal peptide sequences displayed a significant similarity to the family of fibrinogen-related molecules. Degenerate primers were designed and used for PCR with hemocyte cDNA as a template. The sequence of the whole Dorin M cDNA was completed by the method of RACE. The tissue-specific expression investigated by RT-PCR revealed that Dorin M, in addition to hemocytes, is significantly expressed in salivary glands. The derived amino-acid sequence clearly shows that Dorin M has a fibrinogen-like domain, and exhibited the most significant similarity with tachylectins 5A and 5B from a horseshoe crab, Tachypleus tridentatus. In addition, other protein and binding characteristics suggest that Dorin M is closely related to tachylectins-5. Since these lectins have been reported to function as non-self recognizing molecules, we believe that Dorin M may play a similar role in an innate immunity of the tick and, possibly, also in pathogen transmission by this vector.

R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography.

PubMed

Toyo'oka, T; Jin, D; Tomoi, N; Oe, T; Hiranuma, H

2001-02-01

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.

Glycosaminoglycan Chain of Dentin Sialoprotein Proteoglycan

PubMed Central

Zhu, Q.; Sun, Y.; Prasad, M.; Wang, X.; Yamoah, A.K.; Li, Y.; Feng, J.; Qin, C.

2010-01-01

Dentin sialophosphoprotein (DSPP) is processed into dentin sialoprotein (DSP) and dentin phosphoprotein. A molecular variant of rat DSP, referred to as “HMW-DSP”, has been speculated to be a proteoglycan form of DSP. To determine if HMW-DSP is the proteoglycan form of DSP and to identify the glycosaminoglycan side-chain attachment site(s), we further characterized HMW-DSP. Chondroitinase ABC treatment reduced the migration rate for portions of rat HMW-DSP to the level of DSP. Disaccharide analysis showed that rat HMW-DSP contains glycosaminoglycan chains made of chondroitin-4-sulfate and has an average of 31-32 disaccharides/mol. These observations confirmed that HMW-DSP is the proteoglycan form of DSP (renamed “DSP-PG”). Edman degradation and mass spectrometric analyses of tryptic peptides from rat DSP-PG, along with substitution analyses of candidate Ser residues in mouse DSPP, confirmed that 2 glycosaminoglycan chains are attached to Ser241 and Ser253 in the rat, or Ser242 and Ser254 in the mouse DSPP sequence. PMID:20400719

Glycosaminoglycan chain of dentin sialoprotein proteoglycan.

PubMed

Zhu, Q; Sun, Y; Prasad, M; Wang, X; Yamoah, A K; Li, Y; Feng, J; Qin, C

2010-08-01

Dentin sialophosphoprotein (DSPP) is processed into dentin sialoprotein (DSP) and dentin phosphoprotein. A molecular variant of rat DSP, referred to as "HMW-DSP", has been speculated to be a proteoglycan form of DSP. To determine if HMW-DSP is the proteoglycan form of DSP and to identify the glycosaminoglycan side-chain attachment site(s), we further characterized HMW-DSP. Chondroitinase ABC treatment reduced the migration rate for portions of rat HMW-DSP to the level of DSP. Disaccharide analysis showed that rat HMW-DSP contains glycosaminoglycan chains made of chondroitin-4-sulfate and has an average of 31-32 disaccharides/mol. These observations confirmed that HMW-DSP is the proteoglycan form of DSP (renamed "DSP-PG"). Edman degradation and mass spectrometric analyses of tryptic peptides from rat DSP-PG, along with substitution analyses of candidate Ser residues in mouse DSPP, confirmed that 2 glycosaminoglycan chains are attached to Ser(241) and Ser(253) in the rat, or Ser(242) and Ser(254) in the mouse DSPP sequence.

Probing the reactivity of nucleophile residues in human 2,3-diphosphoglycerate/deoxy-hemoglobin complex by aspecific chemical modifications.

PubMed

Scaloni, A; Ferranti, P; De Simone, G; Mamone, G; Sannolo, N; Malorni, A

1999-06-11

The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS-Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pKa and the relative surface accessibility of the nucleophilic residues, calculated from the X-ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.

Isolation and characterization of a new bacteriocin, termed enterocin M, produced by environmental isolate Enterococcus faecium AL41.

PubMed

Mareková, Mária; Lauková, Andrea; Skaugen, Morten; Nes, Ingolf

2007-08-01

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH4)2SO4 precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies

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

Peters, J.; Peters, M.; Lottspeich, F.

1987-11-01

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate (HPI))-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and M/sub r/ estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%)more » of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.« less

Structural characterization of osmoregulator peptides from the brain of the leech Theromyzon tessulatum: IPEPYVWD and IPEPYVWD-amide.

PubMed

Salzet, M; Vandenbulcke, F; Verger-Bocquet, M

1996-12-31

Neurons immunoreactive to an antiserum (a-OT) directed specifically against the C-terminal part (prolyl-leucyl-glycinamide) of vertebrate oxytocin (OT) were detected in the brain of the leech Theromyzon tessulatum. With high pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts, evidence was given of the presence of three peptides (P1, P2, P3) immunoreactive to a-OT. Results of injection experiments in T. tessulatum and of titrations of each peptide at the different physiological stages of the animals which showed a peak in peptide P1 amount at stage 3B, indicated that P1 is the active OT-like peptide. Using three steps of reversed-phase HPLC, Edman degradation and electrospray mass spectrometry, two sequences for P1 (IPEPYVWD and IPEPYVWD-amide) were found. These peptides differ from peptides to the oxytocin/vasopressin family and are unique in the animal kingdom. Confirmation of their action on the hydric balance and their distribution in the CNS were presented.

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

Kem, W.; Dunn, B.; Parten, B.

A 5000 dalton polypeptide neurotoxin (Sh-NI) purified by G50 Sephadex, P-cellulose, and SP-Sephadex chromatography was homogeneous by isoelectric focusing. Sh-NI was highly toxic to crayfish (LD/sub 50/ 0.6 ..mu..g/kg) but without effect upon mice at 15,000 ..mu..g/kg (i.p. injection). The reduced, /sup 3/H-carboxymethylated toxin and its fragments were subjected to automatic Edman degradation and the resulting PTH-amino acids were identified by HPLC, back hydrolysis, and scintillation counting. Peptides resulting from proteolytic (clostripain, staphylococcal protease) and chemical (tryptophan) cleavage were sequenced. The sequence is: AACKCDDEGPDIRTAPLTGTVDLGSCNAGWEKCASYYTIIADCCRKKK. This sequence differs considerably from the homologous Anemonia and Anthopleura toxins; many of the identical residuesmore » (6 half-cystines, G9, P10, R13, G19, G29, W30) are probably critical for folding rather than receptor recognition. However, the Sh-NI sequence closely resembles Radioanthus macrodactylus neurotoxin III and r. paumotensis II. The authors propose that Sh-NI and related Radioanthus toxins act upon a different site on the sodium channel.« less

Structural characterization of thioether-bridged bacteriocins.

PubMed

Lohans, Christopher T; Vederas, John C

2014-01-01

Bacteriocins are a group of ribosomally synthesized antimicrobial peptides produced by bacteria, some of which are extensively post-translationally modified. Some bacteriocins, namely the lantibiotics and sactibiotics, contain one or more thioether bridges. However, these modifications complicate the structural elucidation of these bacteriocins using conventional techniques. This review will discuss the techniques and strategies that have been applied to determine the primary structures of lantibiotics and sactibiotics. A major challenge is to identify the topology of thioether bridges in these peptides (i.e., which amino-acid residues are involved in which bridges). Edman degradation, NMR spectroscopy and tandem MS have all been commonly applied to characterize these bacteriocins, but can be incompatible with the post-translational modifications present. Chemical modifications to the modified residues, such as desulfurization and reduction, make the treated bacteriocins more compatible to analysis by these standard peptide analytical techniques. Despite their differences in structure, similar strategies have proved useful to study the structures of both lantibiotics and sactibiotics.

The amino acid sequences of carboxypeptidases I and II from Aspergillus niger and their stability in the presence of divalent cations.

PubMed

Svendsen, I; Dal Degan, F

1998-09-08

The amino acid sequences of serine carboxypeptidase I (CPD-I) and II (CPD-II), respectively, from Aspergillus niger have been determined by conventional Edman degradation of the reduced and vinylpyridinated enzymes and peptides hereof generated by cleavage with cyanogen bromide, iodobenzoic acid, glutamic acid cleaving enzyme, AspN-endoproteinase and EndoLysC proteinase. CPD-I consists of a single peptide chain of 471 amino acid residues, three disulfide bridges and nine N-glycosylated asparaginyl residues, while CPD-II consists of a single peptide chain of 481 amino acid residues, has three disulfide bridges, one free cysteinyl residue and nine glycosylated asparaginyl residues. The enzymes are closely related to carboxypeptidase S3 from Penicillium janthinellum. Both Ca2+ and Mg2+ stabilize CPD-I as well as CPD-II, at basic pH values, Ca2+ being most effective, while the divalent ions have no effect on the activity of the two enzymes.

[The primary structure of the alpha-amylase inhibitor Hoe 467A from Streptomyces tendae 4158. A new class of inhibitors].

PubMed

Aschauer, H; Vértesy, L; Nesemann, G; Braunitzer, G

1983-10-01

The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.

Porifera Lectins: Diversity, Physiological Roles and Biotechnological Potential.

PubMed

Gardères, Johan; Bourguet-Kondracki, Marie-Lise; Hamer, Bojan; Batel, Renato; Schröder, Heinz C; Müller, Werner E G

2015-08-07

An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest.

A Low Molecular Weight Protein from the Sea Anemone Anemonia viridis with an Anti-Angiogenic Activity

PubMed Central

Loret, Erwann P.; Luis, José; Nuccio, Christopher; Villard, Claude; Mansuelle, Pascal; Lebrun, Régine; Villard, Pierre Henri

2018-01-01

Sea anemones are a remarkable source of active principles due to a decentralized venom system. New blood vessel growth or angiogenesis is a very promising target against cancer, but the few available antiangiogenic compounds have limited efficacy. In this study, a protein fraction, purified from tentacles of Anemonia viridis, was able to limit endothelial cells proliferation and angiogenesis at low concentration (14 nM). Protein sequences were determined with Edman degradation and mass spectrometry in source decay and revealed homologies with Blood Depressing Substance (BDS) sea anemones. The presence of a two-turn alpha helix observed with circular dichroism and a trypsin activity inhibition suggested that the active principle could be a Kunitz-type inhibitor, which may interact with an integrin due to an Arginine Glycin Aspartate (RGD) motif. Molecular modeling showed that this RGD motif was well exposed to solvent. This active principle could improve antiangiogenic therapy from existing antiangiogenic compounds binding on the Vascular Endothelial Growth Factor (VEGF). PMID:29671760

Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan.

PubMed

Fujita, M; Nomura, K; Hong, K; Ito, Y; Asada, A; Nishimuro, S

1993-12-30

A strong fibrinolytic enzyme (nattokinase) was purified from the vegetable cheese natto. Nattokinase was extracted from natto with saline and isolated by sequential use of hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on CM-Toyopearl, and gel-filtration on Sephadex G-50. The isolated protein gave a single sharp band on SDS-PAGE either before or after reduction. The sequence, as determined by automated Edman degradation of the uncleaved molecule and its enzymatically derived peptide, consisted of a total 275 amino acid residues (M.W = 27,728) and exhibited a high homology with the subtilisins. The purified nattokinase digested not only fibrin but also several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. PMSF inhibited both the fibrinolytic activity and the amidolytic activity. The results indicate that nattokinase is a subtilisin-like serine protease.

Porifera Lectins: Diversity, Physiological Roles and Biotechnological Potential

PubMed Central

Gardères, Johan; Bourguet-Kondracki, Marie-Lise; Hamer, Bojan; Batel, Renato; Schröder, Heinz C.; Müller, Werner E. G.

2015-01-01

An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest. PMID:26262628

Unraveling the sequence and structure of the protein osteocalcin from a 42 ka fossil horse

NASA Astrophysics Data System (ADS)

Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Andrews, Philip C.; Leykam, Joseph; Stafford, Thomas W.; Kelly, Robert L.; Walker, Danny N.; Buckley, Mike; Humpula, James

2006-04-01

We report the first complete amino acid sequence and evidence of secondary structure for osteocalcin from a temperate fossil. The osteocalcin derives from a 42 ka equid bone excavated from Juniper Cave, Wyoming. Results were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) and Edman sequencing with independent confirmation of the sequence in two laboratories. The ancient sequence was compared to that of three modern taxa: horse ( Equus caballus), zebra ( Equus grevyi), and donkey ( Equus asinus). Although there was no difference in sequence among modern taxa, MALDI-MS and Edman sequencing show that residues 48 and 49 of our modern horse are Thr, Ala rather than Pro, Val as previously reported (Carstanjen B., Wattiez, R., Armory, H., Lepage, O.M., Remy, B., 2002. Isolation and characterization of equine osteocalcin. Ann. Med. Vet.146(1), 31-38). MALDI-MS and Edman sequencing data indicate that the osteocalcin sequence of the 42 ka fossil is similar to that of modern horse. Previously inaccessible structural attributes for ancient osteocalcin were observed. Glu 39 rather than Gln 39 is consistent with deamidation, a process known to occur during fossilization and aging. Two post-translational modifications were documented: Hyp 9 and a disulfide bridge. The latter suggests at least partial retention of secondary structure. As has been done for ancient DNA research, we recommend standards for preparation and criteria for authenticating results of ancient protein sequencing.

Ligatoxin B, a new cytotoxic protein with a novel helix-turn-helix DNA-binding domain from the mistletoe Phoradendron liga.

PubMed Central

Li, Shi-Sheng; Gullbo, Joachim; Lindholm, Petra; Larsson, Rolf; Thunberg, Eva; Samuelsson, Gunnar; Bohlin, Lars; Claeson, Per

2002-01-01

A new basic protein, designated ligatoxin B, containing 46 amino acid residues has been isolated from the mistletoe Phoradendron liga (Gill.) Eichl. (Viscaceae). The protein's primary structure, determined unambiguously using a combination of automated Edman degradation, trypsin enzymic digestion, and tandem MS analysis, was 1-KSCCPSTTAR-NIYNTCRLTG-ASRSVCASLS-GCKIISGSTC-DSGWNH-46. Ligatoxin B exhibited in vitro cytotoxic activities on the human lymphoma cell line U-937-GTB and the primary multidrug-resistant renal adenocarcinoma cell line ACHN, with IC50 values of 1.8 microM and 3.2 microM respectively. Sequence alignment with other thionins identified a new member of the class 3 thionins, ligatoxin B, which is similar to the earlier described ligatoxin A. As predicted by the method of homology modelling, ligatoxin B shares a three-dimensional structure with the viscotoxins and purothionins and so may have the same mode of cytotoxic action. The novel similarities observed by structural comparison of the helix-turn-helix (HTH) motifs of the thionins, including ligatoxin B, and the HTH DNA-binding proteins, led us to propose the working hypothesis that thionins represent a new group of DNA-binding proteins. This working hypothesis could be useful in further dissecting the molecular mechanisms of thionin cytotoxicity and of thionin opposition to multidrug resistance, and useful in clarifying the physiological function of thionins in plants. PMID:12049612

Characterization of a Cadmium-Binding Complex of Cabbage Leaves 1

PubMed Central

Wagner, George J.

1984-01-01

The chemical nature of a principal, inducible cadmium-binding complex which accumulates in cabbage leaves (Wagner and Trotter 1982 Plant Physiol 69: 804-809) was studied and compared with that of animal metallothionein and copper-binding proteins isolated from various organisms. The apparent molecular weight of native cabbage complex and carboxymethylated ligand of the complex under native conditions as determined by gel filtration was about 10,000 daltons. Under denaturing conditions their apparent molecular weights were about 2000 daltons. Ligand of native complex contained 37, 28, and 9 residue per cent of glutamic acid-glutamine, cysteine, and glycine, respectively, and low aromatic residue, serine and lysine content. The high acidic and low hydrophobic residue content explain the behavior of complex on electrophoresis in the presence and absence of sodium dodecyl sulfate. Its isoelectric point was below 4.0 and it bound 4 to 6 moles cadmium per mole ligand in what appear to be cadmium-mercaptide chromophores. The complex was found to be heat stable, relatively protease insensitive, and lacking in disulfide bonds. Attempts to determine the primary sequence of reduced native complex and carboxymethylated, cleaved ligand using the Edman degradation procedure were unsuccessful. An electrophoretic procedure is described for preparative isolation of purified complex and a method is described for monitoring ligand of complex as its fluorescent dibromobimane adduct. Images Fig. 1 Fig. 3 PMID:16663927

Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing.

PubMed

Odintsova, Tatyana I; Müller, Eva-Christina; Ivanov, Anton V; Egorov, Tsezi A; Bienert, Ralf; Vladimirov, Serguei N; Kostka, Susanne; Otto, Albrecht; Wittmann-Liebold, Brigitte; Karpova, Galina G

2003-04-01

The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.

Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

NASA Astrophysics Data System (ADS)

Barton, Janice S.; Tang, Chung-Fei; Reed, Steven S.

2000-02-01

Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

PubMed

Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

2013-01-01

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

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

James Graham, Robert Leslie; Graham, Ciaren; McClean, Stephen

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin actionmore » at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.« less

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

PubMed

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

2011-07-01

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria. Copyright © 2011 Elsevier Inc. All rights reserved.

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

PubMed Central

Dubois, G C; Robinson, E A; Inman, J K; Perham, R N; Appella, E

1981-01-01

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies. PMID:6803762

An insecticidal toxin from Nephila clavata spider venom.

PubMed

Jin, Lin; Fang, Mingqian; Chen, Mengrou; Zhou, Chunling; Ombati, Rose; Hakim, Md Abdul; Mo, Guoxiang; Lai, Ren; Yan, Xiuwen; Wang, Yumin; Yang, Shilong

2017-07-01

Spiders are the most successful insect predators given that they use their venom containing insecticidal peptides as biochemical weapons for preying. Due to the high specificity and potency of peptidic toxins, discoveries of insecticidal toxins from spider venom have provided an opportunity to obtain natural compounds for agricultural applications without affecting human health. In this study, a novel insecticidal toxin (μ-NPTX-Nc1a) was identified and characterized from the venom of Nephila clavata. Its primary sequence is GCNPDCTGIQCGWPRCPGGQNPVMDKCVSCCPFCPPKSAQG which was determined by automated Edman degradation, cDNA cloning, and MS/MS analysis. BLAST search indicated that Nc1a shows no similarity with known peptides or proteins, indicating that Nc1a belongs to a novel family of insecticidal peptide. Nc1a displayed inhibitory effects on Na V and K V channels in cockroach dorsal unpaired median neurons. The median lethal dose (LD50) of Nc1a on cockroach was 573 ng/g. Herein, a study that identifies a novel insecticidal toxin, which can be a potential candidate and/or template for the development of bioinsecticides, is presented.

Systemic AA amyloidosis in the red fox (Vulpes vulpes).

PubMed

Rising, Anna; Cederlund, Ella; Palmberg, Carina; Uhlhorn, Henrik; Gaunitz, Stefan; Nordling, Kerstin; Ågren, Erik; Ihse, Elisabet; Westermark, Gunilla T; Tjernberg, Lars; Jörnvall, Hans; Johansson, Jan; Westermark, Per

2017-11-01

Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full-length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter-species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model. © 2017 The Protein Society.

Partial amino acid sequence of the branched chain amino acid aminotransferase (TmB) of E. coli JA199 pDU11

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

Feild, M.J.; Armstrong, F.B.

1987-05-01

E. coli JA199 pDU11 harbors a multicopy plasmid containing the ilv GEDAY gene cluster of S. typhimurium. TmB, gene product of ilv E, was purified, crystallized, and subjected to Edman degradation using a gas phase sequencer. The intact protein yielded an amino terminal 31 residue sequence. Both carboxymethylated apoenzyme and (/sup 3/H)-NaBH-reduced holoenzyme were then subjected to digestion by trypsin. The digests were fractionated using reversed phase HPLC, and the peptides isolated were sequenced. The borohydride-treated holoenzyme was used to isolate the cofactor-binding peptide. The peptide is 27 residues long and a comparison with known sequences of other aminotransferases revealedmore » limited homology. Peptides accounting for 211 of 288 predicted residues have been sequenced, including 9 residues of the carboxyl terminus. Comparison of peptides with the inferred amino acid sequence of the E. coli K-12 enzyme has helped determine the sequence of the amino terminal 59 residues; only two differences between the sequences are noted in this region.« less

Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

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

Semelr, B.L.; Anderson, C.W.; Hanecak, R.

1982-02-01

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNAmore » sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.« less

Low molecular weight squash trypsin inhibitors from Sechium edule seeds.

PubMed

Laure, Hélen J; Faça, Vítor M; Izumi, Clarice; Padovan, Júlio C; Greene, Lewis J

2006-02-01

Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.

(+)-Meyeniins A-C, Novel Hexahydroimidazo[1,5-c]thiazole Derivatives from the Tubers of Lepidium meyenii: Complete Structural Elucidation by Biomimetic Synthesis and Racemic Crystallization.

PubMed

Zhou, Min; Ma, Hang-Ying; Liu, Zhi-Hua; Yang, Guang-Yu; Du, Gang; Ye, Yan-Qing; Li, Gan-Peng; Hu, Qiu-Fen

2017-03-08

(+)-Meyeniins A-C (1-3), a novel class of sulfur-containing hexahydroimidazo[1,5-c]thiazole derivatives, were isolated from the tubers of Lepidium meyenii (maca) cultivated in Lijiang, Yunnan province, China. Guided by their biosynthetic hypothesis, a stereocontrolled biomimetic synthesis of meyeniins A-C and their individual enantiomers was efficiently accomplished by a combination of a condensation reaction and Edman degradation. The formation of high-quality crystals for X-ray crystallography occurred much more readily from a racemic mixture of (±)-meyeniin A than with the single enantiomer alone in this case. These extensive strategies, combined with circular dichroism (CD) spectra, allowed the complete structural assignments of (+)-meyeniins A-C. Among them, (+)-meyeniin A showed moderate selective cytotoxicities against the HL-60, A549 and MCF-7 human cell lines with IC 50 values of 14.41, 32.22, and 33.14 μM, respectively. To some extent, these findings support traditional applications of maca as healthy nutritional supplements or functional foods for cancer prevention.

Evaluation of IgE reactivity of active and thermally inactivated actinidin, a biomarker of kiwifruit allergy.

PubMed

Grozdanovic, Milica; Popovic, Milica; Polovic, Natalija; Burazer, Lidija; Vuckovic, Olga; Atanaskovic-Markovic, Marina; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

2012-03-01

Actinidin, an abundant cysteine protease from kiwifruit, is a specific biomarker of isolated allergy to kiwifruit. This study evaluates the IgE-binding properties of biologically active and thermally inactivated actinidin. Employing two different activity assays (caseinolytic assay and zymogram with gelatin) we showed that actinidin obtained from kiwifruit extract under native conditions represents a mixture of inactive and active enzyme. The structural integrity of actinidin was confirmed by SDS-PAGE, Edman degradation, mass fingerprint and Western blot with polyclonal antibodies. Although it was capable of inducing positive skin prick test reactions, we failed to detect IgE reactivity of active actinidin in Western blot with patient sera. Thermally inactivated actinidin exhibited IgE reactivity both in vivo and in vitro, indicating that heat processed kiwifruit products may induce clinical reactivity. These findings imply that apart from the allergenic epitopes on its surface, actinidin also contains hidden epitopes inside the protein which become accessible to IgE upon thermal treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Brain cDNA clone for human cholinesterase

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

McTiernan, C.; Adkins, S.; Chatonnet, A.

1987-10-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum.more » The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase.« less

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: A novel CRF-like peptide with smooth muscle contraction activity.

PubMed

Zhou, Yu; Shaw, Chris; Chen, Tianbao

2015-09-15

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Novel Factor Xa-Inhibiting Peptide from Centipedes Venom.

PubMed

Kong, Yi; Shao, Yu; Chen, Hao; Ming, Xin; Wang, Jin-Bin; Li, Zhi-Yu; Wei, Ji-Fu

2013-01-01

Centipedes have been used as traditional medicine for thousands of years in China. Centipede venoms consist of many biochemical peptides and proteins. Factor Xa (FXa) is a serine endopeptidase that plays the key role in blood coagulation, and has been used as a new target for anti-thrombotic drug development. A novel FXa inhibitor, a natural peptide with the sequence of Thr-Asn-Gly-Tyr-Thr (TNGYT), was isolated from the venom of Scolopendra subspinipes mutilans using a combination of size-exclusion and reverse-phase chromatography. The molecular weight of the TNGYT peptide was 554.3 Da measured by electrospray ionization mass spectrometry. The amino acid sequence of TNGYT was determined by Edman degradation. TNGYT inhibited the activity of FXa in a dose-dependent manner with an IC 50 value of 41.14 mg/ml. It prolonged the partial thromboplastin time and prothrombin time in both in vitro and ex vivo assays. It also significantly prolonged whole blood clotting time and bleeding time in mice. This is the first report that an FXa inhibiting peptide was isolated from centipedes venom.

Crab digestive phospholipase: a new invertebrate member.

PubMed

Cherif, Slim; Ben Bacha, Abir; Ben Ali, Yassine; Horchani, Habib; Rekik, Wiem; Gargouri, Youssef

2010-01-01

Crab digestive phospholipase (CDPL) was purified from the hepatopancreas of Carcinus mediterraneus crabs. Homogeneous enzyme was obtained after two chromatography steps: anion exchange and size exclusion HPLC column. Homogeneous CDPL has a molecular mass of 14 kDa as determined by SDS/PAGE analysis. Unlike known digestive phospholipases like porcine PLA(2) (PPPL), CDPL displayed its maximal activity at 50 degrees C and not at 37 degrees C. A specific activity of 40 U/mg for the purified CDPL was measured using PC as substrate under optimal conditions (pH 8 and 50 degrees C) in the presence of 8 mM sodium deoxycholate (NaDC) and 10 mM CaCl(2). In contrast to PPPL, purified CDPL was completely inactivated at 60 degrees C. The N-terminal sequence was determined by automatic Edman degradation. No similarity between 12 N-terminal amino acid residues of CDPL was found with those of known digestive phospholipases. CDPL appears to be a new member of invertebrate phospholipases, and it is potentially useful for treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds which can be used in the food industry.

A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

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

McIntire, W.S.; Wemmer, D.E.; Chistoserdov, A.

Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectralmore » data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.« less

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella

PubMed Central

Leal, Walter S.; Ishida, Yuko; Pelletier, Julien; Xu, Wei; Rayo, Josep; Xu, Xianzhong; Ames, James B.

2009-01-01

Background The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins. Methodology By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components. Conclusion We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH. PMID:19789654

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

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

Takegami, T.; Semler, B.L.; Anderson, C.W.

1983-01-01

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have alsomore » revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.« less

Carolina View, Vol II, 1986.

ERIC Educational Resources Information Center

Davis, Char W., Ed.; Small, LaVeta T., Ed.

1986-01-01

Diverse issues in higher education are addressed in 19 articles. Titles and authors are as follows: "2001: Formulation of a Vision" (Kenneth L. Schwab); Trustees' Roles and Student Issues" (Davis Powers); "What Do Students and Faculty Talk about When They Eat Meals Together?" (John H. Schuh, Neal Edman); "Student…

Identification of Tumor Suppressor Genes by Genetic and Epigenetic Genome-Scanning

DTIC Science & Technology

2008-04-01

SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U...oncogene-related sequences in human neuroblastomas. Cell 35: 359-67; 1983. 3. Capon, D. J.; Seeburg, P. H.; McGrath, J. P.; Hayflick , J. S.; Edman

Frontoxins, three-finger toxins from Micrurus frontalis venom, decrease miniature endplate potential amplitude at frog neuromuscular junction.

PubMed

Moreira, K G; Prates, M V; Andrade, F A C; Silva, L P; Beirão, P S L; Kushmerick, C; Naves, L A; Bloch, C

2010-08-01

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors. Copyright 2010 Elsevier Ltd. All rights reserved.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

PubMed Central

Boisgerault, F; Khalil, I; Tieng, V; Connan, F; Tabary, T; Cohen, J H; Choppin, J; Charron, D; Toubert, A

1996-01-01

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy. PMID:8622959

Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure).

PubMed

Mendes, Maria Anita; de Souza, Bibiana Monson; Palma, Mario Sergio

2005-01-01

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation.

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

PubMed

Bergmann, D J; Arciero, D M; Hooper, A B

1994-06-01

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.

Antagonistic Activities of Novel Peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and Fusarium oxysporum.

PubMed

Kim, Young Gwon; Kang, Hee Kyoung; Kwon, Kee-Deok; Seo, Chang Ho; Lee, Hyang Burm; Park, Yoonkyung

2015-12-09

Bacillus species have recently drawn attention due to their potential use in the biological control of fungal diseases. This paper reports on the antifungal activity of novel peptides isolated from Bacillus amyloliquefaciens PT14. Reverse-phase high-performance liquid chromatography revealed that B. amyloliquefaciens PT14 produces five peptides (PT14-1, -2, -3, -4a, and -4b) that exhibit antifungal activity but are inactive against bacterial strains. In particular, PT14-3 and PT14-4a showed broad-spectrum antifungal activity against Fusarium solani and Fusarium oxysporum. The PT14-4a N-terminal amino acid sequence was identified through Edman degradation, and a BLAST homology analysis showed it not to be identical to any other protein or peptide. PT14-4a displayed strong fungicidal activity with minimal inhibitory concentrations of 3.12 mg/L (F. solani) and 6.25 mg/L (F. oxysporum), inducing severe morphological deformation in the conidia and hyphae. On the other hand, PT14-4a had no detectable hemolytic activity. This suggests PT14-4a has the potential to serve as an antifungal agent in clinical therapeutic and crop-protection applications.

A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers.

PubMed

Astafieva, A A; Rogozhin, Eugene A; Andreev, Yaroslav A; Odintsova, T I; Kozlov, S A; Grishin, Eugene V; Egorov, Tsezi A

2013-09-01

A novel peptide named ToAMP4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, and RP-HPLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids, and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 does not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4, it was successfully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4-mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and functional diversity of AMPs in plants. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Maltaricin CPN, a new class IIa bacteriocin produced by Carnobacterium maltaromaticum CPN isolated from mould-ripened cheese.

PubMed

Hammi, I; Delalande, F; Belkhou, R; Marchioni, E; Cianferani, S; Ennahar, S

2016-11-01

The purpose of this study was to isolate, characterize and determine the structure and the antibacterial activities of a bacteriocin produced by Carnobacterium maltaromaticum CPN, a strain isolated from unpasteurized milk Camembert cheese. This bacteriocin, termed maltaricin CPN, was produced at higher amounts in MRS broth at temperatures between 15°C and 25°C. It was purified to homogeneity from culture supernatant by using a simple method consisting of cation-exchange and reversed-phase chromatographies. Mass spectrometry showed that maltaricin was a 4427·29 Da bacteriocin. Its amino acid sequence was determined by Edman degradation which showed that it had close similarity with bacteriocins of the class IIa. Maltaricin CPN consisted in fact of 44 unmodified amino acids including two cysteine residues at positions 9 and 14 linked by a disulphide bond. The antimicrobial activity of maltaricin CPN covered a range of bacteria, with strong activity against many species of Gram-positive bacteria, especially the food-borne pathogen Listeria monocytogenes, but no activity against Gram-negative ones. In the studied conditions, C. maltaromaticum CPN produced a new class IIa bacteriocin with strong anti-Listeria activity. The study covers the purification and the structural characterization of a new bacteriocin produced by strain C. maltaromaticum CPN isolated from Camembert cheese. Its activity against strains of L. monocytogenes and higher production rates at relatively low temperatures show potential technological applications to improve the safety of refrigerated food. © 2016 The Society for Applied Microbiology.

Radioiodination of scorpion and snake toxins. [/sup 125/I, /sup 127/I

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

Rochat, H.; Tessier, M.; Miranda, F.

1977-10-01

Several scorpion and snake toxins were radioiodinated using the lactoperoxydase method of (/sup 125/I)iodide oxidation. Two techniques of labeling were set up: Using carrier-free Na/sup 125/I and 5 ..mu..g of toxin, about one iodine atom was incorporated per mole of protein without loss of toxicity. Specific radioactivities about 2,000 Ci/mmol (280 ..mu..Ci/..mu..g) were obtained. The modified toxin, purified by immunoprecipitation with an antiserum prepared against the native toxin, was obtained in a short time (4 hr), with a good yield (50 to 80%), and in a small volume (1 ml). Using Na/sup 127/I traced with Na /sup 125/I and largermore » amounts (200 ..mu..g) of toxin, more than one iodine atom was incorporated per mole of protein without loss of activity. Lower specific radioactivities (1 to 1.5 Ci/mmol) were obtained. The iodinated toxins were purified by gel filtration of the radioiodination mixtures on a column made of two layers of Sephadex (G-15 and G-50). The modified proteins were extensively analyzed by paper electrophoresis and polyacrylamide gel electrophoresis. Their content of monoiodotyrosine and diiodotyrosine was estimated and, in the case of toxin I of Androctonus australis Hector, it was possible to follow the iodination rate of its three tyrosine residues by automatic Edman degradation. The mode of purification of the iodinated scorpion toxins affects their behavior on molecular sieving on Sephadex G-50 and on electrophoresis on polyacrylamide gel. The results are discussed.« less

Preparation and characterization of 5-(4-hydroxy-3-nitrobenzyl)-3-phenyl-2-thiohydantoin, the phenylthiohydantoin derivative of 3-nitrotyrosine.

PubMed

Lilova, A; Kleinschmidt, T; Nedkov, P; Braunitzer, G

1986-10-01

The phenylthiocarbamoyl derivative of 3-nitrotyrosine was synthesized according to the known Edman method and then converted to its phenylthiohydantoin derivative [5-(4-hydroxy-3-nitrobenzyl)-3-phenyl-2-thiohydantion] by incubation in 0.5M HCl for 24 h at room temperature. After drying over P2O5 the chromatographically pure substance could be obtained by double recrystallization from hot acetic acid. It could be established that a shorter incubation time leads to an incomplete conversion and higher temperatures cause polymerization of the product. The compounds could be characterized by thin-layer and high-performance liquid chromatography, melting point, elemental analysis as well as NMR- and absorption spectroscopy.

Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum.

PubMed Central

Gu, K; Linhardt, R J; Laliberté, M; Gu, K; Zimmermann, J

1995-01-01

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers. Images Figure 2 Figure 4 PMID:8526872

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

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

Pimkin, Maxim; Miller, C. Glenn; Blakesley, Lauryn

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the proteinmore » identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.« less

Hemoglobin adducts as biomarkers of 1,3-butadiene in occupationally low exposed Italian workers and a few diesel-exposed miners.

PubMed

Begemann, P; Upton, P B; Ranasinghe, A; Swenberg, J A; Soleo, L; Vimercati, L; Gelormini, A; Fustinoni, S; Zwirner-Baier, I; Neumann, H G

2001-06-01

Hemoglobin adducts were determined as biomarkers of 1,3-butadiene (BD) in 30 workers and 10 controls from an Italian BD plant and in 14 diesel-exposed miners. N-(2,3,4-trihydroxybutyl)valine (THBVal), an N-terminal valine globin adduct of reactive butadiene metabolites, was analyzed by gas chromatography/high resolution mass spectrometry after a modified Edman degradation and further acetylation. The BD exposure for the plant workers was 31 microg/m(3) (personal sampling). Whereas there was no detectable difference in hemoglobin adduct levels (range 17.7-61.4 pmol/g globin) between the total group of exposed and controls, slight but significant differences could be found between two subgroups of workers from different production units as well as one subgroup and controls (P<0.05), between smoking (n=13) and non-smoking exposed workers (n=17; P=0.066) as well as between smoking exposed workers and controls (P=0.055). Adduct levels of the miners (all non-smokers) were in the same range as those of the Italian BD-workers and controls. The internal exposure and strain measured by THBVal levels resulting from a very low occupational BD exposure was in the range of the contribution of moderate smoking.

Angiotensin I-Converting Enzyme Inhibitor Derived from Cross-Linked Oyster Protein

PubMed Central

Xie, Cheng-Liang; Kim, Jin-Soo; Ha, Jong-Myung; Choung, Se-Young

2014-01-01

Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 μM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension. PMID:25140307

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

PubMed

Chang, S-Y; Chen, Y-S; Pan, S-F; Lee, Y-S; Chang, C-H; Chang, C-H; Yu, B; Wu, H-C

2013-09-01

Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative. © 2013 The Society for Applied Microbiology.

Proteomic analysis of the venom from the fish eating coral snake Micrurus surinamensis: novel toxins, their function and phylogeny.

PubMed

Olamendi-Portugal, Timoteo; Batista, Cesar V F; Restano-Cassulini, Rita; Pando, Victoria; Villa-Hernandez, Oscar; Zavaleta-Martínez-Vargas, Alfonso; Salas-Arruz, Maria C; Rodríguez de la Vega, Ricardo C; Becerril, Baltazar; Possani, Lourival D

2008-05-01

The protein composition of the soluble venom from the South American fish-eating coral snake Micrurus surinamensis surinamensis, here abbreviated M. surinamensis, was separated by RP-HPLC and 2-DE, and their components were analyzed by automatic Edman degradation, MALDI-TOF and ESI-MS/MS. Approximately 100 different molecules were identified. Sixty-two components possess molecular masses between 6 and 8 kDa, are basically charged molecules, among which are cytotoxins and neurotoxins lethal to fish (Brachidanios rerio). Six new toxins (abbreviated Ms1-Ms5 and Ms11) were fully sequenced. Amino acid sequences similar to the enzymes phospholipase A2 and amino acid oxidase were identified. Over 20 additional peptides were identified by sequencing minor components of the HPLC separation and from 2-DE gels. A functional assessment of the physiological activity of the six toxins was also performed by patch clamp using muscular nicotinic acetylcholine receptor assays. Variable degrees of blockade were observed, most of them reversible. The structural and functional data obtained were used for phylogenetic analysis, providing information on some evolutionary aspects of the venom components of this snake. This contribution increases by a factor of two the total number of alpha-neurotoxins sequenced from the Micrurus genus in currently available literature.

Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin.

PubMed

Yamashita, H; Theerasilp, S; Aiuchi, T; Nakaya, K; Nakamura, Y; Kurihara, Y

1990-09-15

A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed.

Comparative proteomic analysis of male and female venoms from the Cuban scorpion Rhopalurus junceus.

PubMed

Rodríguez-Ravelo, Rodolfo; Batista, Cesar V F; Coronas, Fredy I V; Zamudio, Fernando Z; Hernández-Orihuela, Lorena; Espinosa-López, Georgina; Ruiz-Urquiola, Ariel; Possani, Lourival D

2015-12-01

A complete mass spectrometry analysis of venom components from male and female scorpions of the species Rhophalurus junceus of Cuba is reported. In the order of 200 individual molecular masses were identified in both venoms, from which 63 are identical in male and females genders. It means that a significant difference of venom components exists between individuals of different sexes, but the most abundant components are present in both sexes. The relative abundance of identical components is different among the genders. Three well defined groups of different peptides were separated and identified. The first group corresponds to peptides with molecular masses of 1000-2000 Da; the second to peptides with 3500-4500 Da molecular weight, and the third with 6500-8000 Da molecular weights. A total of 86 peptides rich in disulfide bridges were found in the venoms, 27 with three disulfide bridges and 59 with four disulfide bridges. LC-MS/MS analysis allowed the identification and amino acid sequence determination of 31 novel peptides in male venom. Two new putative K(+)-channel peptides were sequences by Edman degradation. They contain 37 amino acid residues, packed by three disulfide bridges and were assigned the systematic numbers: α-KTx 1.18 and α-KTx 2.15. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification and characterization of an antibacterial and anti-inflammatory polypeptide from Arca subcrenata.

PubMed

Chen, Yuyan; Li, Chunlei; Zhu, Jianhua; Xie, Wangshi; Hu, Xianjing; Song, Liyan; Zi, Jiachen; Yu, Rongmin

2017-03-01

A polypeptide coded as PGC was isolated from Arca subcrenata muscle using ion exchange, Sephadex G-50 gel chromatography and RP-HPLC. PGC was identified to be a homogeneous compound by Native-PAGE and the purity was more than 98.9% measured by HPLC. The isoelectric point of PGC was determined to be 9.76 by IEF-PAGE. The molecular weight was determined to be 15,973.0Da by ESI-MS/MS. The conformational structure of PGC was characterized by UV-vis, FT-IR and CD spectroscopy. N terminal amino acid sequence of PGC was shown as PSVYDAAAQLTADVKKDLRDSWKVIGGDKKGNGVA by Edman degradation. The results demonstrated that there is a high degree of homology between PGC and the subunit from hemoglobin, and proposed that PGC is the depolymerized polypeptide of Hemoglobin I (HbI) from A. subcrenata. The evaluation of biological activities showed that the diameters of the inhibitory ring of PGC on Escherichia coli and Staphylococcus aureus were 14.5±0.44mm and 16.5±1.15mm, respectively. The IC 50 of inhibition rate for PGC on NO production was 9.60±0.71μg/mL. Therefore, PGC might be developed as one of potential antibacterial and anti-inflammatory agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Asymmetric arginine dimethylation of heterogeneous nuclear ribonucleoprotein K by protein-arginine methyltransferase 1 inhibits its interaction with c-Src.

PubMed

Ostareck-Lederer, Antje; Ostareck, Dirk H; Rucknagel, Karl P; Schierhorn, Angelika; Moritz, Bodo; Huttelmaier, Stefan; Flach, Nadine; Handoko, Lusy; Wahle, Elmar

2006-04-21

Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.

Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

PubMed Central

de Magalhães, Mariana T. Q.; Barbosa, Eder A.; Prates, Maura V.; Verly, Rodrigo M.; Munhoz, Victor Hugo O.; de Araújo, Ivan E.; Bloch, Carlos

2013-01-01

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy 1H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways. PMID:23565145

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado).

PubMed

Konno, K; Hisada, M; Naoki, H; Itagaki, Y; Kawai, N; Miwa, A; Yasuhara, T; Morimoto, Y; Nakata, Y

2000-11-01

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder.

PubMed

Conlon, J M; Davis, M S; Falkmer, S; Thim, L

1987-11-02

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.

Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies.

PubMed

Chelius, Dirk; Jing, Kay; Lueras, Alexis; Rehder, Douglas S; Dillon, Thomas M; Vizel, Alona; Rajan, Rahul S; Li, Tiansheng; Treuheit, Michael J; Bondarenko, Pavel V

2006-04-01

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

Isolation and identification of a cardioactive peptide from Tenebrio molitor and Spodoptera eridania.

PubMed

Furuya, K; Liao, S; Reynolds, S E; Ota, R B; Hackett, M; Schooley, D A

1993-12-01

We isolated several cardioactive peptides from extracts of whole heads of the mealworm, Tenebrio molitor, and the southern armyworm, Spodoptera eridania, using a semi-isolated heart of Manduca sexta for bioassay. We have now isolated from each species the peptide with the strongest effect on rate of contraction of the heart. The peptides were identified using micro Edman sequencing and mass spectrometric methods. This cardioactive peptide has the same primary structure from both species: Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, a cyclic nonapeptide which is identical to crustacean cardioactive peptide (CCAP) originally isolated from the shore crab, Carcinus maenas, and subsequently isolated from Locusta migratoria and Manduca sexta. This is additional evidence that CCAP has widespread occurrence in arthropoda.

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy

PubMed Central

1991-01-01

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity- purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure. PMID:1999465

Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae).

PubMed

Sousa, Juliana C; Berto, Raquel F; Gois, Elicélia A; Fontenele-Cardi, Nauíla C; Honório, José E R; Konno, Katsuhiro; Richardson, Michael; Rocha, Marcos F G; Camargo, Antônio A C M; Pimenta, Daniel C; Cardi, Bruno A; Carvalho, Krishnamurti M

2009-07-01

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Isolation and characterization of a leech neuropeptide in rat brains: coupling to nitric oxide release in leech, rat and human tissues.

PubMed

Salzet, M; Salzet, B; Sáutière, P; Lésage, J; Beauvillain, J C; Bilfinger, T V; Rialas, C; Bjenning, C; Stefano, G B

1998-03-30

The osmoregulator peptide (leech osmoregulatory factor, LORF; IPEPYVWD) was first found in the leech central nervous system (CNS). Given the fact that certain peptides can be found in mammals and invertebrates, e.g., opioid, we examined rat brains to determine if LORF was present. This peptide was found and isolated by successive reversed-phase HPLC purification steps and characterized by electrospray mass spectrometry measurement. It was sequenced by Edman degradation and quantified in different tissues by ELISA. Our results demonstrate the presence of LORF in the hypothalamus, thalamus, and striatum (6 pmol/mg of protein extract) and in other brain areas at lower levels. This octapeptide is also present in the rat duodenum and liver (10 to 14 pmol/mg) and at lower levels in heart, lung, pancreas and caudal spinal cord (< 5 pmol/mg). The testes, adrenals and kidneys have the lowest levels of all the tissues examined (ca. 0.5 pmol/mg of protein). Furthermore, we also demonstrate that LORF is coupled to nitric oxide (NO) release in leech CNS, rat hypothalamus and human saphenous vein in a manner which is inhibited by a nitric oxide synthase inhibitor as well as an antibody directed toward LORF. The study demonstrates that LORF, and its function in relation to NO release, has been conserved over more than 400 million years of evolution.

A broad-spectrum antimicrobial activity of Bacillus subtilis RLID 12.1.

PubMed

Ramachandran, Ramya; Chalasani, Ajay Ghosh; Lal, Ram; Roy, Utpal

2014-01-01

In the present study, an attempt was made to biochemically characterize the antimicrobial substance from the soil isolate designated as RLID 12.1 and explore its potential applications in biocontrol of drug-resistant pathogens. The antimicrobial potential of the wild-type isolate belonging to the genus Bacillus was determined by the cut-well agar assay. The production of antimicrobial compound was recorded maximum at late exponential growth phase. The ultrafiltered concentrate was insensitive to organic solvents, metal salts, surfactants, and proteolytic and nonproteolytic enzymes. The concentrate was highly heat stable and active over a wide range of pH values. Partial purification, zymogram analysis, and TLC were performed to determine the preliminary biochemical nature. The molecular weight of the antimicrobial peptide was determined to be less than 2.5 kDa in 15% SDS-PAGE and in zymogram analysis against Streptococcus pyogenes. The N-terminal amino acid sequence by Edman degradation was partially determined to be T-P-P-Q-S-X-L-X-X-G, which shows very insignificant identity to other antimicrobial peptides from bacteria. The minimum inhibitory concentrations of dialysed and partially purified ion exchange fractions were determined against some selected gram-positive and gram-negative bacteria and some pathogenic yeasts. The presence of three important antimicrobial peptide biosynthesis genes ituc, fend, and bmyb was determined by PCR.

PhcrTx2, a New Crab-Paralyzing Peptide Toxin from the Sea Anemone Phymanthus crucifer

PubMed Central

Garateix, Anoland; Salceda, Emilio; Zaharenko, André Junqueira; Pons, Tirso; Santos, Yúlica; Arreguín, Roberto; Ständker, Ludger; Forssmann, Wolf-Georg; Tytgat, Jan; Vega, Rosario

2018-01-01

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new β-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae. PMID:29414882

Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli.

PubMed

Sporeno, E; Barbato, G; Graziani, R; Pucci, P; Nitti, G; Paonessa, G

1994-05-01

Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells. After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector. Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column. We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay. Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry. Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis).

PubMed

Darville, Lancia N F; Merchant, Mark E; Maccha, Venkata; Siddavarapu, Vivekananda Reddy; Hasan, Azeem; Murray, Kermit K

2012-02-01

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35kDa protein was ~98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates. Copyright © 2011 Elsevier Inc. All rights reserved.

Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

PubMed Central

Marcondes, M.F.M.; Alves, F.M.; Assis, D.M.; Hirata, I.Y.; Juliano, L.; Oliveira, V.; Juliano, M.A.

2015-01-01

The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1′ substrate positions: Ser = Gln > Thr at P1 and Ser > Thr at P1′. Non-polar residues were frequent at the substrate P3, P2, P2′ and P3′ positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1′ substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase. PMID:26082885

Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

NASA Astrophysics Data System (ADS)

Schmidt, Thomas P.; Perna, Anna M.; Fugmann, Tim; Böhm, Manja; Jan Hiss; Haller, Sarah; Götz, Camilla; Tegtmeyer, Nicole; Hoy, Benjamin; Rau, Tilman T.; Neri, Dario; Backert, Steffen; Schneider, Gisbert; Wessler, Silja

2016-03-01

The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

The removal of pyroglutamic acid from monoclonal antibodies without denaturation of the protein chains.

PubMed

Werner, William E; Wu, Sylvia; Mulkerrin, Michael

2005-07-01

Typically, the removal of pyroglutamate from the protein chains of immunoglobulins with the enzyme pyroglutamate aminopeptidase requires the use of chaotropic and reducing agents, quite often with limited success. This article describes a series of optimization experiments using elevated temperatures and detergents to denature and stabilize the heavy chains of immunoglobulins such that the pyroglutamate at the amino terminal was accessible to enzymatic removal using the thermostable protease isolated from Pyrococcus furiosus. The detergent polysorbate 20 (Tween 20) was used successfully to facilitate the removal of pyroglutamate residues. A one-step digestion was developed using elevated temperatures and polysorbate 20, rather than chaotropic and reducing agents, with sample cleanup and preparation for Edman sequencing performed using a commercial cartridge containing the PVDF membrane. All of the immunoglobulins digested with this method yielded heavy chain sequence, but the extent of deblocking was immunglobulin dependent (typically>50%).

Studies on the plasma membrane H sup + -ATPase of oat roots: Preparation and assay, cytological localization, and sulfhydryl chemistry

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

Katz, D.B.

1989-01-01

Biochemical and cytological studies were performed on the plasma membrane proton pump (H{sup +}-ATPase) of oat roots (Avena sativa cv. Stout). H{sup +}-ATPase activity in oat root plasma membranes is inhibited by N-ethylmaleimide (NEM), a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced in the presence of ADP or MgADP. An M{sub r} = 100,000 plasma membrane polypeptide showed reduced labelling by ({sup 3}H)NEM in the presence of ADP. When tryptic peptides from ({sup 3}H)NEM-labeled M{sub r} = 100,000 polypeptide were separated by reverse-phase high-pressure liquid chromatography (HPLC), only one radioactive peak consistently showed labeling inmore » the presence of ADP. In order to determine the location and identity of the NEM-reactive residue, the radioactive peptide in this peak was further purified by HPLC. The amino acid sequence(s) in the resulting sample were then determined by Edman degradation on an automated gas-phase sequenator. The PTH-amino acids released at each cycle of the degradation were separated by HPLC. Analysis of the chromatograms suggested that the radio-labeled residue was located in a peptide of sequence V-E-N-Q-D-A-I-D-A-C{sup *}-M-V-G-M-L-A-D-P-K. The NEM-reactive residue was cysteine, based on the retention time of the radioactivity released. The ATP-hydrolyzing activity observed in electron micrographs by lead-precipitation of enzymically released inorganic phosphate was compared with that observed in in vitro assays of the soluble and plasma membrane fractions of oat root homogenates. Although an ATP-hydrolyzing activity was observed on the plasma membrane in the electron micrographs, its substrate specificity and inhibitor sensitivity was identical to that observed for phosphatase activity.« less

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites.

PubMed

Qin, Chunlin; Brunn, Jan C; Cook, Richard G; Orkiszewski, Ralph S; Malone, James P; Veis, Arthur; Butler, William T

2003-09-05

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

An isotope-dilution UPLC-MS/MS technique for the human biomonitoring of the internal exposure to glycidol via a valine adduct at the N-terminus of hemoglobin.

PubMed

Hielscher, Jan; Monien, Bernhard H; Abraham, Klaus; Jessel, Sönke; Seidel, Albrecht; Lampen, Alfonso

2017-08-01

Fatty acid esters of glycidol (glycidyl esters) are processing contaminants generated as a byproduct of the industrial deodorization of vegetable oils and fats. Oral intake of glycidyl esters leads to the release of glycidol in the gastrointestinal tract. Glycidol is carcinogenic, genotoxic and teratogenic in rodents. It is rated as probably carcinogenic to humans (IARC group 2A). The determination of internal exposure of glycidol may support the assessment of the possible human health risks related to glycidyl ester intake. For this purpose, hemoglobin adducts of glycidol may be suitable biomarkers reflecting the cumulative exposure of up to four months. We applied a modified Edman degradation to assess the glycidol adduct at the N-terminal valine, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), of hemoglobin. The modified valine was cleaved with fluorescein-5-isothiocyanate (FITC), resulting in the formation of N-(2,3-dihydroxypropyl)-valine fluorescein thiohydantoin (DHP-Val-FTH). An isotope-dilution technique was developed for the quantification of the thiohydantoin analyte by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and DHP-Val-d 7 -FTH as reference standard. The limit of detection was 4 fmol DHP-Val-FTH per injection corresponding to 0.7pmol 2,3-diHOPr-Val/g hemoglobin. The adduct levels in blood samples of 12 non-smoking participants were in the range of 2.2-4.9pmol 2,3-diHOPr-Val/g hemoglobin. The current work presents the first isotope-dilution technique using UPLC-MS/MS for the quantification of 2,3-diHOPr-Val at the N-terminus of hemoglobin as a sensitive and convenient alternative to earlier GC-MS methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph.

PubMed

Alpuche, Juan; Pereyra, Ali; Agundis, Concepción; Rosas, Carlos; Pascual, Cristina; Slomianny, Marie-Christine; Vázquez, Lorena; Zenteno, Edgar

2005-06-20

A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL-ligand interactions.

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

Fox, J.W.; Elzinga, M.; Tu, A.T.

The primary structure of myotoxin a, a myotoxin protein from the venom of the North American rattlesnake Crotalus viridis viridis, was determined and the position of the disulfide bonds assigned. The toxin was isolated, carboxymethylated, and cleaved by cyanogen bromide, and the resultant peptides were isolated. The cyanogen bromide peptides were subjected to amino acid sequence analysis. In order to assign the positions of the three disulfide bonds, the native toxin was cleaved sequentially with cyanogen bromide and trypsin. A two peptide unit connected by one disulfide bond was isolated and characterized, and a three-peptide unit connected by two disulfidemore » bonds was isolated. One peptide in the three-peptide unit was identified as Cys-Cys-Lys. In order to establish the linkages between the peptides and Cys-Cys-Lys, one cycle of Edman degradation was carried out such that the Cys-Cys bond was cleaved. Upon isolation and analysis of the cleavage products, the disulfide bonds connecting the three peptides were determined. The positions of the disulfide bridges of myotoxin a were determined to be totally different from those of neurotoxins isolated from snake venoms. The sequence of myotoxin a was compared with the sequences of other snake venom toxins using the computer program RELATE to determine whether myotoxin a is similar to any other types of toxins. From the computer analysis, myotoxin a did not show any close relationship to other toxins except crotamine from the South American rattlesnake Crotalus durissus terrificus.« less

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

PubMed Central

Franz, Charles M. A. P.; Worobo, Randy W.; Quadri, Luis E. N.; Schillinger, Ulrich; Holzapfel, Wilhelm H.; Vederas, John C.; Stiles, Michael E.

1999-01-01

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433. PMID:10224016

Skin secretion peptides: the molecular facet of the deimatic behavior of the four-eyed frog, Physalaemus nattereri (Anura, Leptodactylidae).

PubMed

Barbosa, Eder Alves; Iembo, Tatiane; Martins, Graciella Ribeiro; Silva, Luciano Paulino; Prates, Maura Vianna; Andrade, Alan Carvalho; Bloch, Carlos

2015-11-15

Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri. Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data. Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions. The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frog's deimatic behavior. Copyright © 2015 John Wiley & Sons, Ltd.

Ubiquitylation Functions in the Calcium Carbonate Biomineralization in the Extracellular Matrix

PubMed Central

Fang, Dong; Pan, Cong; Lin, Huijuan; Lin, Ya; Xu, Guangrui; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

2012-01-01

Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes. PMID:22558208

AcT-2: a novel myotropic and antimicrobial type 2 tryptophyllin from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

PubMed

Ge, Lilin; Lyu, Peng; Zhou, Mei; Zhang, Huiling; Wan, Yuantai; Li, Bin; Li, Renjie; Wang, Lei; Chen, Tianbao; Shaw, Chris

2014-01-01

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure--GMRPPWF-NH2--was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μ M). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Perlinhibin, a Cysteine-, Histidine-, and Arginine-Rich Miniprotein from Abalone (Haliotis laevigata) Nacre, Inhibits In Vitro Calcium Carbonate Crystallization

PubMed Central

Mann, Karlheinz; Siedler, Frank; Treccani, Laura; Heinemann, Fabian; Fritz, Monika

2007-01-01

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins. PMID:17496038

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

PubMed Central

2011-01-01

Background The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. Results Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. Conclusion Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans. PMID:21477375

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1.

PubMed

Nicolas, Guillaume G; LaPointe, Gisèle; Lavoie, Marc C

2011-04-10

The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

PubMed Central

Ferreira, Francis Barbosa; Gomes, Mário Sérgio Rocha; Naves de Souza, Dayane Lorena; Gimenes, Sarah Natalie Cirilo; Castanheira, Letícia Eulalio; Borges, Márcia Helena; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Homsi Brandeburgo, Maria Inês; Rodrigues, Veridiana M.

2013-01-01

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A2 (PLA2) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA2-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA2-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA2-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA2-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA2s. The phylogenetic analyses showed that BpPLA2-TXI forms a group with other acidic D49 PLA2s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects. PMID:24304676

Structural characterization and comparative modeling of PD-Ls 1-3, type 1 ribosome-inactivating proteins from summer leaves of Phytolacca dioica L.

PubMed

Di Maro, Antimo; Chambery, Angela; Carafa, Vincenzo; Costantini, Susan; Colonna, Giovanni; Parente, Augusto

2009-03-01

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1-2 and PD-Ls 3-4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1-3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man)(3) (GlcNAc)(2) (Fuc)(1) (Xyl)(1) linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates.

Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.

PubMed Central

Spiess, J; Noe, B D

1985-01-01

A novel 28-residue somatostatin (SS) has been isolated from anglerfish pancreatic islets and characterized by complete Edman degradation, peptide mapping, and amino acid analysis. The primary structure of this anglerfish SS-28 (aSS-28) containing hydroxylysine (Hyl) was established to be H-Ser-Val-Asp-Ser-Thr-Asn-Asn-Leu-Pro-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys- Lys-Asn-Phe-Tyr-Trp-Hyl-Gly-Phe-Thr-Ser-Cys-OH. This sequence (with the exception of hydroxylysine-23, which is replaced by lysine) is identical to the sequence of the COOH-terminal 28 residues of prepro-SS II predicted on the basis of cDNA analysis [Hobart, P., Crawford, R., Shen, L., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141]. This is the first instance in which hydroxylysine (to date characteristically observed in collagen or collagen-like structures) has been found in a potential regulatory peptide. Chromatographic characterization of peptides, radiolabeled in islet culture, revealed that aSS-28 contained 10-12% of the radioactivity incorporated into the 8000- to 1000-dalton SS-like polypeptides, whereas 88-90% of this radioactivity was detected in anglerfish SS-14. It appears probable that aSS-28 represents the predominant primary cleavage product derived from prepro-SS II by cleavage at the COOH-terminal side of a single arginine. Based on knowledge of the collagen biosynthesis, it is speculated that hydroxylation may take place as an early post-translational event. Images PMID:2857489

Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity.

PubMed

Astafieva, Alexandra A; Enyenihi, Atim A; Rogozhin, Eugene A; Kozlov, Sergey A; Grishin, Eugene V; Odintsova, Tatyana I; Zubarev, Roman A; Egorov, Tsezi A

2015-09-01

Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Purification, characterization and molecular cloning of chymotrypsin inhibitor peptides from the venom of Burmese Daboia russelii siamensis.

PubMed

Guo, Chun-Teng; McClean, Stephen; Shaw, Chris; Rao, Ping-Fan; Ye, Ming-Yu; Bjourson, Anthony J

2013-05-01

One novel Kunitz BPTI-like peptide designated as BBPTI-1, with chymotrypsin inhibitory activity was identified from the venom of Burmese Daboia russelii siamensis. It was purified by three steps of chromatography including gel filtration, cation exchange and reversed phase. A partial N-terminal sequence of BBPTI-1, HDRPKFCYLPADPGECLAHMRSF was obtained by automated Edman degradation and a Ki value of 4.77nM determined. Cloning of BBPTI-1 including the open reading frame and 3' untranslated region was achieved from cDNA libraries derived from lyophilized venom using a 3' RACE strategy. In addition a cDNA sequence, designated as BBPTI-5, was also obtained. Alignment of cDNA sequences showed that BBPTI-5 exhibited an identical sequence to BBPTI-1 cDNA except for an eight nucleotide deletion in the open reading frame. Gene variations that represented deletions in the BBPTI-5 cDNA resulted in a novel protease inhibitor analog. Amino acid sequence alignment revealed that deduced peptides derived from cloning of their respective precursor cDNAs from libraries showed high similarity and homology with other Kunitz BPTI proteinase inhibitors. BBPTI-1 and BBPTI-5 consist of 60 and 66 amino acid residues respectively, including six conserved cysteine residues. As these peptides have been reported to have influence on the processes of coagulation, fibrinolysis and inflammation, their potential application in biomedical contexts warrants further investigation. Copyright © 2013 Elsevier Inc. All rights reserved.

Blood-feeding Behaviors of Anopheles stephensi But Not Phlebotomus papatasi are Influenced by Actively Warming Guinea Pigs (Cavia porcellus) Under General Anesthesia

DTIC Science & Technology

2015-01-01

response in mosquitoes. Proc Natl Acad Sci 108:8026–8029. Davis EE, Sokolove PG. 1975. Temperature responses of antennal receptors of the mosquito, Aedes ...books/NBK54050/ Peterson D, Brown A. 1951. Studies of the responses of the female Aedes mosquito. Part III. The response of Aedes aegypti (L.) to a warm...Hyg 33:1232–1238. Walker ED, Edman JD. 1985. Feeding-site selection and blood-feeding behavior of Aedes triseriatus (Diptera: Culicidae) on rodent

Monitoring abacavir bioactivation in humans: screening for an aldehyde metabolite.

PubMed

Grilo, Nádia M; Antunes, Alexandra M M; Caixas, Umbelina; Marinho, Aline T; Charneira, Catarina; Conceição Oliveira, M; Monteiro, Emília C; Matilde Marques, M; Pereira, Sofia A

2013-05-10

The anti-HIV drug abacavir is associated with idiosyncratic hypersensitivity reactions and cardiotoxicity. Although the mechanism underlying abacavir-toxicity is not fully understood, drug bioactivation to reactive metabolites may be involved. This work was aimed at identifying abacavir-protein adducts in the hemoglobin of HIV patients as biomarkers of abacavir bioactivation and protein modification. The protocol received prior approval from the Hospital Ethics Committee, patients gave their written informed consent and adherence was controlled through a questionnaire. Abacavir-derived Edman adducts with the N-terminal valine of hemoglobin were analyzed by an established liquid chromatography-electrospray ionization-tandem mass spectrometry method. Abacavir-valine adducts were detected in three out of ten patients. This work represents the first evidence of abacavir-protein adduct formation in humans. The data confirm the ability of abacavir to modify self-proteins and suggest that the molecular mechanism(s) of some abacavir-induced adverse reactions may require bioactivation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan.

PubMed

Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

2011-04-27

Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

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

Smith, L.I.; Bodwell, J.E.; Mendel, D.B.

1988-05-17

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with (/sup 3/H)dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single (/sup 3/H)dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the (/sup 3/H)dexamethasone 21-mesylate was located at position 5 frommore » the amino terminus. Dual-isotope labeling studies with (/sup 3/H)dexamethasone 21-mesylate and (/sup 35/S)methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of (/sup 3/H)dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells.« less

The adipokinetic hormone of the coleopteran suborder Adephaga: Structure, function, and comparison of distribution in other insects.

PubMed

Gäde, Gerd; Marco, Heather G

2017-07-01

The aim of the current study is to identify the adipokinetic hormone(s) (AKHs) of a basal suborder of the species-rich Coleoptera, the Adephaga, and possibly learn more about the ancestral AKH of beetles. Moreover, we wanted to compare the ancestral AKH with AKHs of more advanced beetles, of which a number are pest insects. This would allow us to assess whether AKH mimetics would be suitable as insecticides, that is, be harmful to the pest species but not to the beneficial species. Nine species of the Adephaga were investigated and all synthesize only one octapeptide in the corpus cardiacum, as revealed by Edman degradation sequencing techniques or by mass spectrometry. The amino acid sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp corresponds to Schgr-AKH-II that was first identified in the desert locust. It is assumed that Schgr-AKH-II-the peptide of a basal beetle clade-is the ancestral AKH for beetles. Some other beetle families, as well as some Hymenoptera (including honey bees) also contain this peptide, whereas most of the pest beetle species have different AKHs. This argues that those peptides and their receptors should be explored for developing mimetics with insecticidal properties. A scenario where Schgr-AKH-II (the only AKH of Adephaga) is used as basic molecular structure to derive almost all other known beetle AKHs via single step mutations is very likely, and supports the interpretation that Schgr-AKH-II is the ancestral AKH of Coleoptera. © 2017 Wiley Periodicals, Inc.

Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus.

PubMed

Valdez-Velázquez, Laura L; Quintero-Hernández, Verónica; Romero-Gutiérrez, Maria Teresa; Coronas, Fredy I V; Possani, Lourival D

2013-01-01

Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecular masses from 259 to 44,392 Da. Most of these components are within the expected molecular masses for Na(+)- and K(+)-channel specific toxic peptides, supporting the clinical findings of intoxication, when humans are stung by this scorpion. From the cDNA library 162 clones were randomly chosen, from which 130 sequences of good quality were identified and were clustered in 28 contigs containing, each, two or more expressed sequence tags (EST) and 49 singlets with only one EST. Deduced amino acid sequence analysis from 53% of the total ESTs showed that 81% (24 sequences) are similar to known toxic peptides that affect Na(+)-channel activity, and 19% (7 unique sequences) are similar to K(+)-channel especific toxins. Out of the 31 sequences, at least 8 peptides were confirmed by direct Edman degradation, using components isolated directly from the venom. The remaining 19%, 4%, 4%, 15% and 5% of the ESTs correspond respectively to proteins involved in cellular processes, antimicrobial peptides, venom components, proteins without defined function and sequences without similarity in databases. Among the cloned genes are those similar to metalloproteinases.

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi.

PubMed

Pinto, Erika Gracielle; Pimenta, Daniel C; Antoniazzi, Marta Maria; Jared, Carlos; Tempone, Andre Gustavo

2013-12-01

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25-0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50=34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX(®) Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies. Copyright © 2013 Elsevier Inc. All rights reserved.

Some biochemical and histochemical properties of human liver serine dehydratase.

PubMed

Kashii, Tatsuhiko; Gomi, Tomoharu; Oya, Takeshi; Ishii, Yoko; Oda, Hirofumi; Maruyama, Muneharu; Kobayashi, Masashi; Masuda, Tohru; Yamazaki, Mitsuaki; Nagata, Takuya; Tsukada, Kazuhiro; Nakajima, Akinori; Tatsu, Kazuhito; Mori, Hisashi; Takusagawa, Fusao; Ogawa, Hirofumi; Pitot, Henry C

2005-03-01

In rat, serine dehydratase (SDH) is abundant in the liver and known to be a gluconeogenic enzyme, while there is little information about the biochemical property of human liver serine dehydratase because of its low content and difficulty in obtaining fresh materials. To circumvent these problems, we purified recombinant enzyme from Escherichia coli, and compared some properties between human and rat liver serine dehydratases. Edman degradation showed that the N-terminal sequence of about 75% of human serine dehydratase starts from MetSTART-Met2-Ser3- and the rest from Ser3-, whereas the N-terminus of rat enzyme begins from the second codon of MetSTART-Ala2-. The heterogeneity of the purified preparation was totally confirmed by mass spectrometry. Accordingly, this observation in part fails to follow the general rule that the first Met is not removed when the side chain of the penultimate amino acid is bulky such as Met, Arg, Lys, etc. There existed the obvious differences in the local structures between the two enzymes as revealed by limited-proteolysis experiments using trypsin and Staphylococcus aureus V8 protease. The most prominent difference was found histochemically: expression of rat liver serine dehydratase is confined to the periportal region in which many enzymes involved in gluconeogenesis and urea cycle are known to coexist, whereas human liver serine dehydratase resides predominantly in the perivenous region. These findings provide an additional support to the previous notion suggested by physiological experiments that contribution of serine dehydratase to gluconeogenesis is negligible or little in human liver.

The human gastrin precursor. Characterization of phosphorylated forms and fragments.

PubMed Central

Varro, A; Desmond, H; Pauwels, S; Gregory, H; Young, J; Dockray, G J

1988-01-01

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product. PMID:3223964

Peroxisomal copper, zinc superoxide dismutase. Characterization of the isoenzyme from watermelon cotyledons.

PubMed Central

Bueno, P; Varela, J; Gimeénez-Gallego, G; del Río, L A

1995-01-01

The biochemical and immunochemical characterization of a superoxide dismutase (SOD, EC 1.15.1.1) from peroxisomal origin has been carried out. The enzyme is a Cu,Zn-containing SOD (CuZn-SOD) located in the matrix of peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons (L.M. Sandalio and L.A. del Río [1988] Plant Physiol 88: 1215-1218). The amino acid composition of the enzyme was determined. Analysis by reversed-phase high-performance liquid chromatography of the peroxisomal CuZn-SOD incubated with 6 M guanidine-HCl indicated that this enzyme contained a noncovalently bound chromophore group that was responsible for the absorbance peak of the native enzyme at 260 nm. The amino acid sequence of the peroxisomal CuZn-SOD was determined by Edman degradation. Comparison of its sequence with those reported for other plant SODs revealed homologies of about 70% with cytosolic CuZn-SODs and of 90% with chloroplastic CuZn-SODs. The peroxisomal SOD has a high thermal stability and resistance to inactivation by hydrogen peroxide. A polyclonal antibody was raised against peroxisomal CuZn-SOD, and by western blotting the antibody cross-reacted with plant CuZn-SODs but did not recognize either plant Mn-SOD or bacterial Fe-SOD. The antiSOD-immunoglobulin G showed a weak cross-reaction with bovine erythrocytes and liver CuZn-SODs, and also with cell-free extracts from trout liver. The possible function of this CuZn-SOD in the oxidative metabolism of peroxisomes is discussed. PMID:7630940

Tripeptidyl peptidase II. An oligomeric protease complex from Arabidopsis.

PubMed

Book, Adam J; Yang, Peizhen; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2005-06-01

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.

Tripeptidyl Peptidase II. An Oligomeric Protease Complex from Arabidopsis1

PubMed Central

Book, Adam J.; Yang, Peizhen; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2005-01-01

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling. PMID:15908606

Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species.

PubMed

Minervini, F; Algaron, F; Rizzello, C G; Fox, P F; Monnet, V; Gobbetti, M

2003-09-01

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine alpha(S1)-casein (alpha(S1)-CN) 24-47 fragment (f24-47), f169-193, and beta-CN f58-76; ovine alpha(S1)-CN f1-6 and alpha(S2)-CN f182-185 and f186-188; caprine beta-CN f58-65 and alpha(S2)-CN f182-187; buffalo beta-CN f58-66; and a mixture of three tripeptides originating from human beta-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 micro g/ml (100 micro mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC(50)) of some of the crude peptide fractions was very low (16 to 100 micro g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC(50)s were confirmed. An antibacterial peptide corresponding to beta-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 micro g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.

Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

PubMed Central

Zha, Hong-Guang; Flowers, V. Lynn; Yang, Min; Chen, Ling-Yang; Sun, Hang

2012-01-01

Background and Aims To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco species. No function was demonstrated in antimicrobial defence. Therefore, floral nectarins in selfing species maintain their functional significance in reproductive organ development. PMID:22271925

UNIT 10.7 Electroblotting from Polyacrylamide Gels

PubMed Central

Goldman, Aaron; Speicher, David W.

2015-01-01

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications. PMID:26521711

Electroblotting from Polyacrylamide Gels.

PubMed

Goldman, Aaron; Ursitti, Jeanine A; Mozdzanowski, Jacek; Speicher, David W

2015-11-02

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications. Copyright © 2015 John Wiley & Sons, Inc.

A cardioactive peptide from the southern armyworm, Spodoptera eridania.

PubMed

Furuya, K; Hackett, M; Cirelli, M A; Schegg, K M; Wang, H; Shabanowitz, J; Hunt, D F; Schooley, D A

1999-01-01

A cardioactive peptide was isolated from extracts of whole heads of the southern armyworm, Spodoptera eridania. This peptide has the sequence ENFAVGCTPGYQRTADGRCKPTF (Mr = 2516.8), determined from both Edman sequencing and tandem mass spectrometry in combination with off-line micropreparative capillary liquid chromatography. This peptide, termed Spoer-CAP23, has excitatory effects on a semi-isolated heart from larval Manduca sexta, causing an inotropic effect at low concentrations of peptide and chronotropic and inotropic effects at high doses. The threshold concentration for stimulatory effects of the synthetic peptide on the semi-isolated heart was about 1 nM, suggesting a physiological role as a neuropeptide.

A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment.

PubMed

Qin, Chunlin; Huang, Bingzhen; Wygant, James N; McIntyre, Bradley W; McDonald, Charles H; Cook, Richard G; Butler, William T

2006-03-24

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser74, located in the Ser74-Gly75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH2-terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser74-Gly75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.

Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks

PubMed Central

Aguilar, Manuel B.; Luna-Ramírez, Karen S.; Echeverría, Daniel; Falcón, Andrés; Olivera, Baldomero M.; Heimer de la Cotera, Edgar P.; Maillo, María

2008-01-01

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPMγDPLγIIRI-nh2, (γ, gamma-carboxyglutamate;-nh2, amidated C-terminus; calculated monoisotopic mass, 1,468.72 Da; experimental monoisotopic mass, 1,468.70 Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms. PMID:18201803

Purification and characterisation of an antifungal protein, MCha-Pr, from the intercellular fluid of bitter gourd (Momordica charantia) leaves.

PubMed

Zhang, Beibei; Xie, Chengjian; Wei, Yunming; Li, Jing; Yang, Xingyong

2015-03-01

An antifungal protein, designated MCha-Pr, was isolated from the intercellular fluid of bitter gourd (Momordica charantia) leaves during a screen for potent antimicrobial proteins from plants. The isolation procedure involved a combination of extraction, ammonium sulphate precipitation, gel filtration on Bio-Gel P-6, ion exchange chromatography on CM-Sephadex, an additional gel filtration on HiLoad 16/60 Superdex 30, and finally, HPLC on a SOURCE 5RPC column. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that the protein had a molecular mass of 25733.46Da. Automated Edman degradation was used to determine the N-terminal sequence of MCha-Pr, and the amino acid sequence was identified as V-E-Y-T-I-T-G-N-A-G-N-T-P-G-G. The MCha-Pr protein has some similarity to the pathogenesis-related proteins from Atropa belladonna (deadly nightshade), Solanum tuberosum (potato), Ricinus communis (castor bean), and Nicotiana tabacum (tobacco). Analysis of the circular dichroism spectra indicated that MCha-Pr predominantly contains α-helix and β-sheet structures. MCha-Pr had inhibitory effects towards a variety of fungal species and the 50% inhibition of fungal growth (IC50) for Alternaria brassicae, Cercospora personata, Fusarium oxysporum, Mucor sp., and Rhizoctonia solani are 33 μM, 42 μM, 37 μM, 40 μM, and 48 μM, respectively. In addition, this antifungal protein can inhibit the germination of A. brassicae spores at 12.5 μM. These results suggest that MCha-Pr in bitter gourd leaves plays a protective role against phytopathogens and has a wide antimicrobial spectrum. Copyright © 2014 Elsevier Inc. All rights reserved.

Heat shock proteins on the human sperm surface.

PubMed

Naaby-Hansen, Soren; Herr, John C

2010-01-01

The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

Characterization of the novel antifungal protein PgAFP and the encoding gene of Penicillium chrysogenum.

PubMed

Rodríguez-Martín, Andrea; Acosta, Raquel; Liddell, Susan; Núñez, Félix; Benito, M José; Asensio, Miguel A

2010-04-01

The strain RP42C from Penicillium chrysogenum produces a small protein PgAFP that inhibits the growth of some toxigenic molds. The molecular mass of the protein determined by electrospray ionization mass spectrometry (ESI-MS) was 6 494Da. PgAFP showed a cationic character with an estimated pI value of 9.22. Upon chemical and enzymatic treatments of PgAFP, no evidence for N- or O-glycosylations was obtained. Five partial sequences of PgAFP were obtained by Edman degradation and by ESI-MS/MS after trypsin and chymotrypsin digestions. Using degenerate primers from these peptide sequences, a segment of 70bp was amplified by PCR from pgafp gene. 5'- and 3'-ends of pgafp were obtained by RACE-PCR with gene-specific primers designed from the 70bp segment. The complete pgafp sequence of 404bp was obtained using primers designed from 5'- and 3'-ends. Comparison of genomic and cDNA sequences revealed a 279bp coding region interrupted by two introns of 63 and 62bp. The precursor of the antifungal protein consists of 92 amino acids and appears to be processed to the mature 58 amino acids PgAFP. The deduced amino acid sequence of the mature protein shares 79% identity to the antifungal protein Anafp from Aspergillus niger. PgAFP is a new protein that belongs to the group of small, cysteine-rich, and basic proteins with antifungal activity produced by ascomycetes. Given that P. chrysogenum is regarded as safe mold commonly found in foods, PgAFP may be useful to prevent growth of toxigenic molds in food and agricultural products. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Comparative analysis of the XopD T3S effector family in plant pathogenic bacteria

PubMed Central

Kim, Jung-Gun; Taylor, Kyle W.; Mudgett, Mary Beth

2011-01-01

SUMMARY XopD is a type III effector protein that is required for Xanthomonas campestris pathovar vesicatoria (Xcv) growth in tomato. It is a modular protein consisting of an N-terminal DNA-binding domain, two EAR transcriptional repressor motifs, and a C-terminal SUMO protease. In tomato, XopD functions as a transcriptional repressor, resulting in the suppression of defense responses at late stages of infection. A survey of available genome sequences for phytopathogenic bacteria revealed that XopD homologs are limited to species within three Genera of Proteobacteria – Xanthomonas, Acidovorax, and Pseudomonas. While the EAR motif(s) and SUMO protease domain are conserved in all the XopD-like proteins, variation exists in the length and sequence identity of the N-terminal domains. Comparative analysis of the DNA sequences surrounding xopD and xopD-like genes led to revised annotation of the xopD gene. Edman degradation sequence analysis and functional complementation studies confirmed that the xopD gene from Xcv encodes a 760 amino acid protein with a longer N-terminal domain than previously predicted. None of the XopD-like proteins studied complemented Xcv ΔxopD mutant phenotypes in tomato leaves suggesting that the N-terminus of XopD defines functional specificity. Xcv ΔxopD strains expressing chimeric fusion proteins containing the N-terminus of XopD fused to the EAR motif(s) and SUMO protease domain of the XopD-like protein from Xanthomonas campestris pathovar campestris strain B100 were fully virulent in tomato demonstrating that the N-terminus of XopD controls specificity in tomato. PMID:21726373

Purification and Characterization of Plantaricin JLA-9: A Novel Bacteriocin against Bacillus spp. Produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a Traditional Chinese Fermented Cabbage.

PubMed

Zhao, Shengming; Han, Jinzhi; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia

2016-04-06

Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry.

Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

PubMed

Cruz, Ana C B; Massena, Fábio S; Migliolo, Ludovico; Macedo, Leonardo L P; Monteiro, Norberto K V; Oliveira, Adeliana S; Macedo, Francisco P; Uchoa, Adriana F; Grossi de Sá, Maria F; Vasconcelos, Ilka M; Murad, Andre M; Franco, Octavio L; Santos, Elizeu A

2013-09-01

The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Characterization of toxin III of the scorpion Leiurus quinquestriatus quinquestriatus: a new type of alpha-toxin highly toxic both to mammals and insects.

PubMed

Kopeyan, C; Mansuelle, P; Martin-Eauclaire, M F; Rochat, H; Miranda, F

1993-01-01

The primary structure of toxin III of Leiurus quinquestriatus quinquestriatus (Lqq III) was elucidated by automatic Edman degradation of the reduced and S-carboxymethylated protein and derived tryptic peptides. Like other scorpion toxins that are active on sodium channels, Lqq III, consisting of 64 amino acids, is a 7 kDa single-chain polypeptide crosslinked by four disulfide bridges. It belongs to the alpha-toxin group, as judged by competition experiments with 125I AaH II for binding to rat brain synaptosomes (K0.5 = 7 x 10(-7) M). Lqq III is the first alpha-toxin to be characterized that is highly toxic to mice [LD50 = 50 micrograms (7.1 nmol)/kg body wt], by subcutaneous injection, insects Blatella germanica [LD50 = 60 ng (8.5 pmol)/g body wt.] and Musca domestica [LD50 = 120 ng (17 pmol)/g body wt]. When tested via the intracerebroventricular route, the toxicity for mice [55 micrograms (8 nmol)/kg] was of the same order as that found by subcutaneous injection, indicating that Lqq III has a higher affinity for peripheral sodium channels that for those of the central nervous system. There are three differences between the sequences of Lqq III and Lqh alpha IT, an alpha-toxin isolated from the venom of Leiurus quinquestriatus hebraeus. These substitutions are found at positions 20, 24, and 64 (Ser-->Ala,Asp-->Glu and His-->Arg, respectively). Surprisingly Lqh alpha IT is only weakly active in mice [LD50 = 5 mg (0.7 mumol)/kg], while in insects its toxicity is similar to that of Lqq III [140 ng (20 pmol)/g body wt blowfly larvae]. These observations are relevant to the definition of scorpion toxin structure-activity relationships.

The Acheta domesticus Densovirus, Isolated from the European House Cricket, Has Evolved an Expression Strategy Unique among Parvoviruses▿†

PubMed Central

Liu, Kaiyu; Li, Yi; Jousset, Françoise-Xavière; Zadori, Zoltan; Szelei, Jozsef; Yu, Qian; Pham, Hanh Thi; Lépine, François; Bergoin, Max; Tijssen, Peter

2011-01-01

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses. PMID:21775445

New APETx-like peptides from sea anemone Heteractis crispa modulate ASIC1a channels.

PubMed

Kalina, Rimma; Gladkikh, Irina; Dmitrenok, Pavel; Chernikov, Oleg; Koshelev, Sergey; Kvetkina, Aleksandra; Kozlov, Sergey; Kozlovskaya, Emma; Monastyrnaya, Margarita

2018-06-01

Sea anemones are an abundant source of various biologically active peptides. The hydrophobic 20% ethanol fraction of tropical sea anemone Heteractis crispa was shown to contain at least 159 peptide compounds including neurotoxins, proteinase and α-amylase inhibitors, as well as modulators of acid-sensing ion channels (ASICs). The three new peptides, π-AnmTX Hcr 1b-2, -3, and -4 (41 aa) (short names Hcr 1b-2, -3, -4), identified by a combination of reversed-phase liquid chromatography and mass spectrometry were found to belong to the class 1b sea anemone neurotoxins. The amino acid sequences of these peptides were determined by Edman degradation and tandem mass spectrometry. The percent of identity of Hcr 1b-2, -3, and -4 with well-known ASIC3 inhibitors Hcr 1b-1 from H. crispa and APETx2 from Anthopleura elegantissima is 95-78% and 46-49%, respectively. Electrophysiological experiments on homomeric ASIC channels expressed in Xenopus laevis oocytes establish that these peptides are the first inhibitors of ASIC1a derived from sea anemone venom. The major peptide, Hcr 1b-2, inhibited both rASIC1a (IC 50 4.8 ± 0.3 μM; nH 0.92 ± 0.05) and rASIC3 (IC 50 15.9 ± 1.1 μM; nH 1.0 ± 0.05). The maximum inhibition at saturating peptide concentrations reached 64% and 81%, respectively. In the model of acid-induced muscle pain Hcr 1b-2 was also shown to exhibit an antihyperalgesic effect, significantly reducing of the pain threshold of experimental animals. Copyright © 2018 Elsevier Inc. All rights reserved.

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus.

PubMed

García-Gómez, B I; Coronas, F I V; Restano-Cassulini, R; Rodríguez, R R; Possani, L D

2011-07-01

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na+ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K+ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na+ currents verified here, this venom certainly warrant further investigation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Turkish scorpion Buthacus macrocentrus: general characterization of the venom and description of Bu1, a potent mammalian Na⁺-channel α-toxin.

PubMed

Caliskan, F; Quintero-Hernández, V; Restano-Cassulini, R; Batista, C V F; Zamudio, F Z; Coronas, F I; Possani, L D

2012-03-01

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200-4500 Da, and 6000-7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country. Copyright © 2012 Elsevier Ltd. All rights reserved.

Purification and Characterization of a Novel Anti-Campylobacter Bacteriocin Produced by Lactobacillus curvatus DN317.

PubMed

Zommiti, Mohamed; Almohammed, Hamdan; Ferchichi, Mounir

2016-12-01

The lactic acid bacteria (LAB) microbiota of Saudi chicken ceca was determined. From 60 samples, 204 isolates of lactic acid bacteria were obtained. Three isolates produced antimicrobial activities against Campylobacter jejuni, Listeria monocytogenes, and Bacillus subtilis. The isolate DN317, which had the highest activity against Campylobacter jejuni ATCC 33560, was identified as Lactobacillus curvatus (GenBank accession numbers: KX353849 and KX353850). Full inhibitory activity was observed after a 2-h incubation with the supernatant at pH values between 4 and 8. Only 16% of the activity was conserved after a treatment at 121 °C for 15 min. The use of proteinase K, pepsin, chymotrypsin, trypsin, papain, and lysozyme drastically reduced the antimicrobial activity. However, lipase, catalase, and lysozyme had no effect on this activity. The active peptide produced by Lactobacillus curvatus DN317 was purified by precipitation with an 80% saturated ammonium sulfate solution, and two steps of reversed phase HPLC on a C18 column. The molecular weight of this peptide was 4448 Da as determined by MALDI-ToF. N-terminal sequence analysis using Edman degradation revealed 47 amino acid residues (UniProt Knowledgebase accession number C0HK82) revealing homology with the amino acid sequences of sakacin P and curvaticin L442. The antimicrobial activity of the bacteriocin, namely curvaticin DN317, was found to be bacteriostatic against Campylobacter jejuni ATCC 33560. The use of microbial antagonism by LAB is one of the best ways to control microorganisms safely in foods. This result constitutes a reasonable advance in the antimicrobial field because of its potential applications in food technology.

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica*

PubMed Central

Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.

2012-01-01

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

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

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins.

PubMed

Lin, A; McNally, J; Wool, I G

1983-09-10

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.

Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

PubMed Central

Shcherbakova, Larisa A.; Odintsova, Tatyana I.; Stakheev, Alexander A.; Fravel, Deborah R.; Zavriev, Sergey K.

2016-01-01

The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed. PMID:26779237

Echinoderm phosphorylated matrix proteins UTMP16 and UTMP19 have different functions in sea urchin tooth mineralization.

PubMed

Alvares, Keith; Dixit, Saryu N; Lux, Elizabeth; Veis, Arthur

2009-09-18

Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 N HCl after prior removal of non-mineralized constituents with 4.0 M guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.

Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2

PubMed Central

Anagón, Alejandro C.; Molinar, Ricardo R.; Possani, Lourival D.; Fletcher, Paul L.; Cronan, John E.; Julia, Jordi Z.

1980-01-01

The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A2. Amino acid analysis has shown that phospholipase A2 (type 1) has 97 residues with a calculated mol.wt. of 10978±11. Phospholipase A2 (type 2) has 96 residues with a mol.wt. of 10959±11. Phospholipase A2 (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042±31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A2 (type 2). ImagesFig. 1. PMID:7387631

Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.

PubMed

Gimenes, Sarah Natalie Cirilo; Ferreira, Francis Barbosa; Silveira, Ana Carolina Portella; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Izabel Dos Santos, Juliana; Fontes, Marcos Roberto de Mattos; de Campos Brites, Vera Lúcia; Santos, André Luiz Quagliatto; Borges, Márcia Helena; Lopes, Daiana Silva; Rodrigues, Veridiana M

2014-04-01

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved. Copyright © 2014 Elsevier Ltd. All rights reserved.

Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in humans.

PubMed

Poposki, Julie A; Klingler, Aiko I; Stevens, Whitney W; Peters, Anju T; Hulse, Kathryn E; Grammer, Leslie C; Schleimer, Robert P; Welch, Kevin C; Smith, Stephanie S; Sidle, Douglas M; Conley, David B; Tan, Bruce K; Kern, Robert C; Kato, Atsushi

2017-05-01

Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of patients with chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. We sought to investigate the mechanisms of truncation of TSLP in NPs and the function of the truncated products. We incubated recombinant human TSLP with NP extracts, and determined the protein sequence of the truncated forms of TSLP using Edman protein sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. We investigated the functional activity of truncated TSLP using a PBMC-based bioassay. Edman sequencing and mass spectrometry results indicated that NP extracts generated 2 major truncated products, TSLP (residues 29-124) and TSLP (131-159). Interestingly, these 2 products remained linked with disulfide bonds and presented as a dimerized form, TSLP (29-124 + 131-159). We identified that members of the proprotein convertase were rate-limiting enzymes in the truncation of TSLP between residues 130 and 131 and generated a heterodimeric unstable metabolite TSLP (29-130 + 131-159). Carboxypeptidase N immediately digested 6 amino acids from the C terminus of the longer subunit of TSLP to generate a stable dimerized form, TSLP (29-124 + 131-159), in NPs. These truncations were homeostatic but primate-specific events. A metabolite TSLP (29-130 + 131-159) strongly activated myeloid dendritic cells and group 2 innate lymphoid cells compared with mature TSLP. Posttranslational modifications control the functional activity of TSLP in humans and overproduction of TSLP may be a key trigger for the amplification of type 2 inflammation in diseases. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

PubMed Central

Minervini, F.; Algaron, F.; Rizzello, C. G.; Fox, P. F.; Monnet, V.; Gobbetti, M.

2003-01-01

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine αS1-casein (αS1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine αS1-CN f1-6 and αS2-CN f182-185 and f186-188; caprine β-CN f58-65 and αS2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC50) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC50s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin. PMID:12957917

Targeted polypeptide degradation

DOEpatents

Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

2008-05-13

This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

Electrical condition monitoring method for polymers

DOEpatents

Watkins, Jr. Kenneth S.; Morris, Shelby J.; Masakowski, Daniel D.; Wong, Ching Ping; Luo, Shijian

2010-02-16

An electrical condition monitoring method utilizes measurement of electrical resistivity of a conductive composite degradation sensor to monitor environmentally induced degradation of a polymeric product such as insulated wire and cable. The degradation sensor comprises a polymeric matrix and conductive filler. The polymeric matrix may be a polymer used in the product, or it may be a polymer with degradation properties similar to that of a polymer used in the product. The method comprises a means for communicating the resistivity to a measuring instrument and a means to correlate resistivity of the degradation sensor with environmentally induced degradation of the product.

Robust PV Degradation Methodology and Application

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

Jordan, Dirk; Deline, Christopher A; Kurtz, Sarah

The degradation rate plays an important role in predicting and assessing the long-term energy generation of PV systems. Many methods have been proposed for extracting the degradation rate from operational data of PV systems, but most of the published approaches are susceptible to bias due to inverter clipping, module soiling, temporary outages, seasonality, and sensor degradation. In this manuscript, we propose a methodology for determining PV degradation leveraging available modeled clear-sky irradiance data rather than site sensor data, and a robust year-over-year (YOY) rate calculation. We show the method to provide reliable degradation rate estimates even in the case ofmore » sensor drift, data shifts, and soiling. Compared with alternate methods, we demonstrate that the proposed method delivers the lowest uncertainty in degradation rate estimates for a fleet of 486 PV systems.« less

Robust PV Degradation Methodology and Application

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

Jordan, Dirk C.; Deline, Chris; Kurtz, Sarah R.

The degradation rate plays an important role in predicting and assessing the long-term energy generation of photovoltaics (PV) systems. Many methods have been proposed for extracting the degradation rate from operational data of PV systems, but most of the published approaches are susceptible to bias due to inverter clipping, module soiling, temporary outages, seasonality, and sensor degradation. In this paper, we propose a methodology for determining PV degradation leveraging available modeled clear-sky irradiance data rather than site sensor data, and a robust year-over-year rate calculation. We show the method to provide reliable degradation rate estimates even in the case ofmore » sensor drift, data shifts, and soiling. Compared with alternate methods, we demonstrate that the proposed method delivers the lowest uncertainty in degradation rate estimates for a fleet of 486 PV systems.« less

Robust PV Degradation Methodology and Application

DOE PAGES

Jordan, Dirk C.; Deline, Chris; Kurtz, Sarah R.; ...

2017-12-21

The degradation rate plays an important role in predicting and assessing the long-term energy generation of photovoltaics (PV) systems. Many methods have been proposed for extracting the degradation rate from operational data of PV systems, but most of the published approaches are susceptible to bias due to inverter clipping, module soiling, temporary outages, seasonality, and sensor degradation. In this paper, we propose a methodology for determining PV degradation leveraging available modeled clear-sky irradiance data rather than site sensor data, and a robust year-over-year rate calculation. We show the method to provide reliable degradation rate estimates even in the case ofmore » sensor drift, data shifts, and soiling. Compared with alternate methods, we demonstrate that the proposed method delivers the lowest uncertainty in degradation rate estimates for a fleet of 486 PV systems.« less

Recovering of images degraded by atmosphere

NASA Astrophysics Data System (ADS)

Lin, Guang; Feng, Huajun; Xu, Zhihai; Li, Qi; Chen, Yueting

2017-08-01

Remote sensing images are seriously degraded by multiple scattering and bad weather. Through the analysis of the radiative transfer procedure in atmosphere, an image atmospheric degradation model considering the influence of atmospheric absorption multiple scattering and non-uniform distribution is proposed in this paper. Based on the proposed model, a novel recovering method is presented to eliminate atmospheric degradation. Mean-shift image segmentation and block-wise deconvolution are used to reduce time cost, retaining a good result. The recovering results indicate that the proposed method can significantly remove atmospheric degradation and effectively improve contrast compared with other removal methods. The results also illustrate that our method is suitable for various degraded remote sensing, including images with large field of view (FOV), images taken in side-glance situations, image degraded by atmospheric non-uniform distribution and images with various forms of clouds.

Statistical Bayesian method for reliability evaluation based on ADT data

NASA Astrophysics Data System (ADS)

Lu, Dawei; Wang, Lizhi; Sun, Yusheng; Wang, Xiaohong

2018-05-01

Accelerated degradation testing (ADT) is frequently conducted in the laboratory to predict the products’ reliability under normal operating conditions. Two kinds of methods, degradation path models and stochastic process models, are utilized to analyze degradation data and the latter one is the most popular method. However, some limitations like imprecise solution process and estimation result of degradation ratio still exist, which may affect the accuracy of the acceleration model and the extrapolation value. Moreover, the conducted solution of this problem, Bayesian method, lose key information when unifying the degradation data. In this paper, a new data processing and parameter inference method based on Bayesian method is proposed to handle degradation data and solve the problems above. First, Wiener process and acceleration model is chosen; Second, the initial values of degradation model and parameters of prior and posterior distribution under each level is calculated with updating and iteration of estimation values; Third, the lifetime and reliability values are estimated on the basis of the estimation parameters; Finally, a case study is provided to demonstrate the validity of the proposed method. The results illustrate that the proposed method is quite effective and accuracy in estimating the lifetime and reliability of a product.

Method of radiation degradation of PTFE under vacuum conditions

NASA Astrophysics Data System (ADS)

Korenev, Sergey

2004-09-01

A new method of radiation degradation of Polytetrafluoroethylene (PTFE) under vacuum conditions is considered in this report. The combination of glow gas discharge and electrical surface discharge (on surface and inside PTFE) increases the efficiency of thermal-radiation degradation. The main mechanism of this degradation method consists of the breaking of C-C and C-F bonds. The vacuum conditions allow decreasing of the concentration of toxic compounds, such as a HF. Experimental results for degradation of PTFE are presented.

A hybrid degradation tendency measurement method for mechanical equipment based on moving window and Grey-Markov model

NASA Astrophysics Data System (ADS)

Jiang, Wei; Zhou, Jianzhong; Zheng, Yang; Liu, Han

2017-11-01

Accurate degradation tendency measurement is vital for the secure operation of mechanical equipment. However, the existing techniques and methodologies for degradation measurement still face challenges, such as lack of appropriate degradation indicator, insufficient accuracy, and poor capability to track the data fluctuation. To solve these problems, a hybrid degradation tendency measurement method for mechanical equipment based on a moving window and Grey-Markov model is proposed in this paper. In the proposed method, a 1D normalized degradation index based on multi-feature fusion is designed to assess the extent of degradation. Subsequently, the moving window algorithm is integrated with the Grey-Markov model for the dynamic update of the model. Two key parameters, namely the step size and the number of states, contribute to the adaptive modeling and multi-step prediction. Finally, three types of combination prediction models are established to measure the degradation trend of equipment. The effectiveness of the proposed method is validated with a case study on the health monitoring of turbine engines. Experimental results show that the proposed method has better performance, in terms of both measuring accuracy and data fluctuation tracing, in comparison with other conventional methods.

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

PubMed Central

Eberhardt, S; Korn, S; Lottspeich, F; Bacher, A

1997-01-01

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. PMID:9139911

Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme.

PubMed

Mukherjee, J J; Dekker, E E

1987-10-25

Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase). This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000. Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093. Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids. It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm. Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction. Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5. Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.

Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions.

PubMed

Gusmão, Karla A G; Dos Santos, Daniel M; Santos, Virgílio M; Cortés, María Esperanza; Reis, Pablo V M; Santos, Vera L; Piló-Veloso, Dorila; Verly, Rodrigo M; de Lima, Maria Elena; Resende, Jarbas M

2017-01-01

The availability of antimicrobial peptides from several different natural sources has opened an avenue for the discovery of new biologically active molecules. To the best of our knowledge, only two peptides isolated from the frog Leptodactylus labyrinthicus , namely pentadactylin and ocellatin-F1, have shown antimicrobial activities. Therefore, in order to explore the antimicrobial potential of this species, we have investigated the biological activities and membrane interactions of three peptides isolated from the anuran skin secretion. Three peptide primary structures were determined by automated Edman degradation. These sequences were prepared by solid-phase synthesis and submitted to activity assays against gram-positive and gram-negative bacteria and against two fungal strains. The hemolytic properties of the peptides were also investigated in assays with rabbit blood erythrocytes. The conformational preferences of the peptides and their membrane interactions have been investigated by circular dichroism spectroscopy and liposome dye release assays. The amino acid compositions of three ocellatins were determined and the sequences exhibit 100% homology for the first 22 residues (ocellatin-LB1 sequence). Ocellatin-LB2 carries an extra Asn residue and ocellatin-F1 extra Asn-Lys-Leu residues at C-terminus. Ocellatin-F1 presents a stronger antibiotic potential and a broader spectrum of activities compared to the other peptides. The membrane interactions and pore formation capacities of the peptides correlate directly with their antimicrobial activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. All peptides acquire high helical contents in membrane environments. However, ocellatin-F1 shows in average stronger helical propensities. The obtained results indicate that the three extra amino acid residues at the ocellatin-F1 C-terminus play an important role in promoting stronger peptide-membrane interactions and antimicrobial properties. The extra Asn-23 residue present in ocellatin-LB2 sequence seems to decrease its antimicrobial potential and the strength of the peptide-membrane interactions.

Structural characterization of a novel neuropeptide from the central nervous system of the leech Erpobdella octoculata. The leech osmoregulator factor.

PubMed

Salzet, M; Bulet, P; Weber, W M; Clauss, W; Verger-Bocquet, M; Malecha, J

1996-03-22

Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme-linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 19 kDa for in vitro central nervous system-translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin), confirming that LORF did not belong to the oxytocin/vasopressin family.

Investigation of the protein osteocalcin of Camelops hesternus: Sequence, structure and phylogenetic implications

NASA Astrophysics Data System (ADS)

Humpula, James F.; Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Stafford, Thomas W.; Smith, James J.; Voorhies, Michael R.; George Corner, R.; Andrews, Phillip C.

2007-12-01

Ancient DNA sequences offer an extraordinary opportunity to unravel the evolutionary history of ancient organisms. Protein sequences offer another reservoir of genetic information that has recently become tractable through the application of mass spectrometric techniques. The extent to which ancient protein sequences resolve phylogenetic relationships, however, has not been explored. We determined the osteocalcin amino acid sequence from the bone of an extinct Camelid (21 ka, Camelops hesternus) excavated from Isleta Cave, New Mexico and three bones of extant camelids: bactrian camel ( Camelus bactrianus); dromedary camel ( Camelus dromedarius) and guanaco ( Llama guanacoe) for a diagenetic and phylogenetic assessment. There was no difference in sequence among the four taxa. Structural attributes observed in both modern and ancient osteocalcin include a post-translation modification, Hyp 9, deamidation of Gln 35 and Gln 39, and oxidation of Met 36. Carbamylation of the N-terminus in ancient osteocalcin may result in blockage and explain previous difficulties in sequencing ancient proteins via Edman degradation. A phylogenetic analysis using osteocalcin sequences of 25 vertebrate taxa was conducted to explore osteocalcin protein evolution and the utility of osteocalcin sequences for delineating phylogenetic relationships. The maximum likelihood tree closely reflected generally recognized taxonomic relationships. For example, maximum likelihood analysis recovered rodents, birds and, within hominins, the Homo-Pan-Gorilla trichotomy. Within Artiodactyla, character state analysis showed that a substitution of Pro 4 for His 4 defines the Capra-Ovis clade within Artiodactyla. Homoplasy in our analysis indicated that osteocalcin evolution is not a perfect indicator of species evolution. Limited sequence availability prevented assigning functional significance to sequence changes. Our preliminary analysis of osteocalcin evolution represents an initial step towards a complete character analysis aimed at determining the evolutionary history of this functionally significant protein. We emphasize that ancient protein sequencing and phylogenetic analyses using amino acid sequences must pay close attention to post-translational modifications, amino acid substitutions due to diagenetic alteration and the impacts of isobaric amino acids on mass shifts and sequence alignments.

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog*

PubMed Central

Jayakar, Selwyn S.; Dailey, William P.; Eckenhoff, Roderic G.; Cohen, Jonathan B.

2013-01-01

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [3H]AziPm, and labeled amino acids were identified by Edman degradation. [3H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [3H]AziPm photolabeling at αM2-10′. Propofol inhibited [3H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 μm) than it inhibited ion channel photolabeling (IC50 = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site. PMID:23300078

Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

PubMed

Skubatz, Hanna; Howald, William N

2013-03-01

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

Novel haemoglobin-derived antimicrobial peptides from chicken (Gallus gallus) blood: purification, structural aspects and biological activity.

PubMed

Vasilchenko, A S; Rogozhin, E A; Vasilchenko, A V; Kartashova, O L; Sycheva, M V

2016-12-01

To purify and characterize antimicrobial peptides derived from the acid extract of Gallus gallus blood cells. Two polypeptides (i.e. CHb-1 and CHb-2) with antibacterial activity were detected in the acidic extract of blood cells from chicken (G. gallus). The isolated peptides that possessed a potent antibacterial activity were purified using a two-step chromatography procedure that involved solid-phase extraction of a total protein/peptide extract followed by thin fractionation by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the purified peptides were similar and were 4824·4 and 4825·2 Da, which have been measured by matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS). Their amino acid sequences were determined by Edman degradation and showed that the peptides were fully identical to the two fragments of G. gallus α-haemoglobin localized into different subunits (A and D respectively). The peptides were active in micromolar concentrations against Gram-negative Escherichia coli K12 TG1. Using the 1-N-phenylnaphthylamine, the FITC-dextran labelled probes and the live/dead staining allowed to show the hemocidin mode of action and estimate the pore size. In this study, for the first time, α-haemoglobin from chicken (G. gallus) has been investigated as a donor of the two high homologous native peptide fragments that possess potent antibacterial activity in vitro. These are membrane-active peptides and their mechanism of action against E. coli involves a toroidal pore formation. The obtained results expand the perception of the role of haemoglobin in a living system, describing it as a source of multifunction substances. Additionally, the data presented in this paper may contribute to the development of new, cost-effective, antimicrobial agents. © 2016 The Society for Applied Microbiology.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.

PubMed

Silva, Fredy D A; Vasconcelos, Ilka M; Lobo, Marina D P; de Castro, Patrícia G; Magalhães, Vladimir G; de Freitas, Cléverson D T; Carlini, Célia R R S; Pinto, Paulo M; Beltramini, Leila M; Filho, José H A; Barros, Eduardo B; Alencar, Luciana M R; Grangeiro, Thalles B; Oliveira, José T A

2012-07-01

Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.

Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

PubMed Central

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

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

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

PubMed

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

2013-01-01

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

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco.

PubMed

Achemchem, F; Martínez-Bueno, M; Abrini, J; Valdivia, E; Maqueda, M

2005-01-01

Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.

DEPRESSION AND INTERNALLY DIRECTED AGGRESSION: GENETIC AND ENVIRONMENTAL CONTRIBUTIONS

PubMed Central

Haddad, Suzanne K.; Neiderhiser, Jenae M.; Spotts, Erica L.; Ganiban, Jody; Lichtenstein, Paul; Reiss, David

2013-01-01

This study uses behavior genetic (BG) methodology to investigate Freud’s theory of depression as aggression directed toward the self (1930) and the extent to which genetically and environmentally influenced aggressive tendencies contribute to depressive symptoms. Data from the Twin and Offspring Study in Sweden (TOSS) is used to demonstrate how, in estimating shared and unique environmental influences, BG methods can inform psychoanalytic theory and practice, particularly because of their shared emphasis on the importance of individual experience in development. The TOSS sample consists of 909 pairs of adult twins, their partners, and one adolescent child per couple. The Center for Epidemiologic Studies Depression Scale (Radloff 1977) was used to measure depressive symptoms and the Karolinska Scales of Personality (Schalling and Edman 1993) to measure internally directed aggression. Genetic analyses indicated that for both men and women, their unique experiences as well as genetic factors contributed equally to the association between internally directed aggression and depressive symptoms. These findings support Freud’s theory that constitutionally based differences in aggression, along with individual experiences, contribute to a person’s depressive symptoms. Establishing that an individual’s unique, not shared, experiences and perceptions contribute to depressive symptoms and internally directed aggression reinforces the use of patient-specific treatment approaches implemented in psychoanalytic psychotherapy or psychoanalysis. PMID:18515705

Validated stability-indicating spectrophotometric methods for the determination of Silodosin in the presence of its degradation products.

PubMed

Boltia, Shereen A; Abdelkawy, Mohammed; Mohammed, Taghreed A; Mostafa, Nahla N

2018-09-05

Five simple, rapid, accurate, and precise spectrophotometric methods are developed for the determination of Silodosin (SLD) in the presence of its acid induced and oxidative induced degradation products. Method A is based on dual wavelength (DW) method; two wavelengths are selected at which the absorbance of the oxidative induced degradation product is the same, so wavelengths 352 and 377 nm are used to determine SLD in the presence of its oxidative induced degradation product. Method B depends on induced dual wavelength theory (IDW), which is based on selecting two wavelengths on the zero-order spectrum of SLD where the difference in absorbance between them for the spectrum of acid induced degradation products is not equal to zero so through multiplying by the equality factor, the absorption difference is made to be zero for the acid induced degradation product while it is still significant for SLD. Method C is first derivative ( 1 D) spectrophotometry of SLD and its degradation products. Peak amplitudes are measured at 317 and 357 nm. Method D is ratio difference spectrophotometry (RD) where the drug is determined by the difference in amplitude between two selected wavelengths, at 350 and 277 nm for the ratio spectrum of SLD and its acid induced degradation products while for the ratio spectrum of SLD and its oxidative induced degradation products the difference in amplitude is measured at 345 and 292 nm. Method E depends on measuring peak amplitudes of the first derivative of the ratio ( 1 DD) where peak amplitudes are measured at 330 nm in the presence of the acid induced degradation product and measured by peak to peak technique at 326 and 369 nm in the presence of the oxidative induced degradation product. The proposed methods are validated according to ICH recommendations. The calibration curves for all the proposed methods are linear over a concentration range of 5-70 μg/mL. The selectivity of the proposed methods was tested using different laboratory prepared mixtures of SLD with either its acid induced or oxidative induced degradation products showing specificity of SLD with accepted recovery values. The proposed methods have been successfully applied to the analysis of SLD in pharmaceutical dosage forms without interference from additives. Copyright © 2018 Elsevier B.V. All rights reserved.

Remaining useful life prediction of degrading systems subjected to imperfect maintenance: Application to draught fans

NASA Astrophysics Data System (ADS)

Wang, Zhao-Qiang; Hu, Chang-Hua; Si, Xiao-Sheng; Zio, Enrico

2018-02-01

Current degradation modeling and remaining useful life prediction studies share a common assumption that the degrading systems are not maintained or maintained perfectly (i.e., to an as-good-as new state). This paper concerns the issues of how to model the degradation process and predict the remaining useful life of degrading systems subjected to imperfect maintenance activities, which can restore the health condition of a degrading system to any degradation level between as-good-as new and as-bad-as old. Toward this end, a nonlinear model driven by Wiener process is first proposed to characterize the degradation trajectory of the degrading system subjected to imperfect maintenance, where negative jumps are incorporated to quantify the influence of imperfect maintenance activities on the system's degradation. Then, the probability density function of the remaining useful life is derived analytically by a space-scale transformation, i.e., transforming the constructed degradation model with negative jumps crossing a constant threshold level to a Wiener process model crossing a random threshold level. To implement the proposed method, unknown parameters in the degradation model are estimated by the maximum likelihood estimation method. Finally, the proposed degradation modeling and remaining useful life prediction method are applied to a practical case of draught fans belonging to a kind of mechanical systems from steel mills. The results reveal that, for a degrading system subjected to imperfect maintenance, our proposed method can obtain more accurate remaining useful life predictions than those of the benchmark model in literature.

Validated stability-indicating spectrophotometric methods for the determination of cefixime trihydrate in the presence of its acid and alkali degradation products.

PubMed

Mostafa, Nadia M; Abdel-Fattah, Laila; Weshahy, Soheir A; Hassan, Nagiba Y; Boltia, Shereen A

2015-01-01

Five simple, accurate, precise, and economical spectrophotometric methods have been developed for the determination of cefixime trihydrate (CFX) in the presence of its acid and alkali degradation products without prior separation. In the first method, second derivative (2D) and first derivative (1D) spectrophotometry was applied to the absorption spectra of CFX and its acid (2D) or alkali (1D) degradation products by measuring the amplitude at 289 and 308 nm, respectively. The second method was a first derivative (1DD) ratio spectrophotometric method where the peak amplitudes were measured at 311 nm in presence of the acid degradation product, and 273 and 306 nm in presence of its alkali degradation product. The third method was ratio subtraction spectrophotometry where the drug is determined at 286 nm in laboratory-prepared mixtures of CFX and its acid or alkali degradation product. The fourth method was based on dual wavelength analysis; two wavelengths were selected at which the absorbances of one component were the same, so wavelengths 209 and 252 nm were used to determine CFX in presence of its acid degradation product and 310 and 321 nm in presence of its alkali degradation product. The fifth method was bivariate spectrophotometric calibration based on four linear regression equations obtained at the wavelengths 231 and 290 nm, and 231 and 285 nm for the binary mixture of CFX with either its acid or alkali degradation product, respectively. The developed methods were successfully applied to the analysis of CFX in laboratory-prepared mixtures and pharmaceutical formulations with good recoveries, and their validation was carried out following the International Conference on Harmonization guidelines. The results obtained were statistically compared with each other and showed no significant difference with respect to accuracy and precision.

De novo prediction of the genomic components and capabilities for microbial plant biomass degradation from (meta-)genomes

PubMed Central

2013-01-01

Background Understanding the biological mechanisms used by microorganisms for plant biomass degradation is of considerable biotechnological interest. Despite of the growing number of sequenced (meta)genomes of plant biomass-degrading microbes, there is currently no technique for the systematic determination of the genomic components of this process from these data. Results We describe a computational method for the discovery of the protein domains and CAZy families involved in microbial plant biomass degradation. Our method furthermore accurately predicts the capability to degrade plant biomass for microbial species from their genome sequences. Application to a large, manually curated data set of microbial degraders and non-degraders identified gene families of enzymes known by physiological and biochemical tests to be implicated in cellulose degradation, such as GH5 and GH6. Additionally, genes of enzymes that degrade other plant polysaccharides, such as hemicellulose, pectins and oligosaccharides, were found, as well as gene families which have not previously been related to the process. For draft genomes reconstructed from a cow rumen metagenome our method predicted Bacteroidetes-affiliated species and a relative to a known plant biomass degrader to be plant biomass degraders. This was supported by the presence of genes encoding enzymatically active glycoside hydrolases in these genomes. Conclusions Our results show the potential of the method for generating novel insights into microbial plant biomass degradation from (meta-)genome data, where there is an increasing production of genome assemblages for uncultured microbes. PMID:23414703

A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant.

PubMed

Lalitha Devi, M; Chandrasekhar, K B

2009-12-05

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

Liquid chromatography/tandem mass spectrometry study of forced degradation of azilsartan medoxomil potassium.

PubMed

Swain, Debasish; Patel, Prinesh N; Palaniappan, Ilayaraja; Sahu, Gayatri; Samanthula, Gananadhamu

2015-08-15

Azilsartan medoxomil potassium (AZM) is a new antihypertensive drug introduced in the year 2011. The presence of degradation products not only affects the quality, but also the safety aspects of the drug. Thus, it is essential to develop an efficient analytical method which could be useful to selectively separate and identify the degradation products of azilsartan medoxomil potassium. AZM was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions. Separation of the drug and degradation products was achieved by a liquid chromatography (LC) method using an Acquity UPLC(®) C18 CSH column with mobile phase consisting of 0.02% trifluoroacetic acid and acetonitrile using a gradient method. Identification and characterization of the degradation products was carried out using LC/electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). A total of five degradation products (DP 1 to DP 5) were formed under various stress conditions and their structures were proposed with the help of tandem mass spectrometry (MS/MS) experiments and accurate mass data. A common degradation product (DP 4) was observed under all the degradation conditions. DP 1, DP 2 and DP 5 were observed under acid hydrolytic conditions whereas DP 3 was observed under alkaline conditions. AZM was found to degrade under hydrolytic, oxidative and photolytic stress conditions. The structures of all the degradation products were proposed. The degradation pathway for the formation of degradation products was also hypothesized. A selective method was developed to quantify the drug in the presence of degradation products which is useful to monitor the quality of AZM. Copyright © 2015 John Wiley & Sons, Ltd.

A stability indicating HPLC method for determination of mebeverine in the presence of its degradation products and kinetic study of its degradation in oxidative condition

PubMed Central

Souri, E.; Aghdami, A. Negahban; Adib, N.

2014-01-01

An HPLC method for determination of mebeverine hydrochloride (MH) in the presence of its degradation products was developed. The degradation of MH was studied under hydrolysis, oxidative and photolysis stress conditions. Under alkaline, acidic and oxidative conditions, degradation of MH was observed. The separation was performed using a Symmetry C18 column and a mixture of 50 mM KH2PO4, acetonitrile and tetrahydrfuran (THF) (63:35:2; v/v/v) as the mobile phase. No interference peaks from degradation products in acidic, alkaline and oxidative conditions were observed. The linearity, accuracy and precision of the method were studied. The method was linear over the range of 1-100 μg/ml MH (r2>0.999) and the CV values for intra-day and inter-day variations were in the range of 1.0-1.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method were 1.0 and 0.2 μg/ml, respectively. Determination of MH in pharmaceutical dosage forms was performed using the developed method. Furthermore the kinetics of the degradation of MH in the presence of hydrogen peroxide was investigated. The proposed method could be a suitable method for routine quality control studies of mebeverine dosage forms. PMID:25657790

A stability indicating HPLC method for determination of mebeverine in the presence of its degradation products and kinetic study of its degradation in oxidative condition.

PubMed

Souri, E; Aghdami, A Negahban; Adib, N

2014-01-01

An HPLC method for determination of mebeverine hydrochloride (MH) in the presence of its degradation products was developed. The degradation of MH was studied under hydrolysis, oxidative and photolysis stress conditions. Under alkaline, acidic and oxidative conditions, degradation of MH was observed. The separation was performed using a Symmetry C18 column and a mixture of 50 mM KH2PO4, acetonitrile and tetrahydrfuran (THF) (63:35:2; v/v/v) as the mobile phase. No interference peaks from degradation products in acidic, alkaline and oxidative conditions were observed. The linearity, accuracy and precision of the method were studied. The method was linear over the range of 1-100 μg/ml MH (r(2)>0.999) and the CV values for intra-day and inter-day variations were in the range of 1.0-1.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method were 1.0 and 0.2 μg/ml, respectively. Determination of MH in pharmaceutical dosage forms was performed using the developed method. Furthermore the kinetics of the degradation of MH in the presence of hydrogen peroxide was investigated. The proposed method could be a suitable method for routine quality control studies of mebeverine dosage forms.

Comparing Standard and Selective Degradation DNA Extraction Methods: Results from a Field Experiment with Sexual Assault Kits^.

PubMed

Campbell, Rebecca; Pierce, Steven J; Sharma, Dhruv B; Shaw, Jessica; Feeney, Hannah; Nye, Jeffrey; Schelling, Kristin; Fehler-Cabral, Giannina

2017-01-01

A growing number of U.S. cities have large numbers of untested sexual assault kits (SAKs) in police property facilities. Testing older kits and maintaining current case work will be challenging for forensic laboratories, creating a need for more efficient testing methods. We evaluated selective degradation methods for DNA extraction using actual case work from a sample of previously unsubmitted SAKs in Detroit, Michigan. We randomly assigned 350 kits to either standard or selective degradation testing methods and then compared DNA testing rates and CODIS entry rates between the two groups. Continuation-ratio modeling showed no significant differences, indicating that the selective degradation method had no decrement in performance relative to customary methods. Follow-up equivalence tests indicated that CODIS entry rates for the two methods could differ by more than ±5%. Selective degradation methods required less personnel time for testing and scientific review than standard testing. © 2016 American Academy of Forensic Sciences.

A novel mechanochemical method for reconstructing the moisture-degraded HKUST-1.

PubMed

Sun, Xuejiao; Li, Hao; Li, Yujie; Xu, Feng; Xiao, Jing; Xia, Qibin; Li, Yingwei; Li, Zhong

2015-07-11

A novel mechanochemical method was proposed to reconstruct quickly moisture-degraded HKUST-1. The degraded HKUST-1 can be restored within minutes. The reconstructed samples were characterized, and confirmed to have 95% surface area and 92% benzene capacity of the fresh HKUST-1. It is a simple and effective strategy for degraded MOF reconstruction.

Methods And Systms For Analyzing The Degradation And Failure Of Mechanical Systems

DOEpatents

Jarrell, Donald B.; Sisk, Daniel R.; Hatley, Darrel D.; Kirihara, Leslie J.; Peters, Timothy J.

2005-02-08

Methods and systems for identifying, understanding, and predicting the degradation and failure of mechanical systems are disclosed. The methods include measuring and quantifying stressors that are responsible for the activation of degradation mechanisms in the machine component of interest. The intensity of the stressor may be correlated with the rate of physical degradation according to some determinable function such that a derivative relationship exists between the machine performance, degradation, and the underlying stressor. The derivative relationship may be used to make diagnostic and prognostic calculations concerning the performance and projected life of the machine. These calculations may be performed in real time to allow the machine operator to quickly adjust the operational parameters of the machinery in order to help minimize or eliminate the effects of the degradation mechanism, thereby prolonging the life of the machine. Various systems implementing the methods are also disclosed.

Current remote sensing approaches to monitoring forest degradation in support of countries measurement, reporting and verification (MRV) systems for REDD.

PubMed

Mitchell, Anthea L; Rosenqvist, Ake; Mora, Brice

2017-12-01

Forest degradation is a global phenomenon and while being an important indicator and precursor to further forest loss, carbon emissions due to degradation should also be accounted for in national reporting within the frame of UN REDD+. At regional to country scales, methods have been progressively developed to detect and map forest degradation, with these based on multi-resolution optical, synthetic aperture radar (SAR) and/or LiDAR data. However, there is no one single method that can be applied to monitor forest degradation, largely due to the specific nature of the degradation type or process and the timeframe over which it is observed. The review assesses two main approaches to monitoring forest degradation: first, where detection is indicated by a change in canopy cover or proxies, and second, the quantification of loss (or gain) in above ground biomass (AGB). The discussion only considers degradation that has a visible impact on the forest canopy and is thus detectable by remote sensing. The first approach encompasses methods that characterise the type of degradation and track disturbance, detect gaps in, and fragmentation of, the forest canopy, and proxies that provide evidence of forestry activity. Progress in these topics has seen the extension of methods to higher resolution (both spatial and temporal) data to better capture the disturbance signal, distinguish degraded and intact forest, and monitor regrowth. Improvements in the reliability of mapping methods are anticipated by SAR-optical data fusion and use of very high resolution data. The second approach exploits EO sensors with known sensitivity to forest structure and biomass and discusses monitoring efforts using repeat LiDAR and SAR data. There has been progress in the capacity to discriminate forest age and growth stage using data fusion methods and LiDAR height metrics. Interferometric SAR and LiDAR have found new application in linking forest structure change to degradation in tropical forests. Estimates of AGB change have been demonstrated at national level using SAR and LiDAR-assisted approaches. Future improvements are anticipated with the availability of next generation LiDAR sensors. Improved access to relevant satellite data and best available methods are key to operational forest degradation monitoring. Countries will need to prioritise their monitoring efforts depending on the significance of the degradation, balanced against available resources. A better understanding of the drivers and impacts of degradation will help guide monitoring and restoration efforts. Ultimately we want to restore ecosystem service and function in degraded forests before the change is irreversible.

Practical Approaches to Forced Degradation Studies of Vaccines.

PubMed

Hasija, Manvi; Aboutorabian, Sepideh; Rahman, Nausheen; Ausar, Salvador F

2016-01-01

During the early stages of vaccine development, forced degradation studies are conducted to provide information about the degradation properties of vaccine formulations. In addition to supporting the development of analytical methods for the detection of degradation products, these stress studies are used to identify optimal long-term storage conditions and are part of the regulatory requirements for the submission of stability data. In this chapter, we provide detailed methods for forced degradation analysis under thermal, light, and mechanical stress conditions.

A quantitative PCR approach for quantification of functional genes involved in the degradation of polycyclic aromatic hydrocarbons in contaminated soils.

PubMed

Shahsavari, Esmaeil; Aburto-Medina, Arturo; Taha, Mohamed; Ball, Andrew S

2016-01-01

Polycyclic aromatic hydrocarbons (PAHs) are major pollutants globally and due to their carcinogenic and mutagenic properties their clean-up is paramount. Bioremediation or using PAH degrading microorganisms (mainly bacteria) to degrade the pollutants represents cheap, effective methods. These PAH degraders harbor functional genes which help microorganisms use PAHs as source of food and energy. Most probable number (MPN) and plate counting methods are widely used for counting PAHs degraders; however, as culture based methods only count a small fraction (<1%) of microorganisms capable of carrying out PAH degradation, the use of culture-independent methodologies is desirable.•This protocol presents a robust, rapid and sensitive qPCR method for the quantification of the functional genes involved in the degradation of PAHs in soil samples.•This protocol enables us to screen a vast number of PAH contaminated soil samples in few hours.•This protocol provides valuable information about the natural attenuation potential of contaminated soil and can be used to monitor the bioremediation process.

Comparison of oxytetracycline degradation behavior in pig manure with different antibiotic addition methods.

PubMed

Wang, Yan; Chen, Guixiu; Liang, Juanboo; Zou, Yongde; Wen, Xin; Liao, Xindi; Wu, Yinbao

2015-12-01

Using manure collected from swine fed with diet containing antibiotics and antibiotic-free swine manure spiked with antibiotics are the two common methods of studying the degradation behavior of veterinary antibiotic in manure in the environment. However, few studies had been conducted to co-compare these two different antibiotic addition methods. This study used oxytetracycline (OTC) as a model antibiotic to study antibiotic degradation behavior in manure under the above two OTC addition methods. In addition, the role of microorganisms present in the manure on degradation behavior was also examined. The results showed that degradation half-life of OTC in manure from swine fed OTC (9.04 days) was significantly shorter than that of the manure directly treated with OTC (9.65 days). Concentration of 4-epi-OTC in manure from swine fed OTC peaked earlier than that in manure spiked with OTC, and the degradation rates of 4-epi-OTC and α-apo-OTC in the manure from swine fed OTC were faster, but the peak concentrations were lower, than those in manure spiked with OTC. Bacterial diversity and relative abundance of Bacillus cereus data demonstrated that sterilization of the manure before experiment significantly decreased OTC degradation rate in both of the addition methods. Results of the present study demonstrated that the presence of the metabolites (especially 4-epi-OTC) and microorganisms had significant influence on OTC degradation.

Stability-indicating spectrophotometric methods for determination of the anticoagulant drug apixaban in the presence of its hydrolytic degradation product

NASA Astrophysics Data System (ADS)

Tantawy, Mahmoud A.; El-Ragehy, Nariman A.; Hassan, Nagiba Y.; Abdelkawy, Mohamed

2016-04-01

Apixaban (a novel anticoagulant agent) was subjected to a stress stability study including acid, alkali, oxidative, photolytic, and thermal degradation. The drug was found to be only liable to acidic and alkaline hydrolysis. The degradation product was then isolated and identified by IR and GC-mass spectrometry. Four spectrophotometric methods, namely; first derivative (D1), derivative ratio (DR), ratio difference (RD) and mean centering of ratio spectra (MCR), have been suggested for the determination of apixaban in presence of its hydrolytic degradation product. The proposed methods do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined, and the methods were validated as per ICH guidelines and the specificity was assessed by analyzing synthetic mixtures containing different percentages of the degradation product with the drug. The developed methods were successfully applied for the determination of apixaban in bulk powder and its tablet dosage form.

Stability-indicating spectrophotometric methods for determination of the anticoagulant drug apixaban in the presence of its hydrolytic degradation product.

PubMed

Tantawy, Mahmoud A; El-Ragehy, Nariman A; Hassan, Nagiba Y; Abdelkawy, Mohamed

2016-04-15

Apixaban (a novel anticoagulant agent) was subjected to a stress stability study including acid, alkali, oxidative, photolytic, and thermal degradation. The drug was found to be only liable to acidic and alkaline hydrolysis. The degradation product was then isolated and identified by IR and GC-mass spectrometry. Four spectrophotometric methods, namely; first derivative (D(1)), derivative ratio (DR), ratio difference (RD) and mean centering of ratio spectra (MCR), have been suggested for the determination of apixaban in presence of its hydrolytic degradation product. The proposed methods do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined, and the methods were validated as per ICH guidelines and the specificity was assessed by analyzing synthetic mixtures containing different percentages of the degradation product with the drug. The developed methods were successfully applied for the determination of apixaban in bulk powder and its tablet dosage form. Copyright © 2016 Elsevier B.V. All rights reserved.

Bivariate versus multivariate smart spectrophotometric calibration methods for the simultaneous determination of a quaternary mixture of mosapride, pantoprazole and their degradation products.

PubMed

Hegazy, M A; Yehia, A M; Moustafa, A A

2013-05-01

The ability of bivariate and multivariate spectrophotometric methods was demonstrated in the resolution of a quaternary mixture of mosapride, pantoprazole and their degradation products. The bivariate calibrations include bivariate spectrophotometric method (BSM) and H-point standard addition method (HPSAM), which were able to determine the two drugs, simultaneously, but not in the presence of their degradation products, the results showed that simultaneous determinations could be performed in the concentration ranges of 5.0-50.0 microg/ml for mosapride and 10.0-40.0 microg/ml for pantoprazole by bivariate spectrophotometric method and in the concentration ranges of 5.0-45.0 microg/ml for both drugs by H-point standard addition method. Moreover, the applied multivariate calibration methods were able for the determination of mosapride, pantoprazole and their degradation products using concentration residuals augmented classical least squares (CRACLS) and partial least squares (PLS). The proposed multivariate methods were applied to 17 synthetic samples in the concentration ranges of 3.0-12.0 microg/ml mosapride, 8.0-32.0 microg/ml pantoprazole, 1.5-6.0 microg/ml mosapride degradation products and 2.0-8.0 microg/ml pantoprazole degradation products. The proposed bivariate and multivariate calibration methods were successfully applied to the determination of mosapride and pantoprazole in their pharmaceutical preparations.

Effect of actinobacteria agent inoculation methods on cellulose degradation during composting based on redundancy analysis.

PubMed

Zhao, Yue; Lu, Qian; Wei, Yuquan; Cui, Hongyang; Zhang, Xu; Wang, Xueqin; Shan, Si; Wei, Zimin

2016-11-01

In this study, actinobacteria agent including Streptomyces sp. and Micromonospora sp. were inoculated during chicken manure composting by different inoculation methods. The effect of different treatments on cellulose degradation and the relationship between inoculants and indigenous actinobacteria were investigated during composting. The results showed that inoculation in different stages of composting all improved the actinobacteria community diversity particularly in the cooling stage of composting (M3). Moreover, inoculation could distinctly accelerate the degradation of organic matters (OM) especially celluloses. Redundancy analysis indicated that the correlation between indigenous actinobacteria and degradation of OM and cellulose were regulated by inoculants and there were significant differences between different inoculation methods. Furthermore, synergy between indigenous actinobacteria and inoculants for degradation of OM and cellulose in M3 was better than other treatments. Conclusively, we suggested an inoculation method to regulate the indigenous actinobacteria based on the relationship between inoculants and indigenous actinobacteria and degradation content. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of proteasomal degradation by modulating proteasomal initiation regions

PubMed Central

Takahashi, Kazunobu; Matouschek, Andreas; Inobe, Tomonao

2016-01-01

Methods for regulating the concentrations of specific cellular proteins are valuable tools for biomedical studies. Artificial regulation of protein degradation by the proteasome is receiving increasing attention. Efficient proteasomal protein degradation requires a degron with two components: a ubiquitin tag that is recognized by the proteasome and a disordered region at which the proteasome engages the substrate and initiates degradation. Here we show that degradation rates can be regulated by modulating the disordered initiation region by the binding of modifier molecules, in vitro and in vivo. These results suggest that artificial modulation of proteasome initiation is a versatile method for conditionally inhibiting the proteasomal degradation of specific proteins. PMID:26278914

Preparation and characterization of two new forced degradation products of letrozole and development of a stability-indicating RP-LC method for its determination.

PubMed

Elkady, Ehab Farouk; Fouad, Marwa Ahmed

2015-11-01

Two new hydrolytic products of letrozole were identified and proved to be true degradation products obtained by alkaline and acidic degradation of the drug. The acid and amide forms of the nitrile groups of letrozole were prepared and identified by IR and mass spectroscopic techniques. Subsequently, a simple, precise and selective stability-indicating RPLC method was developed and validated for the determination of letrozole in the presence of its degradation products. Letrozole was subjected to alkali and acid hydrolysis, oxidation, thermal degradation and photo-degradation. The degradation products were well isolated from letrozole. The chromatographic method was achieved using gradient elution of the drug and its degradation products on a reversed phase Zorbax Eclipse C18 column (100mm x 4.6mm, 3.5 μm) using a mobile phase consisting of 0.01M KH₂PO₄and methanol at a flow rate of 1 mL min⁻¹. Quantitation was achieved with UV detection at 230 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 0.01-80 μgmL⁻¹. The proposed method was successfully applied to the determination of letrozole in bulk, plasma and in its pharmaceutical preparation.

Degradation of Remazol Red in batik dye waste water by contact glow discharge electrolysis method using NaOH and NaCl electrolytes

NASA Astrophysics Data System (ADS)

Saksono, Nelson; Putri, Dita Amelia; Suminar, Dian Ratna

2017-03-01

Contact Glow Discharge Electrolysis (CGDE) method is one of Plasma Electrolysis technology which has been approved to degrade organic waste water because it is very productive in producing hydroxyl radical. This study aims to degrade Remazol Red by CGDE method and evaluate important parameters that have influent in degradation process of Remazol Red in Batik dye waste water in batch system. The kind of electrolyte (acid and base) and the addition of metal ion such as Fe2+ have affected Remazol Red degradation percentage. Ultraviolet-Visible (UV-Vis) absorption spectra were used to monitor the degradation process. The result of study showed that percentage degradation was 99.97% which obtained by using NaCl 0.02 M with addition Fe2+ 20 ppm, applied voltage 700 volt, anode depth 0.5 cm, initial concentration of Remazol Red 250 ppm and the temperature of solutions was maintained 50-60 ˚C.

Stability indicating methods for the analysis of cefprozil in the presence of its alkaline induced degradation product

NASA Astrophysics Data System (ADS)

Attia, Khalid A. M.; Nassar, Mohammed W. I.; El-Zeiny, Mohamed B.; Serag, Ahmed

2016-04-01

Three simple, specific, accurate and precise spectrophotometric methods were developed for the determination of cefprozil (CZ) in the presence of its alkaline induced degradation product (DCZ). The first method was the bivariate method, while the two other multivariate methods were partial least squares (PLS) and spectral residual augmented classical least squares (SRACLS). The multivariate methods were applied with and without variable selection procedure (genetic algorithm GA). These methods were tested by analyzing laboratory prepared mixtures of the above drug with its alkaline induced degradation product and they were applied to its commercial pharmaceutical products.

Adaptation of the Conditions of US EPA Method 538 for the ...

EPA Pesticide Factsheets

Report The objective of this study was to evaluate U.S. EPA’s Method 538 for the assessment of drinking water exposure to the nerve agent degradation product, EA2192, the most toxic degradation product of nerve agent VX. As a result of the similarities in sample preparation and analysis that Method 538 uses for nonvolatile chemicals, this method is applicable to the nonvolatile Chemical Warfare Agent (CWA) degradation product, EA2192, in drinking water. The method may be applicable to other nonvolatile CWAs and their respective degradation products as well, but the method will need extensive testing to verify compatibility. Gaps associated with the need for analysis methods capable of analyzing such analytes were addressed by adapting the EPA 538 method for this CWA degradation product. Many laboratories have the experience and capability to run the already rigorous method for nonvolatile compounds in drinking water. Increasing the number of laboratories capable of carrying out these methods serves to significantly increase the surge laboratory capacity to address sample throughput during a large exposure event. The approach desired for this study was to start with a proven high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) method for nonvolatile chemicals in drinking water and assess the inclusion of a similar nonvolatile chemical, EA2192.

Method of phorbol ester degradation in Jatropha curcas L. seed cake using rice bran lipase.

PubMed

Hidayat, Chusnul; Hastuti, Pudji; Wardhani, Avita Kusuma; Nadia, Lana Santika

2014-03-01

A novel enzymatic degradation of phorbol esters (PE) in the jatropha seed cake was developed using lipase. Cihera rice bran lipase had the highest ability to hydrolyze PE, and reduced PE to a safe level after 8 h of incubation. Enzymatic degradation may be a promising method for PE degradation. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate.

PubMed

Toporisic, Rebeka; Mlakar, Anita; Hvala, Jernej; Prislan, Iztok; Zupancic-Kralj, Lucija

2010-06-05

Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC-MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 microg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 microg/mL and LOQ was 0.5 microg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC-MS/MS(n), Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity. Copyright (c) 2010. Published by Elsevier B.V.

Stability-indicating UPLC method for determination of Valsartan and their degradation products in active pharmaceutical ingredient and pharmaceutical dosage forms.

PubMed

Krishnaiah, Ch; Reddy, A Raghupathi; Kumar, Ramesh; Mukkanti, K

2010-11-02

A simple, precise, accurate stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the quantitative determination of purity of Valsartan drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. The method was developed using Waters Aquity BEH C18 (100 mm x 2.1 mm, 1.7 microm) column with mobile phase containing a gradient mixture of solvents A and B. The eluted compounds were monitored at 225 nm, the run time was within 9.5 min, which Valsartan and its seven impurities were well separated. Valsartan was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Valsartan was found to degrade significantly in acid and oxidative stress conditions and stable in base, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of Valsartan in pharmaceutical dosage forms.

Stability-indicating RP-HPLC method for the simultaneous determination of escitalopram oxalate and clonazepam.

PubMed

Kakde, Rajendra B; Satone, Dinesh D; Gadapayale, Kamalesh K; Kakde, Megha G

2013-07-01

The objective of the current study was to develop a validated, specific stability-indicating reversed-phase liquid chromatographic (LC) method for the quantitative determination of escitalopram oxalate and clonazepam and their related substances in bulk drugs and pharmaceutical dosage forms in the presence of degradation products. Forced degradation studies were performed on the pure drugs of escitalopram oxalate and clonazepam, as per the stress conditions prescribed by the International Conference on Harmonization (ICH) using acid, base, oxidation, thermal stress and photolytic degradation to show the stability-indicating power of the method. Significant degradation was observed during acid and alkaline hydrolysis and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies. Good resolution between the peaks corresponded to the active pharmaceutical ingredients, escitalopram oxalate and clonazepam, and degradation products from the analyte were achieved on an ODS Hypersil C18 column (250 × 4.6 mm) using a mobile phase consisting of a mixture of acetonitrile-50 mM phosphate buffer + 10 mM triethylamine (70:30, v/v). The detection was conducted at 268 nm. The limit of detection and the limit of quantitation for escitalopram oxalate and clonazepam were established. The stress test solutions were assayed against the qualified working standards of escitalopram oxalate and clonazepam, which indicated that the developed LC method was stability-indicating. Validation of the developed LC method was conducted as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of escitalopram oxalate and clonazepam.

A validated stability-indicating UPLC method for desloratadine and its impurities in pharmaceutical dosage forms.

PubMed

Rao, Dantu Durga; Satyanarayana, N V; Malleswara Reddy, A; Sait, Shakil S; Chakole, Dinesh; Mukkanti, K

2010-02-05

A novel stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of desloratadine in presence of its impurities and forced degradation products. The method was developed using Waters Aquity BEH C18 column with mobile phase containing a gradient mixture of solvents A and B. The eluted compounds were monitored at 280nm. The run time was 8min within which desloratadine and its five impurities were well separated. Desloratadine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Desloratadine was found to degrade significantly in oxidative and thermal stress conditions and stable in acid, base, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of desloratadine in pharmaceutical dosage forms.

[BiOBr promoted the photocatalytic degradation of beta-cypermethrin under visible light].

PubMed

Peng, Yi-Zhu; Zhao, Xiao-Rong; Jia, Man-Ke; Zhou, Wei; Huang, Ying-Ping

2014-05-01

As a visible light photocatalyst, bismuth oxide bromide (BiOBr) was used to catalyze the degradation of beta-cypermethrin (beta-CP). The photocatalytic degradation of beta-CP was studied with gas chromatography. The effects of pH and catalyst dose on the photocatalytic degradation efficiency were discussed. The oxidization and mineralization of beta-CP were detected by chemical oxygen demand (COD) analyzer. The results showed that beta-CP could be effectively degraded under visible light irradiation using BiOBr as the catalyst. At given experimental conditions, the degradation rate of beta-CP reached 94. 68% after 10 h and the COD removal rate reached 67. 99% after 36 h. With the increase of catalyst dose and pH value, the degradation rate was improved. The photocatalytic oxidation species was determined by peroxidase method and terephthalic acid fluorescence method. These results suggested that the photocatalytic degradation process mainly referred to hydroxyl radical ( OH) mechanism.

Engineered enzymatically active bacteriophages and methods of uses thereof

DOEpatents

Collins, James J [Newton, MA; Kobayashi, Hideki [Yokohama, JP; Kearn, Mads [Ottawa, CA; Araki, Michihiro [Minatoku, JP; Friedland, Ari [Boston, MA; Lu, Timothy Kuan-Ta [Palo Alto, CA

2012-05-22

The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; a method supplement for the determination of Fipronil and degradates in water by gas chromatography/mass spectrometry

USGS Publications Warehouse

Madsen, James F.; Sandstrom, Mark W.; Zaugg, Steven D.

2002-01-01

A method for the isolation and detemrination of fipronil and four of its degradates has been developed. This method adapts an analytical method created by the U.S. Geological Survey National Water Quality Laboratory in 1995 for the determination of a broad range of high-use pesticides typically found in filtered natural-water samples. In 2000, fipronil and four of its degradates were extracted, analyzed, and validated using this method. The recoveries for these five compounds in reagent-water samples fortified at 1 microgram per liter (ug/L) avereraged 98 percent. Initial method detection limits averaged 0.0029 ug/L. The performance of these five new compounds is consistent with the performance of the compounds in the initial method, making it possible to include them in addition to the other 41 pesticides and pesticide degradates in the original method.

Antioxidant potential of mulberry and non-mulberry silk sericin and its implications in biomedicine.

PubMed

Kumar, Jadi Praveen; Mandal, Biman B

2017-07-01

Sericin, a principal constituent of silk, is widely used in various biomedical applications. In addition, conferring protection against free radicals and oxidative damage add more value to its therapeutic potential. However, the antioxidant (AO) properties of silk sericin (SS) remains contingent on extraction procedures. In the present study, we have evaluated the effect of different extraction methods (conventional, autoclaving, urea, alkali and acid-degradation) on AO properties of SS from three Indian silk varieties [Antheraea assamensis (AA), Philosamia ricini (PR) and Bombyx mori (BM)]. The physico-chemical characterization studies revealed that the molecular weight of SS isolates of each method ranged from 10 to 220kDa along with varied protein structural biochemistry. SS extracts using urea-degradation (BM, PR and AA), conventional method and alkali-degradation (BM) displayed high percentage of β-sheets, random coils and turns. Acid-degraded SS (PR, followed by AA and BM) showed the highest total flavonoid content while conventional method (PR), autoclaving (AA) and alkali-degradation (BM) displayed lowest flavonoid levels. Interestingly, SS extracted by autoclaving (BM and AA), acid-degradation (PR), conventional and alkali-degradation (BM, AA and PR) methods exhibited 50% reduction of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. Moreover, the efficacy of antioxidant potential of SS extracted by different methods was found to be in the order of "alkali>autoclaving>conventional" as demonstrated in L929 cells. Correspondingly, the anti-lipid peroxidation activity of SS extracted by alkali method (AA, BM and PR) further confirmed better AO properties amid others. Thus, the present study demonstrates that the extraction methods may significantly affect AO activity of SS which might be of importance for potential cosmetic applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Degradation Kinetics Study of Alogliptin Benzoate in Alkaline Medium by Validated Stability-Indicating HPTLC Method.

PubMed

Bodiwala, Kunjan Bharatkumar; Shah, Shailesh; Thakor, Jeenal; Marolia, Bhavin; Prajapati, Pintu

2016-11-01

A rapid, sensitive, and stability-indicating high-performance thin-layer chromatographic method was developed and validated to study degradation kinetics of Alogliptin benzoate (ALG) in an alkaline medium. ALG was degraded under acidic, alkaline, oxidative, and thermal stress conditions. The degraded samples were chromatographed on silica gel 60F254-TLC plates, developed using a quaternary-solvent system (chloroform-methanol-ethyl acetate-triethyl amine, 9+1+1+0.5, v/v/v/v), and scanned at 278 nm. The developed method was validated per International Conference on Harmonization guidelines using validation parameters such as specificity, linearity and range, precision, accuracy, LOD, and LOQ. The linearity range for ALG was 100-500 ng/band (correlation coefficient = 0.9997) with an average recovery of 99.47%. The LOD and LOQ for ALG were 9.8 and 32.7 ng/band, respectively. The developed method was successfully applied for the quantitative estimation of ALG in its synthetic mixture with common excipients. Degradation kinetics of ALG in an alkaline medium was studied by degrading it under three different temperatures and three different concentrations of alkali. Degradation of ALG in the alkaline medium was found to follow first-order kinetics. Contour plots have been generated to predict degradation rate constant, half-life, and shelf life of ALG in various combinations of temperature and concentration of alkali using Design Expert software.

An efficient and environment-friendly method of removing graphene oxide in wastewater and its degradation mechanisms.

PubMed

Zhang, Chao-Zhi; Li, Ting; Yuan, Yang; Xu, Jianqiang

2016-06-01

Graphene and graphene oxide (GO) have already existed in air, water and soil due to their popular application in functional materials. However, degradation of graphene and GO in wastewater has not been reported. Degradation of GO plays a key role in the elimination of graphene and GO in wastewater due to graphene being easily oxidized to GO. In this paper, GO was completely degraded to give CO2 by Photo-Fenton. The degradation intermediates were determined by UV-vis absorption spectra, elemental analysis (EA), fourier transform infrared (FT-IR) and liquid chromatography-mass spectrometry (LC-MS). Experimental results showed that graphene oxide was completely degraded to give CO2 after 28 days. Based on UV, FT-IR, LC-MS spectra and EA data of these degradation intermediates, the degradation mechanisms of GO were supposed. This paper suggests an efficient and environment-friendly method to degrade GO and graphene. Copyright © 2016 Elsevier Ltd. All rights reserved.

Degradation of learned skills: Effectiveness of practice methods on visual approach and landing skill retention

NASA Technical Reports Server (NTRS)

Sitterley, T. E.; Zaitzeff, L. P.; Berge, W. A.

1972-01-01

Flight control and procedural task skill degradation, and the effectiveness of retraining methods were evaluated for a simulated space vehicle approach and landing under instrument and visual flight conditions. Fifteen experienced pilots were trained and then tested after 4 months either without the benefits of practice or with static rehearsal, dynamic rehearsal or with dynamic warmup practice. Performance on both the flight control and procedure tasks degraded significantly after 4 months. The rehearsal methods effectively countered procedure task skill degradation, while dynamic rehearsal or a combination of static rehearsal and dynamic warmup practice was required for the flight control tasks. The quality of the retraining methods appeared to be primarily dependent on the efficiency of visual cue reinforcement.

Assessment of chloroethene degradation rates based on ratios of daughter/parent compounds in groundwater plumes

NASA Astrophysics Data System (ADS)

Höhener, Patrick

2014-05-01

Chlorinated solvent spills at industrial and urban sites create groundwater plumes where tetrachloro- and trichloroethene may degrade to their daughter compounds, dichloroethenes, vinyl chloride and ethane. The assessment of degradation and natural attenuation at such sites may be based on the analysis and inverse modelling of concentration data, on the calculation of mass fluxes in transsects, and/or on the analysis of stable isotope ratios in the ethenes. Relatively few work has investigated the possibility of using ratio of concentrations for gaining information on degradation rates. The use of ratios bears the advantage that dilution of a single sample with contaminant-free water does not matter. It will be shown that molar ratios of daughter to parent compounds measured along a plume streamline are a rapid and robust mean of determining whether degradation rates increase or decrease along the degradation chain, and allow furthermore a quantitation of the relative magnitude of degradation rates compared to the rate of the parent compound. Furthermore, ratios of concentration will become constant in zones where degradation is absent, and this allows to sketching the extension of actively degrading zones. The assessment is possible for pure sources and also for mixed sources. A quantification method is proposed in order to estimate first-order degradation rates in zones of constant degradation activity. This quantification method includes corrections that are needed due to longitudinal and transversal dispersivity. The method was tested on a number of real field sites from literature. At the majority of these sites, the first-order degradation rates were decreasing along the degradation chain from tetrachloroethene to vinyl chloride, meaning that the latter was often reaching important concentrations. This is bad news for site owners due to the increased toxicity of vinyl chloride compared to its parent compounds.

Identification and characterization of stress degradants of lacosamide by LC-MS and ESI-Q-TOF-MS/MS: development and validation of a stability indicating RP-HPLC method.

PubMed

Ramisetti, Nageswara Rao; Kuntamukkala, Ramakrishna; Lakshetti, Sridhar; Sripadi, Prabhakar

2014-07-01

The current study dealt with the degradation behavior of lacosamide (LAC) under ICH prescribed stress conditions. LAC was found to be labile under acid and base hydrolytic stress conditions, while it was stable to neutral hydrolytic, oxidative, photolytic and thermal stress. In total, seven degradation products (DPs) were formed, which were separated on a C18 column using a stability-indicating method. LC-MS analyses indicated that one of the DPs had the same molecular mass as that of the drug. Structural characterization of DPs was carried out using ESI-Q-TOF-MS/MS technique. The degradation pathways and mechanisms of degradation of the drug were delineated by carrying out the degradation in different co-solvents viz. methanol, deuterated methanol, ethanol, 1-propanol and acetonitrile. The developed LC method was validated for the determination of related substances and assay of LAC as per ICH guidelines. This study demonstrates a comprehensive approach of LAC degradation studies during its development phase. Copyright © 2014. Published by Elsevier B.V.

Rheological techniques for determining degradation of polylactic acid in bioresorbable medical polymer systems

NASA Astrophysics Data System (ADS)

Choong, Gabriel Y. H.; Parsons, Andrew J.; Grant, David M.; De Focatiis, Davide S. A.

2015-05-01

A method developed in the 1980s for the conversion of linear rheological data to molar mass distribution is revisited in the context of degradable polymers. The method is first applied using linear rheology for a linear polystyrene, for which all conversion parameters are known. A proof of principle is then carried out on four polycarbonate grades. Finally, preliminary results are shown on degradable polylactides. The application of this method to degrading polymer systems, and to systems containing nanofillers, is also discussed. This work forms part of a wider study of bioresorbable nanocomposites using polylactides, novel hydroxyapatite nanoparticles and tailored dispersants for medical applications.

Kinetic study and mechanism of Niclosamide degradation.

PubMed

Zaazaa, Hala E; Abdelrahman, Maha M; Ali, Nouruddin W; Magdy, Maimana A; Abdelkawy, M

2014-11-11

A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function of drug concentration, alkaline concentration and temperature has been established utilizing double divisor-ratio spectra spectrophotometric method. The developed method allowed determination of Niclosamide in presence of its alkaline degradation products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG II) with characterization of its degradation mechanism. It was found that degradation kinetic of Niclosamide followed pseudo-first order under the established experimental conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t1/2) of 8.35 h. The overall degradation rate constant as a function of the temperature under the given conditions obeyed Arrhenius equation where the activation energy was calculated to be 3.41 kcal/mol. Copyright © 2014 Elsevier B.V. All rights reserved.

Methods for cellobiosan utilization

DOEpatents

Linger, Jeffrey; Beckham, Gregg T.

2017-07-11

Disclosed herein are enzymes useful for the degradation of cellobiosan in materials such a pyrolysis oils. Methods of degrading cellobiosan using enzymes or organisms expressing the same are also disclosed.

Effect of different oxytetracycline addition methods on its degradation behavior in soil.

PubMed

Chen, Gui-Xiu; He, Wei-Wei; Wang, Yan; Zou, Yong-De; Liang, Juan-Boo; Liao, Xin-Di; Wu, Yin-Bao

2014-05-01

The degradation behavior of veterinary antibiotics in soil is commonly studied using the following methods of adding antibiotics to the soil: (i) adding manure collected from animals fed with a diet containing antibiotics, (ii) adding antibiotic-free animal manure spiked with antibiotics and (iii) directly adding antibiotics. No research simultaneously comparing different antibiotic addition methods was found. Oxytetracycline (OTC) was used as a model antibiotic to compare the effect of the three commonly used antibiotic addition methods on OTC degradation behavior in soil. The three treatment methods have similar trends, though OTC degradation half-lives show the following significant differences (P<0.05): manure from swine fed OTC (treatment A)

Method of degrading pollutants in soil

DOEpatents

Hazen, Terry C.; Lopez-De-Victoria, Geralyne

1994-01-01

A method and system for enhancing the motility of microorganisms by placing an effective amount of chlorinated hydrocarbons, preferably chlorinated alkenes, and most preferably trichloroethylene in spaced relation to the microbes so that the surprisingly strong, monomodal, chemotactic response of the chlorinated hydrocarbon on subsurface microbes can draw the microbes away from or towards and into a substance, as desired. In remediation of groundwater pollution, for example, TCE can be injected into the plume to increase the population of microbes at the plume whereby the plume can be more quickly degraded. A TCE-degrading microbe, such as Welchia alkenophilia, can be used to degrade the TCE following the degradation of the original pollutant.

Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge.

PubMed

Kayashima, Takakazu; Suzuki, Hisako; Maeda, Toshinari; Ogawa, Hiroaki I

2013-05-01

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Degradation trend estimation of slewing bearing based on LSSVM model

NASA Astrophysics Data System (ADS)

Lu, Chao; Chen, Jie; Hong, Rongjing; Feng, Yang; Li, Yuanyuan

2016-08-01

A novel prediction method is proposed based on least squares support vector machine (LSSVM) to estimate the slewing bearing's degradation trend with small sample data. This method chooses the vibration signal which contains rich state information as the object of the study. Principal component analysis (PCA) was applied to fuse multi-feature vectors which could reflect the health state of slewing bearing, such as root mean square, kurtosis, wavelet energy entropy, and intrinsic mode function (IMF) energy. The degradation indicator fused by PCA can reflect the degradation more comprehensively and effectively. Then the degradation trend of slewing bearing was predicted by using the LSSVM model optimized by particle swarm optimization (PSO). The proposed method was demonstrated to be more accurate and effective by the whole life experiment of slewing bearing. Therefore, it can be applied in engineering practice.

A novel accelerated oxidative stability screening method for pharmaceutical solids.

PubMed

Zhu, Donghua Alan; Zhang, Geoff G Z; George, Karen L S T; Zhou, Deliang

2011-08-01

Despite the fact that oxidation is the second most frequent degradation pathway for pharmaceuticals, means of evaluating the oxidative stability of pharmaceutical solids, especially effective stress testing, are still lacking. This paper describes a novel experimental method for peroxide-mediated oxidative stress testing on pharmaceutical solids. The method utilizes urea-hydrogen peroxide, a molecular complex that undergoes solid-state decomposition and releases hydrogen peroxide vapor at elevated temperatures (e.g., 30°C), as a source of peroxide. The experimental setting for this method is simple, convenient, and can be operated routinely in most laboratories. The fundamental parameter of the system, that is, hydrogen peroxide vapor pressure, was determined using a modified spectrophotometric method. The feasibility and utility of the proposed method in solid form selection have been demonstrated using various solid forms of ephedrine. No degradation was detected for ephedrine hydrochloride after exposure to the hydrogen peroxide vapor for 2 weeks, whereas both anhydrate and hemihydrate free base forms degraded rapidly under the test conditions. In addition, both the anhydrate and the hemihydrate free base degraded faster when exposed to hydrogen peroxide vapor at 30°C under dry condition than at 30°C/75% relative humidity (RH). A new degradation product was also observed under the drier condition. The proposed method provides more relevant screening conditions for solid dosage forms, and is useful in selecting optimal solid form(s), determining potential degradation products, and formulation screening during development. Copyright © 2011 Wiley-Liss, Inc.

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

PubMed

Tomita, Toshio; Mizumachi, Yoshihiro; Chong, Kang; Ogawa, Kanako; Konishi, Norihide; Sugawara-Tomita, Noriko; Dohmae, Naoshi; Hashimoto, Yohichi; Takio, Koji

2004-12-24

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Venom gland transcriptomic and venom proteomic analyses of the scorpion Megacormus gertschi Díaz-Najera, 1966 (Scorpiones: Euscorpiidae: Megacorminae).

PubMed

Santibáñez-López, Carlos E; Cid-Uribe, Jimena I; Zamudio, Fernando Z; Batista, Cesar V F; Ortiz, Ernesto; Possani, Lourival D

2017-07-01

The soluble venom from the Mexican scorpion Megacormus gertschi of the family Euscorpiidae was obtained and its biological effects were tested in several animal models. This venom is not toxic to mice at doses of 100 μg per 20 g of mouse weight, while being lethal to arthropods (insects and crustaceans), at doses of 20 μg (for crickets) and 100 μg (for shrimps) per animal. Samples of the venom were separated by high performance liquid chromatography and circa 80 distinct chromatographic fractions were obtained from which 67 components have had their molecular weights determined by mass spectrometry analysis. The N-terminal amino acid sequence of seven protein/peptides were obtained by Edman degradation and are reported. Among the high molecular weight components there are enzymes with experimentally-confirmed phospholipase activity. A pair of telsons from this scorpion species was dissected, from which total RNA was extracted and used for cDNA library construction. Massive sequencing by the Illumina protocol, followed by de novo assembly, resulted in a total of 110,528 transcripts. From those, we were able to annotate 182, which putatively code for peptides/proteins with sequence similarity to previously-reported venom components available from different protein databases. Transcripts seemingly coding for enzymes showed the richest diversity, with 52 sequences putatively coding for proteases, 20 for phospholipases, 8 for lipases and 5 for hyaluronidases. The number of different transcripts potentially coding for peptides with sequence similarity to those that affect ion channels was 19, for putative antimicrobial peptides 19, and for protease inhibitor-like peptides, 18. Transcripts seemingly coding for other venom components were identified and described. The LC/MS analysis of a trypsin-digested venom aliquot resulted in 23 matches with the translated transcriptome database, which validates the transcriptome. The proteomic and transcriptomic analyses reported here constitute the first approach to study the venom components from a scorpion species belonging to the family Euscorpiidae. The data certainly show that this venom is different from all the ones described thus far in the literature. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag.

PubMed

Batdorj, B; Dalgalarrondo, M; Choiset, Y; Pedroche, J; Métro, F; Prévost, H; Chobert, J-M; Haertlé, T

2006-10-01

The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

PubMed Central

Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

2012-01-01

Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba. PMID:23284650

Purification and Characterization of Suicin 65, a Novel Class I Type B Lantibiotic Produced by Streptococcus suis.

PubMed

Vaillancourt, Katy; LeBel, Geneviève; Frenette, Michel; Fittipaldi, Nahuel; Gottschalk, Marcelo; Grenier, Daniel

2015-01-01

Bacteriocins are antimicrobial peptides of bacterial origin that are considered as a promising alternative to the use of conventional antibiotics. Recently, our laboratory reported the purification and characterization of two lantibiotics, suicin 90-1330 and suicin 3908, produced by the swine pathogen and zoonotic agent Streptococcus suis (serotype 2). In this study, a novel bacteriocin produced by S. suis has been identified and characterized. The producing strain S. suis 65 (serotype 2) was found to belong to the sequence type 28, that includes strains known to be weakly or avirulent in a mouse model. The bacteriocin, whose production was only possible following growth on solid culture medium, was purified to homogeneity by cationic exchange and reversed-phase high-pressure liquid chromatography. The bacteriocin, named suicin 65, was heat, pH and protease resistant. Suicin 65 was active against all S. suis isolates tested, including antibiotic resistant strains. Amino acid sequencing of the purified bacteriocin by Edman degradation revealed the presence of modified amino acids suggesting a lantibiotic. Using the partial sequence obtained, a blast was performed against published genomes of S. suis and allowed to identify a putative lantibiotic locus in the genome of S. suis 89-1591. From this genome, primers were designed and the gene cluster involved in the production of suicin 65 by S. suis 65 was amplified by PCR. Sequence analysis revealed the presence of ten open reading frames, including a duplicate of the structural gene. The structural genes (sssA and sssA') of suicin 65 encodes a 25-amino acid residue leader peptide and a 26-amino acid residue mature peptide yielding an active bacteriocin with a deducted molecular mass of 3,005 Da. Mature suicin 65 showed a high degree of identity with class I type B lantibiotics (globular structure) produced by Streptococcus pyogenes (streptococcin FF22; 84.6%), Streptococcus macedonicus (macedocin ACA-DC 198; 84.6%), and Lactococcus lactis subsp. lactis (lacticin 481; 74.1%). Further studies will evaluate the ability of suicin 65 or the producing strain to prevent experimental S. suis infections in pigs.

Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

PubMed Central

Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin

2016-01-01

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306

Venoms of Centruroides and Tityus species from Panama and their main toxic fractions.

PubMed

Salazar, Marcos H; Arenas, Iván; Corrales-García, Ligia L; Miranda, Roberto; Vélez, Sara; Sánchez, Jairo; Mendoza, Karla; Cleghorn, John; Zamudio, Fernando Z; Castillo, Adolfo; Possani, Lourival D; Corzo, Gerardo; Acosta, Hildaura

2018-01-01

The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na + -channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of the first K⁺ channel blockers from the venom of the Moroccan scorpion Buthus occitanus Paris.

PubMed

Martin-Eauclaire, Marie-France; Céard, Brigitte; Belghazi, Maya; Lebrun, Régine; Bougis, Pierre E

2013-12-01

The availability of a large variety of specific blockers, which inhibit different K(+) currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca(2+)-activated K(+) channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using (125)I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the (125)I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family. Copyright © 2013 Elsevier Ltd. All rights reserved.

Purification and Characterization of Suicin 65, a Novel Class I Type B Lantibiotic Produced by Streptococcus suis

PubMed Central

Vaillancourt, Katy; LeBel, Geneviève; Frenette, Michel; Fittipaldi, Nahuel; Gottschalk, Marcelo; Grenier, Daniel

2015-01-01

Bacteriocins are antimicrobial peptides of bacterial origin that are considered as a promising alternative to the use of conventional antibiotics. Recently, our laboratory reported the purification and characterization of two lantibiotics, suicin 90–1330 and suicin 3908, produced by the swine pathogen and zoonotic agent Streptococcus suis (serotype 2). In this study, a novel bacteriocin produced by S. suis has been identified and characterized. The producing strain S. suis 65 (serotype 2) was found to belong to the sequence type 28, that includes strains known to be weakly or avirulent in a mouse model. The bacteriocin, whose production was only possible following growth on solid culture medium, was purified to homogeneity by cationic exchange and reversed-phase high-pressure liquid chromatography. The bacteriocin, named suicin 65, was heat, pH and protease resistant. Suicin 65 was active against all S. suis isolates tested, including antibiotic resistant strains. Amino acid sequencing of the purified bacteriocin by Edman degradation revealed the presence of modified amino acids suggesting a lantibiotic. Using the partial sequence obtained, a blast was performed against published genomes of S. suis and allowed to identify a putative lantibiotic locus in the genome of S. suis 89–1591. From this genome, primers were designed and the gene cluster involved in the production of suicin 65 by S. suis 65 was amplified by PCR. Sequence analysis revealed the presence of ten open reading frames, including a duplicate of the structural gene. The structural genes (sssA and sssA’) of suicin 65 encodes a 25-amino acid residue leader peptide and a 26-amino acid residue mature peptide yielding an active bacteriocin with a deducted molecular mass of 3,005 Da. Mature suicin 65 showed a high degree of identity with class I type B lantibiotics (globular structure) produced by Streptococcus pyogenes (streptococcin FF22; 84.6%), Streptococcus macedonicus (macedocin ACA-DC 198; 84.6%), and Lactococcus lactis subsp. lactis (lacticin 481; 74.1%). Further studies will evaluate the ability of suicin 65 or the producing strain to prevent experimental S. suis infections in pigs. PMID:26709705

6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases.

PubMed Central

Bouma, C L; Reizer, J; Reizer, A; Robrish, S A; Thompson, J

1997-01-01

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum. PMID:9209025

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2. PMID:9693111

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes*

PubMed Central

Tashima, Alexandre K.; Zelanis, André; Kitano, Eduardo S.; Ianzer, Danielle; Melo, Robson L.; Rioli, Vanessa; Sant'anna, Sávio S.; Schenberg, Ana C. G.; Camargo, Antônio C. M.; Serrano, Solange M. T.

2012-01-01

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 l-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4′ sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. PMID:22869554

Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.

PubMed Central

Calvete, J J; Rivas, G; Maruri, M; Alvarez, M V; McGregor, J L; Hew, C L; Gonzalez-Rodriguez, J

1988-01-01

Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2455507

Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

PubMed

Lehr, S; Kotzka, J; Herkner, A; Klein, E; Siethoff, C; Knebel, B; Noelle, V; Brüning, J C; Klein, H W; Meyer, H E; Krone, W; Müller-Wieland, D

1999-01-05

Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.

Application of a new integrated sediment quality assessment method to Huelva estuary and its littoral of influence (Southwestern Spain).

PubMed

Rosado, Daniel; Usero, José; Morillo, José

2015-09-15

A new integrated sediment quality assessment method composed of several assays (particle size profile, total metal content, protease K extraction, total organic carbon, toxicity bioassay with Photobacterium phosphoreum and macrobenthic community alteration) that provides a single result, the environmental degradation index (EDI), has been developed. The new method was tested on the Huelva estuary (southwest of Spain), a highly polluted area where metals dissolved in the water of the Tinto and Odiel rivers precipitate after flowing through the Iberian Pyrite Belt, one of the largest metallogenic areas of massive sulphide deposits in the world. The proposed method satisfactorily was able to reflect different degrees of pollution on the environmental degradation index. Thus, EDI categorized littoral samples as slightly degraded and all the Tinto and some of the Odiel as very highly degraded, emphasizing the lower zone of the Tinto estuary as the most deeply degraded of the entire study area. Copyright © 2015 Elsevier Ltd. All rights reserved.

Validation of UV spectrophotometric methods for the determination of dothiepin hydrochloride in pharmaceutical dosage form and stress degradation studies

NASA Astrophysics Data System (ADS)

Abdulrahman, Sameer A. M.; Basavaiah, K.; Cijo, M. X.; Vinay, K. B.

2012-11-01

Spectrophotometric methods have been developed for the determination of dothiepin hydrochloride (DOTH) in both pure and tablet dosage form and their limits of detection and quantification have been evaluated. The methods are based on the measurement of the absorbance of a DOTH solution either in 0.1 N HCl at 229 nm (method A) or in methanol at 231 nm (method B). Beer's law is obeyed over a concentration range of 1-16 μg/ml DOTH for both methods. Molar absorptivity values are calculated to be 2.48 × 104 and 2.42 × 104 l/(mol × cm) with Sandell sensitivity values of 0.0134 and 0.0137 μg/cm2 for methods A and B, respectively. The degradation behavior of DOTH was investigated under different stress conditions such as acid hydrolysis, alkaline hydrolysis, water hydrolysis, oxidation, dry heat treatment, and UV-degradation. The drug undergoes significant degradation under oxidative conditions only.

Effect of drying temperatures on structural performance and photocatalytic activity of BiOCl synthesized by a soft chemical method

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

Guang, Lu, E-mail: lu_g@163.com; Hui, Wang; Xuejun, Zou

2016-07-15

A group of BiOCl photocatalysts with different drying temperatures were prepared by a soft chemical method. The effects of drying temperatures on the crystalline phase, morphology, surface area and optical property of as-prepared samples were investigated in detail by XRD, SEM, N{sub 2} absorption–desorption and DRS. Moreover, their photocatalytic activities on the degradation of rhodamine B were evaluated under visible light irradiation. It was found that the sample dried at 120 °C had the best photocatalytic activity, which was mainly attributed to the highest exposing proportion of {001} facets correspond to BiOCl, largest BET and minimum bandgap. The degradation mechanismmore » was explored that superoxide radicals were mainly contributed to the degradation of chromophore, however, holes and hydroxyl were mainly contributed to the photo degradation. Moreover, holes and hydroxyl dominated the degradation of RhB. - Graphical abstract: Holes, hydroxyl and superoxide radicals can attribute to the degradation process but take different degradation pathways. Superoxide radicals mainly contribute to the degradation of chromophore, however, holes and hydroxyl mainly contribute to the photo degradation. Display Omitted - Highlights: • BiOCl nanosheets were prepared by a soft chemical method. • Effect of drying temperatures on as-prepared BiOCl samples was studied. • The highest removal efficiency of RhB was obtained over the sample dried at 120 °C.« less

Poly(fumaric-co-sebacic anhydride). A degradation study as evaluated by FTIR, DSC, GPC and X-ray diffraction.

PubMed

Santos, C A; Freedman, B D; Leach, K J; Press, D L; Scarpulla, M; Mathiowitz, E

1999-06-28

The degradation of three poly(fumaric-co-sebacic anhydride) [P(FA:SA)] copolymers is examined in a composition of microspheres made by the hot melt encapsulation process. The emergence of low molecular weight oligomers occurs during degradation of the copolymer microspheres, as evidenced by a variety of characterization methods. Characterization was conducted to determine the extent of degradation of the polyanhydride microspheres using Fourier-transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and X-ray diffraction. It is demonstrated that degradation of P(FA:SA) is greatly accelerated at basic pH, yet there is little difference between degradation in neutral and acidic buffers. A good correlation exists between the results of each characterization method, which allows a better understanding of the degradation process and the resulting formation of low molecular weight oligomers in poly(fumaric-co-sebacic anhydride).

Application of plasma electrolysis method for simultaneous phenol and Cr(VI) wastewater degradation using Na2SO4 electrolyte

NASA Astrophysics Data System (ADS)

Harianti, Aulia Rahmi; Saksono, Nelson

2017-11-01

Phenol and Cr (VI) are two types of wastewater known as dangerous and difficult to degrade. Through this study, phenol and Cr (VI) metal wastewater were degraded simultaneously using plasma electrolysis method by reactive species, •OH and H•. The variation of anode depth and position of plasma formation as independent variables correlated with yield of hydroxyl radical, percentage of phenol and Cr (VI) degradation, and specific energy. Within 30 minutes, phenol was degraded to 98.4% and Cr (VI) was degraded to 93.35% with 171.05 kJ/mmol in specific energy, and 174.53 ppm in COD. The optimum condition was obtained in anodic plasma and 1.5 cm in anode depth. The highest degradation percentage of phenol and Cr (VI) were 99.79% and 97.33% achieved during 180 minutes of plasma electrolysis process.

UPLC and LC-MS studies on degradation behavior of irinotecan hydrochloride and development of a validated stability-indicating ultra-performance liquid chromatographic method for determination of irinotecan hydrochloride and its impurities in pharmaceutical dosage forms.

PubMed

Kumar, Navneet; Sangeetha, Dhanaraj; Reddy, Sunil P

2012-10-01

The objective of the current investigation was to study the degradation behavior of irinotecan hydrochloride under different International Conference on Harmonization (ICH) recommended stress conditions using ultra-performance liquid chromatography and liquid chromatography-mass spectrometry and to establish a validated stability-indicating reverse-phase ultra-performance liquid chromatographic method for the quantitative determination of irinotecan hydrochloride and its seven impurities and degradation products in pharmaceutical dosage forms. Irinotecan hydrochloride was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Irinotecan hydrochloride was found to degrade significantly in oxidative and base hydrolysis and photolytic degradation conditions. The degradation products were well resolved from the main peak and its impurities, thus proving the stability-indicating power of the method. Chromatographic separation was achieved on a Waters Acquity BEH C8 (100 × 2.1 mm) 1.7-µm column with a mobile phase containing a gradient mixture of solvent A (0.02M KH(2)PO(4) buffer, pH 3.4) and solvent B (a mixture of acetonitrile and methanol in the ratio of 62:38 v/v). The mobile phase was delivered at a flow rate of 0.3 mL/min with ultraviolet detection at 220 nm. The run time was 8 min, within which irinotecan and its seven impurities and degradation products were satisfactorily separated. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of irinotecan hydrochloride in pharmaceutical dosage forms.

Stability-Indicating HPTLC Method for Studying Stress Degradation Behavior of Sulbutiamine HCl

PubMed Central

Farid, Nehal F.; Abdelwahab, Nada S.

2016-01-01

Sulbutiamine (SUL) is an ester of thiazides with neurotropic action. A new stability indicating HPTLC method has been developed and validated for the determination of SUL in the presence of different degradation products. The drug was subjected to different stress conditions following ICH strategy such as hydrolytic degradation (neutral, alkaline and acidic hydrolysis), oxidation, photodegradation and dry heat degradation. The drug demonstrated degradation under all decomposition conditions except neutral hydrolysis and dry heat, where the drug was completely degraded with 0.1 N NaOH, 1 N HCl and 30% H2O2 while it was partially degradaed by 0.1 N HCl, 3% H2O2 and UV light. Structure elucidation of the resulting degradation products was performed using ESI-Q-MS–MS. A well-defined peak for SUL was obtained at Rf = 0.46 and was completely separated from all obtained degradation products. Chromatographic separation was carried out on HPTLC aluminum plates precoated with silica gel 60 F254 using acetone–methylene chloride–ammonia buffer (pH 8.5 ± 0.2) (7:3:0.5, v/v) as a developing system. Densitometric scanning of the separated peaks was performed at 254 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed method. The method was validated with respect to USP guidelines regarding accuracy, precision, specificity, robustness and ruggedness. Good correlation coefficients were achieved in the range of 0.4–5.0 µg/band, and the limit of detection and limit of quantitation were found to be 0.11 and 0.33 µg/band, respectively. The utility of the suggested method was verified by application to Arcalion forte® tablets where no interference from additives was found. PMID:26759487

Solar cell radiation handbook

NASA Technical Reports Server (NTRS)

Carter, J. R., Jr.; Tada, H. Y.

1973-01-01

A method is presented for predicting the degradation of a solar array in a space radiation environment. Solar cell technology which emphasizes the cell parameters that degrade in a radiation environment, is discussed along with the experimental techniques used in the evaluation of radiation effects. Other topics discussed include: theoretical aspects of radiation damage, methods for developing relative damage coefficients, nature of the space radiation environment, method of calculating equivalent fluence from electron and proton energy spectrums and relative damage coefficients, and comparison of flight data with estimated degradation.

A scientific and statistical analysis of accelerated aging for pharmaceuticals. Part 1: accuracy of fitting methods.

PubMed

Waterman, Kenneth C; Swanson, Jon T; Lippold, Blake L

2014-10-01

Three competing mathematical fitting models (a point-by-point estimation method, a linear fit method, and an isoconversion method) of chemical stability (related substance growth) when using high temperature data to predict room temperature shelf-life were employed in a detailed comparison. In each case, complex degradant formation behavior was analyzed by both exponential and linear forms of the Arrhenius equation. A hypothetical reaction was used where a drug (A) degrades to a primary degradant (B), which in turn degrades to a secondary degradation product (C). Calculated data with the fitting models were compared with the projected room-temperature shelf-lives of B and C, using one to four time points (in addition to the origin) for each of three accelerated temperatures. Isoconversion methods were found to provide more accurate estimates of shelf-life at ambient conditions. Of the methods for estimating isoconversion, bracketing the specification limit at each condition produced the best estimates and was considerably more accurate than when extrapolation was required. Good estimates of isoconversion produced similar shelf-life estimates fitting either linear or nonlinear forms of the Arrhenius equation, whereas poor isoconversion estimates favored one method or the other depending on which condition was most in error. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Detecting dryland degradation through the use of Time Series Segmentation and Residual Trend analysis (TSS-RESTREND)

NASA Astrophysics Data System (ADS)

Burrell, A. L.; Evans, J. P.; Liu, Y.

2017-12-01

Dryland degradation is an issue of international significance as dryland regions play a substantial role in global food production. Remotely sensed data provide the only long term, large scale record of changes within dryland ecosystems. The Residual Trend, or RESTREND, method is applied to satellite observations to detect dryland degradation. Whilst effective in most cases, it has been shown that the RESTREND method can fail to identify degraded pixels if the relationship between vegetation and precipitation has broken-down as a result of severe or rapid degradation. This study presents an extended version of the RESTREND methodology that incorporates the Breaks For Additive Seasonal and Trend method to identify step changes in the time series that are related to significant structural changes in the ecosystem, e.g. land use changes. When applied to Australia, this new methodology, termed Time Series Segmentation and Residual Trend analysis (TSS-RESTREND), was able to detect degradation in 5.25% of pixels compared to only 2.0% for RESTREND alone. This modified methodology was then assessed in two regions with known histories of degradation where it was found to accurately capture both the timing and directionality of ecosystem change.

Method of degrading pollutants in soil

DOEpatents

Hazen, T.C.; Lopez-De-Victoria, G.

1994-07-05

Disclosed are a method and system for enhancing the motility of microorganisms. This is accomplished by placing an effective amount of chlorinated hydrocarbons, preferably chlorinated alkenes, and most preferably trichloroethylene in spaced relation to the microbes so that the surprisingly strong, monomodal, chemotactic response of the chlorinated hydrocarbon on subsurface microbes can draw the microbes away from or towards and into a substance, as desired. In remediation of groundwater pollution, for example, TCE can be injected into the plume to increase the population of microbes at the plume whereby the plume can be more quickly degraded. A TCE-degrading microbe, such as Welchia alkenophilia, can be used to degrade the TCE following the degradation of the original pollutant. 5 figures.

Preparation of Diatomite Supported Nano Zinc Oxide Composite Photocatalytic Material and Study on its Formaldehyde Degradation

NASA Astrophysics Data System (ADS)

Xiao, Liguang; Pang, Bo

2017-09-01

This experiment used zinc nitrate as precursor, ethanol as solvent and polyethylene glycol as dispersant, diatomite as carrier, diatomite loaded nano Zinc Oxide was prepared by sol-gel method, in addition, the formaldehyde degradation was studied by two kinds of experimental methods: preparation and loading, preparation and post loading, The samples were characterized by SEM, XRD, BET and IR. Experimental results showed that: Diatomite based nano Zinc Oxide had a continuous adsorption and degradation of formaldehyde, formaldehyde gas with initial concentration was 0.7mg/m3, after 36h degradation, the concentration reached 0.238mg/m3, the degradation rate reached to 66%.

Characterization of degradation products of silodosin under stress conditions by liquid chromatography/Fourier transform mass spectrometry.

PubMed

Pandeti, Sukanya; Narender, Tadigoppula; Prabhakar, Sripadi; Reddy, Thota Jagadeswar

2017-03-30

Silodosin (SDN) is a novel α 1 -adrenoceptor antagonist in the treatment of benign prostatic hyperplasia (BPH). The presence of degradation products in a drug affects not only the quality, but also the safety and efficacy of drug formulation. Thus, it is essential to develop an efficient analytical method which could be useful to selectively separate, identify and characterise of all possible degradation products of SDN which is mandatory in drug development processes. SDN was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions. Separation of the drug and degradation products was achieved by a liquid chromatography (LC) method using an Acquity UPLC® BEH C18 (2.1 × 100 mm, 1.7 μm; Waters) column with mobile phase consisting of 0.1% formic acid (FA) in water (A) and 0.1% FA in acetonitrile (ACN) and methanol (MeOH) (1:1) (B) as organic modifier at a flow rate of 0.15 mL min -1 in gradient elution mode. Identification and characterization of the degradation products was performed by mass spectrometry methods using an LTQ-Orbitrap mass spectrometer. A total of five degradation products (DP1 to DP5) were formed under various stress conditions and their structures were proposed with the help of tandem mass spectrometry (MS/MS) experiments and high-resolution mass spectral data. A common degradation product (DP1) was observed under acidic and basic degradation conditions. DP2 was observed under acidic, DP4 and DP5 were observed under basic hydrolytic conditions, whereas DP3 was observed under oxidative conditions. SDN was found to be labile under hydrolytic and oxidative conditions. The structures of all the degradation products were proposed. The most rational mechanisms for the formation of the degradation products under different stress conditions have been established. The proposed method can be effectively used to carry out the determination and detection of SDN and its degradation products. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Methods of analysis by the U.S. Geological Survey Organic Geochemistry Research Group : determination of selected herbicides and their degradation products in water using solid-phase extraction and gas chromatography/mass spectrometry

USGS Publications Warehouse

Kish, J.L.; Thurman, E.M.; Scribner, E.A.; Zimmerman, L.R.

2000-01-01

A method for the extraction and analysis of eight herbicides and five degradation products using solid-phase extraction from natural water samples followed by gas chromatography/mass spectrometry is presented in this report. This method was developed for dimethenamid; flufenacet; fluometuron and its degradation products, demethylfluometuron (DMFM), 3-(trifluromethyl)phenylurea (TFMPU), 3-(trifluromethyl)-aniline (TFMA); molinate; norflurazon and its degradation product, demethylnorflurazon; pendamethalin; the degradation product of prometryn, deisopropylprometryn; propanil; and trifluralin. The eight herbicides are used primarily in the southern United States where cotton, rice, and soybeans are produced. The exceptions are dimethenamid and flufenacet, which are used on corn in the Midwest. Water samples received by the U.S. Geological Survey's Organic Geochemistry Research Group in Lawrence, Kansas, are filtered to remove suspended particulate matter and then passed through disposable solid-phase extraction columns containing octadecyl-bonded porous silica (C-18) to extract the compounds. The herbicides and their degradation products are removed from the column by ethyl acetate elution. The eluate is evaporated under nitrogen, and components then are separated, identified, and quantified by injecting an aliquot of the concentrated extract into a high-resolution, fused-silica capillary column of a gas chromatograph/mass spectrometer under selected-ion mode. Method detection limits ranged from 0.02 to 0.05 ?g/L for all compounds with the exception of TFMPU, which has a method detection limit of 0.32 ?g/L. The mean absolute recovery is 107 percent. This method for the determination of herbicides and their degradation products is valuable for acquiring information about water quality and compound fate and transport in water.

A Study of Method Development, Validation, and Forced Degradation for Simultaneous Quantification of Paracetamol and Ibuprofen in Pharmaceutical Dosage Form by RP-HPLC Method

PubMed Central

Jahan, Md. Sarowar; Islam, Md. Jahirul; Begum, Rehana; Kayesh, Ruhul; Rahman, Asma

2014-01-01

A rapid and stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous quantification of paracetamol and ibuprofen in their combined dosage form especially to get some more advantages over other methods already developed for this combination. The method was validated according to United States Pharmacopeia (USP) guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, and system suitability. Forced degradation study was validated according to International Conference on Harmonisation (ICH). For this, an isocratic condition of mobile phase comprising phosphate buffer (pH 6.8) and acetonitrile in a ratio of 65:35, v/v at a flow rate of 0.7 mL/minute over RP C18 (octadecylsilane (ODS), 150 × 4.6 mm, 5 μm, Phenomenex Inc.) column at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R2) values of 0.999 and 1.0 for paracetamol and ibuprofen respectively, which were within the limit of correlation coefficient (R2 > 0.995). The percent recoveries for two drugs were found within the acceptance limit of (97.0–103.0%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. Forced degradation of the drug product was carried out as per the ICH guidelines with a view to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions. The degradation of ibuprofen was within the limit (5–20%, according to the guideline of ICH), while paracetamol showed <20% degradation in oxidation and basic condition. PMID:25452691

A Study of Method Development, Validation, and Forced Degradation for Simultaneous Quantification of Paracetamol and Ibuprofen in Pharmaceutical Dosage Form by RP-HPLC Method.

PubMed

Jahan, Md Sarowar; Islam, Md Jahirul; Begum, Rehana; Kayesh, Ruhul; Rahman, Asma

2014-01-01

A rapid and stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous quantification of paracetamol and ibuprofen in their combined dosage form especially to get some more advantages over other methods already developed for this combination. The method was validated according to United States Pharmacopeia (USP) guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, and system suitability. Forced degradation study was validated according to International Conference on Harmonisation (ICH). For this, an isocratic condition of mobile phase comprising phosphate buffer (pH 6.8) and acetonitrile in a ratio of 65:35, v/v at a flow rate of 0.7 mL/minute over RP C18 (octadecylsilane (ODS), 150 × 4.6 mm, 5 μm, Phenomenex Inc.) column at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R (2)) values of 0.999 and 1.0 for paracetamol and ibuprofen respectively, which were within the limit of correlation coefficient (R (2) > 0.995). The percent recoveries for two drugs were found within the acceptance limit of (97.0-103.0%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. Forced degradation of the drug product was carried out as per the ICH guidelines with a view to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions. The degradation of ibuprofen was within the limit (5-20%, according to the guideline of ICH), while paracetamol showed <20% degradation in oxidation and basic condition.

Development and Validation of a Stability-indicating Reversed-phase UPLC-UV Method for the Assay of Imidacloprid and Estimation of its Related Compounds.

PubMed

Tian, Jingzhi; Rustum, Abu

2018-02-01

Imidacloprid is used as an active pharmaceutical ingredient (API) in veterinary drugs to control fleas and ticks for dogs and cats. Here we are reporting for the first time a validated stability-indicating reversed-phase UPLC-UV method for the assay of imidacloprid and estimation of its related compounds. The stability-indicating capability of this method has been demonstrated by a forced degradation study. All related compounds including processing impurities, imidacloprid API and degradates from stressed samples were well separated from each other. Structures of major degradates from forced degradation study were elucidated through UPLC-MS/MS and key degradation pathways were proposed from the proposed chemical structures of major degradates. The UPLC-UV method is carried out using an HSS T3 column (C18, 2.1 × 30 mm, 1.8 μm particle size) maintained at 30°C with mobile phase A (0.05% v/v of phosphoric acid in water) and mobile phase B (methanol/acetonitrile 75/25 v/v). Analytes are separated by a gradient elution and detected at 270 nm. The UPLC method is green and fast with only 6.5 min run time and about 3.5 ml mobile phase consumption for each sample analysis. The UPLC-UV method was validated according to ICH guidelines. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Stress degradation studies and development of stability-indicating TLC-densitometry method for determination of prednisolone acetate and chloramphenicol in their individual and combined pharmaceutical formulations

PubMed Central

2012-01-01

A rapid and reproducible stability indicating TLC method was developed for the determination of prednisolone acetate and chloramphenicol in presence of their degraded products. Uniform degradation conditions were maintained by refluxing sixteen reaction mixtures for two hours at 80°C using parallel synthesizer including acidic, alkaline and neutral hydrolysis, oxidation and wet heating degradation. Oxidation at room temperature, photochemical and dry heating degradation studies were also carried out. Separation was done on TLC glass plates, pre-coated with silica gel 60F-254 using chloroform: methanol (14:1 v/v). Spots at Rf 0.21 ± 0.02 and Rf 0.41 ± 0.03 were recognized as chloramphenicol and prednisolone acetate, respectively. Quantitative analysis was done through densitometric measurements at multiwavelength (243 nm, λmax of prednisolone acetate and 278 nm, λmax of chloramphenicol), simultaneously. The developed method was optimized and validated as per ICH guidelines. Method was found linear over the concentration range of 200-6000 ng/spot with the correlation coefficient (r2 ± S.D.) of 0.9976 ± 3.5 and 0.9920 ± 2.5 for prednisolone acetate and chloramphenicol, respectively. The developed TLC method can be applied for routine analysis of prednisolone acetate and chloramphenicol in presence of their degraded products in their individual and combined pharmaceutical formulations. PMID:22264235

Bearing performance degradation assessment based on time-frequency code features and SOM network

NASA Astrophysics Data System (ADS)

Zhang, Yan; Tang, Baoping; Han, Yan; Deng, Lei

2017-04-01

Bearing performance degradation assessment and prognostics are extremely important in supporting maintenance decision and guaranteeing the system’s reliability. To achieve this goal, this paper proposes a novel feature extraction method for the degradation assessment and prognostics of bearings. Features of time-frequency codes (TFCs) are extracted from the time-frequency distribution using a hybrid procedure based on short-time Fourier transform (STFT) and non-negative matrix factorization (NMF) theory. An alternative way to design the health indicator is investigated by quantifying the similarity between feature vectors using a self-organizing map (SOM) network. On the basis of this idea, a new health indicator called time-frequency code quantification error (TFCQE) is proposed to assess the performance degradation of the bearing. This indicator is constructed based on the bearing real-time behavior and the SOM model that is previously trained with only the TFC vectors under the normal condition. Vibration signals collected from the bearing run-to-failure tests are used to validate the developed method. The comparison results demonstrate the superiority of the proposed TFCQE indicator over many other traditional features in terms of feature quality metrics, incipient degradation identification and achieving accurate prediction. Highlights • Time-frequency codes are extracted to reflect the signals’ characteristics. • SOM network served as a tool to quantify the similarity between feature vectors. • A new health indicator is proposed to demonstrate the whole stage of degradation development. • The method is useful for extracting the degradation features and detecting the incipient degradation. • The superiority of the proposed method is verified using experimental data.

Promotion by humus-reducing bacteria for the degradation of UV254 absorbance in reverse-osmosis concentrates pretreated with O3-assisted UV-Fenton method.

PubMed

Xia, Jiaohui; Zhang, Hui; Ding, Shaoxuan; Li, Changyu; Ding, Jincheng; Lu, Jie

2017-07-12

The primary pollutants in reverse-osmosis concentrates (ROC) are the substances with the UV absorbance at 254 nm (UV 254 ), which is closely related to humic substances that can be degraded by humus-reducing bacteria. This work studied the degradation characteristics of humus-reducing bacteria in ROC treatment. The physiological and biochemical characteristics of humus-reducing bacteria were investigated, and the effects of pH values and electron donors on the reduction of humic analog, antraquinone-2, 6-disulfonate were explored to optimize the degradation. Furthermore, the O 3 -assisted UV-Fenton method was applied for the pretreatment of ROC, and the degradation of UV 254 absorbance was apparently promoted with their removal rate, reaching 84.2% after 10 days of degradation by humus-reducing bacteria.

Differentiating climate- and human-induced drivers of grassland degradation in the Liao River Basin, China.

PubMed

He, Chunyang; Tian, Jie; Gao, Bin; Zhao, Yuanyuan

2015-01-01

Quantitatively distinguishing grassland degradation due to climatic variations from that due to human activities is of great significance to effectively governing degraded grassland and realizing sustainable utilization. The objective of this study was to differentiate these two types of drivers in the Liao River Basin during 1999-2009 using the residual trend (RESTREND) method and to evaluate the applicability of the method in semiarid and semihumid regions. The relationship between the normalized difference vegetation index (NDVI) and each climatic factor was first determined. Then, the primary driver of grassland degradation was identified by calculating the change trend of the normalized residuals between the observed and the predicted NDVI assuming that climate change was the only driver. We found that the RESTREND method can be used to quantitatively and effectively differentiate climate and human drivers of grassland degradation. We also found that the grassland degradation in the Liao River Basin was driven by both natural processes and human activities. The driving factors of grassland degradation varied greatly across the study area, which included regions having different precipitation and altitude. The degradation in the Horqin Sandy Land, with lower altitude, was driven mainly by human activities, whereas that in the Kungl Prairie, with higher altitude and lower precipitation, was caused primarily by climate change. Therefore, the drivers of degradation and local conditions should be considered in an appropriate strategy for grassland management to promote the sustainability of grasslands in the Liao River Basin.

Restoration of motion blurred images

NASA Astrophysics Data System (ADS)

Gaxiola, Leopoldo N.; Juarez-Salazar, Rigoberto; Diaz-Ramirez, Victor H.

2017-08-01

Image restoration is a classic problem in image processing. Image degradations can occur due to several reasons, for instance, imperfections of imaging systems, quantization errors, atmospheric turbulence, relative motion between camera or objects, among others. Motion blur is a typical degradation in dynamic imaging systems. In this work, we present a method to estimate the parameters of linear motion blur degradation from a captured blurred image. The proposed method is based on analyzing the frequency spectrum of a captured image in order to firstly estimate the degradation parameters, and then, to restore the image with a linear filter. The performance of the proposed method is evaluated by processing synthetic and real-life images. The obtained results are characterized in terms of accuracy of image restoration given by an objective criterion.

First derivative spectrophotometric and LC determination of benoxinate hydrochloride and its degradation products.

PubMed

El-Gindy, A

2000-03-01

Two methods are presented for the determination of benoxinate HCI and its acid and alkali-induced degradation products using first derivative (1D) spectrophotometry with zero-crossing measurements and liquid chromatography. Benoxinate HCl was determined by measurement of its first derivative amplitude in mcllvaine's-citric acid phosphate buffer pH 7.0 at 268.4 and 272.4 nm in the presence of its alkali- and acid-induced degradation products, respectively. The acid- and alkali-induced, degradation products were determined by measurement of their first derivative amplitude in the same solvent at 307.5 nm. The LC method depends upon using a mu bondapak CN column with a mobile phase consisting of acetonitrile-water triethylamine (60:40:0.01, v/v) and adjusted to apparent pH 7. Quantitation was achieved with UV detection at 310 nm based on peak area. The proposed methods were utilized to investigate the kinetics of the acidic and alkaline degradation processes at different temperatures. The pH-rate profile of degradation of benoxinate HCl in Britton-Robinson buffer solutions was studied.

The cell-in-series method: A technique for accelerated electrode degradation in redox flow batteries

DOE PAGES

Pezeshki, Alan M.; Sacci, Robert L.; Veith, Gabriel M.; ...

2015-11-21

Here, we demonstrate a novel method to accelerate electrode degradation in redox flow batteries and apply this method to the all-vanadium chemistry. Electrode performance degradation occurred seven times faster than in a typical cycling experiment, enabling rapid evaluation of materials. This method also enables the steady-state study of electrodes. In this manner, it is possible to delineate whether specific operating conditions induce performance degradation; we found that both aggressively charging and discharging result in performance loss. Post-mortem x-ray photoelectron spectroscopy of the degraded electrodes was used to resolve the effects of state of charge (SoC) and current on the electrodemore » surface chemistry. For the electrode material tested in this work, we found evidence that a loss of oxygen content on the negative electrode cannot explain decreased cell performance. Furthermore, the effects of decreased electrode and membrane performance on capacity fade in a typical cycling battery were decoupled from crossover; electrode and membrane performance decay were responsible for a 22% fade in capacity, while crossover caused a 12% fade.« less

Building and degradation of secondary cell walls: are there common patterns of lamellar assembly of cellulose microfibrils and cell wall delamination?

PubMed

De Micco, Veronica; Ruel, Katia; Joseleau, Jean-Paul; Aronne, Giovanna

2010-08-01

During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments.

Photocatalytic and Photoelectrochemically Degradation of Chlorsulfuron herbicide

NASA Astrophysics Data System (ADS)

Guo, Xu; Liu, Hongwei; Miao, Jinjie; Ma, Zhen

2017-12-01

Photocatalytic and photo electrochemical (PEC) degradation of chlorsulfuron herbicide were studied. Two novel PEC electrodes Ti/IrO2-Pt-WO3 (TIW) and Ti/IrO2-Pt-Ag3PO4 (TIA) were designed and some important factors were studied. Lower current density showed lower removal efficiency than higher conditions by electrochemical method. Furthermore, PEC showed higher degradation efficiency than the sum of individual EO and photocatalytic methode.

Lacticin LC14, a new bacteriocin produced by Lactococcus lactis BMG6.14: isolation, purification and partial characterization.

PubMed

Lasta, Samar; Ouzari, Hadda; Andreotti, Nicolas; Fajloun, Ziad; Mansuelle, Pascal; Boudabous, Abdellatif; Sampieri, Francois; Sabatier, Jean Marc

2012-08-01

A new bacteriocin, lacticin LC14, produced by Lactococcus lactis BMG6.14, was isolated and characterized. It was purified to homogeneity from overnight broth culture by ammonium sulfate precipitation, Sep-Pak chromatography, and two steps of reversed-phase HPLC. Lacticin LC14 showed bactericidal-type antimicrobial activity against several lactic acid bacteria and pathogenic strains including Listeria monocytogenes. It was inactivated by proteinase K and pronase E, but was resistant to papain, lysozyme, lipase and catalase. Lacticin LC14 was heat resistant, stable over a wide range of pH (2-10) and after treatment by solvents and detergents. Its N-terminal end was found unreactive towards Edman sequencing. Based on MALDI-TOF mass spectrometry, its molecular mass was 3333.7 Da. LC14 amino acid composition revealed a high proportion of hydrophobic residues, but no modified ones. LC14 may be able to challenge other well known other bacteriocins in probiotic and therapeutic applications.

Alternate seal configuration for lithium primary cells

NASA Technical Reports Server (NTRS)

Kelley, J. A.

1982-01-01

The problem of glass degradation in the glass-to-metal seals in lithium/sulfur dioxide cells is discussed. The glass degradation mechanism is attributed to lithium reacting with glass which is a result of deposition of lithium at the glass/metal/electrolyte interface. The worst degradation was observed when cells were stored in the inverted position. Alternate sealing methods were examined and a modified Ziegler seal is considered to be one of the best possible methods. The seal consists of a crimp type soft seal using a plastic annulus and a metal tube. Results of degradation tests are presented.

Recommendations for the treatment of aging in standard technical specifications

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

Orton, R.D.; Allen, R.P.

1995-09-01

As part of the US Nuclear Regulatory Commission`s Nuclear Plant Aging Research Program, Pacific Northwest Laboratory (PNL) evaluated the standard technical specifications for nuclear power plants to determine whether the current surveillance requirements (SRs) were effective in detecting age-related degradation. Nuclear Plant Aging Research findings for selected systems and components were reviewed to identify the stressors and operative aging mechanisms and to evaluate the methods available to detect, differentiate, and trend the resulting aging degradation. Current surveillance and testing requirements for these systems and components were reviewed for their effectiveness in detecting degraded conditions and for potential contributions to prematuremore » degradation. When the current surveillance and testing requirements appeared ineffective in detecting aging degradation or potentially could contribute to premature degradation, a possible deficiency in the SRs was identified that could result in undetected degradation. Based on this evaluation, PNL developed recommendations for inspection, surveillance, trending, and condition monitoring methods to be incorporated in the SRs to better detect age- related degradation of these selected systems and components.« less

Study on degrading graphene oxide in wastewater under different conditions for developing an efficient and economical degradation method.

PubMed

Li, Ting; Zhang, Chao-Zhi; Gu, Chengyue

2017-12-01

With popular application of graphene and graphene oxide (GO), they have been discharged into water. Graphene and GO harm organisms. However, an efficient and economical method for removing graphene and GO in wastewater has seldom been reported. Graphene can be oxidized by hydrogen peroxide to give GO; therefore, degradation of graphene oxide is an important step in the procedure of removal of graphene from water. In this paper, GO degradation via photo-Fenton reaction under different conditions was carried out. Experimental results suggested that GO in wastewater can be efficiently and economically degraded into carbon dioxide and H 2 O when pH value is 3, concentration of H 2 O 2 and FeCl 3 are 35 mM and 5 ppm, respectively. Degradation mechanism of GO was suggested based on UV-vis absorption spectra, scanning electron microscopy, X-ray diffraction and liquid chromatography-mass spectra data of degradation intermediates. This paper suggests an efficient and economical degradation way of GO in wastewater.

A validated specific stability-indicating RP-HPLC assay method for Ambrisentan and its related substances.

PubMed

Narayana, M B V; Chandrasekhar, K B; Rao, B M

2014-09-01

A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability). © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A General Accelerated Degradation Model Based on the Wiener Process.

PubMed

Liu, Le; Li, Xiaoyang; Sun, Fuqiang; Wang, Ning

2016-12-06

Accelerated degradation testing (ADT) is an efficient tool to conduct material service reliability and safety evaluations by analyzing performance degradation data. Traditional stochastic process models are mainly for linear or linearization degradation paths. However, those methods are not applicable for the situations where the degradation processes cannot be linearized. Hence, in this paper, a general ADT model based on the Wiener process is proposed to solve the problem for accelerated degradation data analysis. The general model can consider the unit-to-unit variation and temporal variation of the degradation process, and is suitable for both linear and nonlinear ADT analyses with single or multiple acceleration variables. The statistical inference is given to estimate the unknown parameters in both constant stress and step stress ADT. The simulation example and two real applications demonstrate that the proposed method can yield reliable lifetime evaluation results compared with the existing linear and time-scale transformation Wiener processes in both linear and nonlinear ADT analyses.

A General Accelerated Degradation Model Based on the Wiener Process

PubMed Central

Liu, Le; Li, Xiaoyang; Sun, Fuqiang; Wang, Ning

2016-01-01

Accelerated degradation testing (ADT) is an efficient tool to conduct material service reliability and safety evaluations by analyzing performance degradation data. Traditional stochastic process models are mainly for linear or linearization degradation paths. However, those methods are not applicable for the situations where the degradation processes cannot be linearized. Hence, in this paper, a general ADT model based on the Wiener process is proposed to solve the problem for accelerated degradation data analysis. The general model can consider the unit-to-unit variation and temporal variation of the degradation process, and is suitable for both linear and nonlinear ADT analyses with single or multiple acceleration variables. The statistical inference is given to estimate the unknown parameters in both constant stress and step stress ADT. The simulation example and two real applications demonstrate that the proposed method can yield reliable lifetime evaluation results compared with the existing linear and time-scale transformation Wiener processes in both linear and nonlinear ADT analyses. PMID:28774107

Atomistic modeling of water diffusion in hydrolytic biomaterials.

PubMed

Gautieri, Alfonso; Mezzanzanica, Andrea; Motta, Alberto; Redealli, Alberto; Vesentini, Simone

2012-04-01

One of the most promising applications of hydrolytically degrading biomaterials is their use as drug release carriers. These uses, however, require that the degradation and diffusion of drug are reliably predicted, which is complex to achieve through present experimental methods. Atomistic modeling can help in the knowledge-based design of degrading biomaterials with tuned drug delivery properties, giving insights on the small molecules diffusivity at intermediate states of the degradation process. We present here an atomistic-based approach to investigate the diffusion of water (through which hydrolytic degradation occurs) in degrading bulk models of poly(lactic acid) or PLA. We determine the water diffusion coefficient for different swelling states of the polymeric matrix (from almost dry to pure water) and for different degrees of degradation. We show that water diffusivity is highly influenced by the swelling degree, while little or not influenced by the degradation state. This approach, giving water diffusivity for different states of the matrix, can be combined with diffusion-reaction analytical methods in order to predict the degradation path on longer time scales. Furthermore, atomistic approach can be used to investigate diffusion of other relevant small molecules, eventually leading to the a priori knowledge of degradable biomaterials transport properties, helping the design of the drug delivery systems.

Method for modeling the gradual physical degradation of a porous material

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

Flach, Greg

Cementitious and other engineered porous materials encountered in waste disposals may degrade over time due to one or more mechanisms. Physical degradation may take the form of cracking (fracturing) and/or altered (e.g. increased) porosity, depending on the material and underlying degradation mechanism. In most cases, the hydraulic properties of degrading materials are expected to evolve due to physical changes occurring over roughly the pore to decimeter scale, which is conducive to calculating equivalent or effective material properties. The exact morphology of a degrading material in its end-state may or may not be known. In the latter case, the fully-degraded conditionmore » can be assumed to be similar to a more-permeable material in the surrounding environment, such as backfill soil. Then the fully-degraded waste form or barrier material is hydraulically neutral with respect to its surroundings, constituting neither a barrier to nor conduit for moisture flow and solute transport. Unless the degradation mechanism is abrupt, a gradual transition between the intact initial and fully-degraded final states is desired. Linear interpolation through time is one method for smoothly blending hydraulic properties between those of an intact matrix and those of a soil or other surrogate for the end-state.« less

A new approach to accelerated drug-excipient compatibility testing.

PubMed

Sims, Jonathan L; Carreira, Judith A; Carrier, Daniel J; Crabtree, Simon R; Easton, Lynne; Hancock, Stephen A; Simcox, Carol E

2003-01-01

The purpose of this study was to develop a method of qualitatively predicting the most likely degradants in a formulation or probing specific drug-excipient interactions in a significantly shorter time frame than the typical 1 month storage testing. In the example studied, accelerated storage testing of a solid dosage form at 50 degrees C, the drug substance SB-243213-A degraded via the formation of two oxidative impurities. These impurities reached a level of 1% PAR after 3 months. Various stressing methods were examined to try to recreate this degradation and in doing so provide a practical and reliable method capable of predicting drug-excipient interactions. The technique developed was able to mimic the 1-month's accelerated degradation in just 1 hr. The method was suitable for automated analysis, capable of multisample stressing, and ideal for use in drug-excipient compatibility screening.

HPLC & NMR-based forced degradation studies of ifosfamide: The potential of NMR in stability studies.

PubMed

Salman, D; Peron, J-M R; Goronga, T; Barton, S; Swinden, J; Nabhani-Gebara, S

2016-03-01

The aim of this study is to conduct a forced degradation study on ifosfamide under several stress conditions to investigate the robustness of the developed HPLC method. It also aims to provide further insight into the stability of ifosfamide and its degradation profile using both HPLC and NMR. Ifosfamide solutions (20mg/mL; n=15, 20mL) were stressed in triplicate by heating (70°C), under acidic (pH 1 & 4) and alkaline (pH 10 & 12) conditions. Samples were analysed periodically using HPLC and FT-NMR. Ifosfamide was most stable under weakly acidic conditions (pH 4). NMR results suggested that the mechanism of ifosfamide degradation involves the cleavage of the PN bond. For all stress conditions, HPLC was not able to detect ifosfamide degradation products that were detected by NMR. These results suggest that the developed HPLC method for ifosfamide did not detect the degradation products shown by NMR. It is possible that degradation products co-elute with ifosfamide, do not elute altogether or are not amenable to the detection method employed. Therefore, investigation of ifosfamide stability requires additional techniques that do not suffer from the aforementioned shortcomings. Copyright © 2015 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.

A Novel Degradation Identification Method for Wind Turbine Pitch System

NASA Astrophysics Data System (ADS)

Guo, Hui-Dong

2018-04-01

It’s difficult for traditional threshold value method to identify degradation of operating equipment accurately. An novel degradation evaluation method suitable for wind turbine condition maintenance strategy implementation was proposed in this paper. Based on the analysis of typical variable-speed pitch-to-feather control principle and monitoring parameters for pitch system, a multi input multi output (MIMO) regression model was applied to pitch system, where wind speed, power generation regarding as input parameters, wheel rotation speed, pitch angle and motor driving currency for three blades as output parameters. Then, the difference between the on-line measurement and the calculated value from the MIMO regression model applying least square support vector machines (LSSVM) method was defined as the Observed Vector of the system. The Gaussian mixture model (GMM) was applied to fitting the distribution of the multi dimension Observed Vectors. Applying the model established, the Degradation Index was calculated using the SCADA data of a wind turbine damaged its pitch bearing retainer and rolling body, which illustrated the feasibility of the provided method.

Modelling accelerated degradation data using Wiener diffusion with a time scale transformation.

PubMed

Whitmore, G A; Schenkelberg, F

1997-01-01

Engineering degradation tests allow industry to assess the potential life span of long-life products that do not fail readily under accelerated conditions in life tests. A general statistical model is presented here for performance degradation of an item of equipment. The degradation process in the model is taken to be a Wiener diffusion process with a time scale transformation. The model incorporates Arrhenius extrapolation for high stress testing. The lifetime of an item is defined as the time until performance deteriorates to a specified failure threshold. The model can be used to predict the lifetime of an item or the extent of degradation of an item at a specified future time. Inference methods for the model parameters, based on accelerated degradation test data, are presented. The model and inference methods are illustrated with a case application involving self-regulating heating cables. The paper also discusses a number of practical issues encountered in applications.

TiO2 used as photocatalyst for rhodamine B degradation under solar radiation

NASA Astrophysics Data System (ADS)

Ariyanti, Dessy; Maillot, Mathilde; Gao, Wei

2017-07-01

Transition metal oxide photocatalysis is a relatively new method representing advanced oxidation process to be applied in industrial wastewater treatment especially for degradation of organic pollutants. We investigate TiO2 as a photocatalyst for the photocatalytic degradation of Rhodamine B (RhB) under simulated sunlight. Various parameters and their effectiveness have been studied. The effects of processing parameters including catalyst loading and feed concentration were investigated; and the degradation pathway was proposed based on the UHPLC-MS analysis. The result showed that a higher kinetic rate can be obtained by employing low catalyst loading and feed concentration, i.e., 0.5 g/L of TiO2 loading and 5 ppm of RhB concentration, respectively. For this particular system, the optimum degradation rate (k) can achieve 0.297/min. The effectiveness of solar light-TiO2 system for RhB degradation shows this method can be used for wastewater treatment.

Stability indicating spectrophotometric and spectrodensitometric methods for the determination of diatrizoate sodium in presence of its degradation product

NASA Astrophysics Data System (ADS)

Abd El-Rahman, Mohamed K.; Riad, Safaa M.; Abdel Gawad, Sherif A.; Fawaz, Esraa M.; Shehata, Mostafa A.

2015-02-01

Three sensitive, selective, and precise stability indicating methods for the determination of the X-ray contrast agent, diatrizoate sodium (DTA), in the presence of its acidic degradation product (highly cytotoxic 3,5 diamino metabolite) and in pharmaceutical formulation were developed and validated. The first method is a first derivative (D1) spectrophotometric one, which allows the determination of DTA in the presence of its degradate at 231.2 nm (corresponding to zero crossing of the degradate) over a concentration range of 2-24 μg/mL with mean percentage recovery 99.95 ± 0.97%. The second method is the first derivative of the ratio spectra (DD1) by measuring the peak amplitude at 227 nm over the same concentration range as D1 spectrophotometric method, with mean percentage recovery 99.99 ± 1.15%. The third method is a TLC-densitometric one, where DTA was separated from its degradate on silica gel plates using chloroform:methanol:ammonium hydroxide (20:10:2 by volume) as a developing system. This method depends on quantitative densitometric evaluation of thin layer chromatogram of DTA at 238 nm over a concentration range of 4-20 μg/spot, with mean percentage recovery 99.88 ± 0.89%. The selectivity of the proposed methods was tested using laboratory-prepared mixtures. The proposed methods have been successfully applied to the analysis of DTA in pharmaceutical dosage forms without interference from other dosage form additives. The results were statistically compared with the official US pharmacopeial method. No significant difference for either accuracy or precision was observed.

Development of RP-HPLC, Stability Indicating Method for Degradation Products of Linagliptin in Presence of Metformin HCl by Applying 2 Level Factorial Design; and Identification of Impurity-VII, VIII and IX and Synthesis of Impurity-VII.

PubMed

Jadhav, Sushant B; Reddy, P Sunil; Narayanan, Kalyanaraman L; Bhosale, Popatrao N

2017-06-27

The novel reverse phase-high performance liquid chromatography (RP-HPLC), stability indicating method was developed for determination of linagliptin (LGP) and its related substances in linagliptin and metformin HCl (MET HCl) tablets by implementing design of experiment to understand the critical method parameters and their relation with critical method attributes; to ensure robustness of the method. The separation of nine specified impurities was achieved with a Zorbax SB-Aq 250 × 4.6 mm, 5 µm column, using gradient elution and a detector wavelength of 225 nm, and validated in accordance with International Conference on Harmonization (ICH) guidelines and found to be accurate, precise, reproducible, robust, and specific . The drug was found to be degrading extensively in heat, humidity, basic, and oxidation conditions and was forming degradation products during stability studies. After slight modification in the buffer and the column, the same method was used for liquid chromatography-mass spectrometry (LC-MS) and ultra-performance liquid chromatography -time-of-flight/mass spectrometry UPLC-TOF/MS analysis, to identify m/z and fragmentation of maximum unspecified degradation products i.e., Impurity-VII ( 7 ), Impurity-VIII ( 8 ), and Impurity-IX ( 9 ) formed during stability studies. Based on the results, a degradation pathway for the drug has been proposed and synthesis of Impurity-VII ( 7 ) is also discussed to ensure an in-depth understanding of LGP and its related degradation products and optimum performance during the lifetime of the product.

Stability-Indicating HPTLC Method for Studying Stress Degradation Behavior of Sulbutiamine HCl.

PubMed

Farid, Nehal F; Abdelwahab, Nada S

2016-04-01

Sulbutiamine (SUL) is an ester of thiazides with neurotropic action. A new stability indicating HPTLC method has been developed and validated for the determination of SUL in the presence of different degradation products. The drug was subjected to different stress conditions following ICH strategy such as hydrolytic degradation (neutral, alkaline and acidic hydrolysis), oxidation, photodegradation and dry heat degradation. The drug demonstrated degradation under all decomposition conditions except neutral hydrolysis and dry heat, where the drug was completely degraded with 0.1 N NaOH, 1 N HCl and 30% H2O2 while it was partially degradaed by 0.1 N HCl, 3% H2O2 and UV light. Structure elucidation of the resulting degradation products was performed using ESI-Q-MS-MS. A well-defined peak for SUL was obtained at Rf = 0.46 and was completely separated from all obtained degradation products. Chromatographic separation was carried out on HPTLC aluminum plates precoated with silica gel 60 F254 using acetone-methylene chloride-ammonia buffer (pH 8.5 ± 0.2) (7:3:0.5, v/v) as a developing system. Densitometric scanning of the separated peaks was performed at 254 nm. System suitability testing parameters were calculated to ascertain the quality performance of the developed method. The method was validated with respect to USP guidelines regarding accuracy, precision, specificity, robustness and ruggedness. Good correlation coefficients were achieved in the range of 0.4-5.0 µg/band, and the limit of detection and limit of quantitation were found to be 0.11 and 0.33 µg/band, respectively. The utility of the suggested method was verified by application to Arcalion forte® tablets where no interference from additives was found. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

First derivative ratio spectrophotometric, HPTLC-densitometric, and HPLC determination of nicergoline in presence of its hydrolysis-induced degradation product.

PubMed

Ahmad, Abdel Kader S; Kawy, M Abdel; Nebsen, M

2002-10-15

Three methods are presented for the determination of Nicergoline in presence of its hydrolysis-induced degradation product. The first method was based on measurement of the first derivative of ratio spectra amplitude of Nicergoline at 291 nm. The second method was based on separation of Nicergoline from its degradation product followed by densitometric measurement of the spots at 287 nm. The separation was carried out on HPTLC silica gel F(254) plates, using methanol-ethyl acetate-glacial acetic acid (5:7:3, v/v/v) as mobile phase. The third method was based on high performance liquid chromatographic (HPLC) separation and determination of Nicergoline from its degradation product on a reversed phase, nucloesil C(18) column using a mobile phase of methanol-water-glacial acetic acid (80:20:0.1, v/v/v) with UV detection at 280 nm. Chlorpromazine hydrochloride was used as internal standard. Laboratory prepared mixtures containing different percentages of the degradation product were analysed by the proposed methods and satisfactory results were obtained. These methods have been successfully applied to the analysis of Nicergoline in Sermion tablets. The validities of these methods were ascertained by applying standard addition technique, the mean percentage recovery +/- R.S.D.% was found to be 99.47 +/- 0.752, 100.01 +/- 0.940, 99.75 +/- 0.740 for the first derivative of ratio spectra method, the HPTLC method and the HPLC method, respectively. The proposed methods were statistically compared with the manufacturer's HPLC method of analysis of Nicergoline and no significant difference was found with respect to both precision and accuracy. They have the advantage of being stability indicating. Therefore, they can be used for routine analysis of the drug in quality control laboratories. Copyright 2002 Elsevier Science B.V.

Chemotactic selection of pollutant degrading soil bacteria

DOEpatents

Hazen, T.C.

1991-03-04

A method is described for identifying soil microbial strains which may be bacterial degraders of pollutants. This method includes: Placing a concentration of a pollutant in a substantially closed container; placing the container in a sample of soil for a period of time ranging from one minute to several hours; retrieving the container and collecting its contents; microscopically determining the identity of the bacteria present. Different concentrations of the pollutant can be used to determine which bacteria respond to each concentration. The method can be used for characterizing a polluted site or for looking for naturally occurring biological degraders of the pollutant. Then bacteria identified as degraders of the pollutant and as chemotactically attracted to the pollutant are used to innoculate contaminated soil. To enhance the effect of the bacteria on the pollutant, nutrients are cyclicly provided to the bacteria then withheld to alternately build up the size of the bacterial colony or community and then allow it to degrade the pollutant.

[Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation].

PubMed

Li, Jinlong; Zhang, Yidan; Zhou, Xin; Ji, Wenjie; Zhao, Jihong; Wei, Luqing; Li, Yuming

2014-09-01

To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis. Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen. HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees. This work established an efficient method for in vitro generation and degradation of human NETs.

A LDR-PCR approach for multiplex polymorphisms genotyping of severely degraded DNA with fragment sizes <100 bp.

PubMed

Zhang, Zhen; Wang, Bao-Jie; Guan, Hong-Yu; Pang, Hao; Xuan, Jin-Feng

2009-11-01

Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini-short tandem repeat-polymerase chain reaction (PCR) and mini-sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.

Development and Validation of a Stability-Indicating Assay of Etofenamate by RP-HPLC and Characterization of Degradation Products

PubMed Central

Peraman, Ramalingam; Nayakanti, Devanna; Dugga, Hari Hara Theja; Kodikonda, Sudhakara

2013-01-01

A validated stability-indicating RP-HPLC method for etofenamate (ETF) was developed by separating its degradation products on a C18 (250 mm × 4.6 mm 5 μm) Qualisil BDS column using a phosphate buffer (pH-adjusted to 6.0 with orthophosphoric acid) and methanol in the ratio of 20:80 % v/v as the mobile phase at a flow rate of 1.0 mL/min. The column effluents were monitored by a photodiode array detector set at 286 nm. The method was validated in terms of specificity, linearity, accuracy, precision, detection limit, quantification limit, and robustness. Forced degradation of etofenamate was carried out under acidic, basic, thermal, photo, and peroxide conditions and the major degradation products of acidic and basic degradation were isolated and characterized by 1H-NMR, 13C-NMR, and mass spectral studies. The mass balance of the method varied between 92–99%. PMID:24482770

Stochastic Modeling and Analysis of Multiple Nonlinear Accelerated Degradation Processes through Information Fusion

PubMed Central

Sun, Fuqiang; Liu, Le; Li, Xiaoyang; Liao, Haitao

2016-01-01

Accelerated degradation testing (ADT) is an efficient technique for evaluating the lifetime of a highly reliable product whose underlying failure process may be traced by the degradation of the product’s performance parameters with time. However, most research on ADT mainly focuses on a single performance parameter. In reality, the performance of a modern product is usually characterized by multiple parameters, and the degradation paths are usually nonlinear. To address such problems, this paper develops a new s-dependent nonlinear ADT model for products with multiple performance parameters using a general Wiener process and copulas. The general Wiener process models the nonlinear ADT data, and the dependency among different degradation measures is analyzed using the copula method. An engineering case study on a tuner’s ADT data is conducted to demonstrate the effectiveness of the proposed method. The results illustrate that the proposed method is quite effective in estimating the lifetime of a product with s-dependent performance parameters. PMID:27509499

Stochastic Modeling and Analysis of Multiple Nonlinear Accelerated Degradation Processes through Information Fusion.

PubMed

Sun, Fuqiang; Liu, Le; Li, Xiaoyang; Liao, Haitao

2016-08-06

Accelerated degradation testing (ADT) is an efficient technique for evaluating the lifetime of a highly reliable product whose underlying failure process may be traced by the degradation of the product's performance parameters with time. However, most research on ADT mainly focuses on a single performance parameter. In reality, the performance of a modern product is usually characterized by multiple parameters, and the degradation paths are usually nonlinear. To address such problems, this paper develops a new s-dependent nonlinear ADT model for products with multiple performance parameters using a general Wiener process and copulas. The general Wiener process models the nonlinear ADT data, and the dependency among different degradation measures is analyzed using the copula method. An engineering case study on a tuner's ADT data is conducted to demonstrate the effectiveness of the proposed method. The results illustrate that the proposed method is quite effective in estimating the lifetime of a product with s-dependent performance parameters.

Nondestructive testing methods to predict effect of degradation on wood : a critical assessment

Treesearch

J. Kaiserlik

1978-01-01

Results are reported for an assessment of methods for predicting strength of wood, wood-based, or related material. Research directly applicable to nondestructive strength prediction was very limited. In wood, strength prediction research is limited to vibration decay, wave attenuation, and multiparameter "degradation models." Nonwood methods with potential...

Ratio manipulating spectrophotometry versus chemometry as stability indicating methods for cefquinome sulfate determination

NASA Astrophysics Data System (ADS)

Yehia, Ali M.; Arafa, Reham M.; Abbas, Samah S.; Amer, Sawsan M.

2016-01-01

Spectral resolution of cefquinome sulfate (CFQ) in the presence of its degradation products was studied. Three selective, accurate and rapid spectrophotometric methods were performed for the determination of CFQ in the presence of either its hydrolytic, oxidative or photo-degradation products. The proposed ratio difference, derivative ratio and mean centering are ratio manipulating spectrophotometric methods that were satisfactorily applied for selective determination of CFQ within linear range of 5.0-40.0 μg mL- 1. Concentration Residuals Augmented Classical Least Squares was applied and evaluated for the determination of the cited drug in the presence of its all degradation products. Traditional Partial Least Squares regression was also applied and benchmarked against the proposed advanced multivariate calibration. Experimentally designed 25 synthetic mixtures of three factors at five levels were used to calibrate and validate the multivariate models. Advanced chemometrics succeeded in quantitative and qualitative analyses of CFQ along with its hydrolytic, oxidative and photo-degradation products. The proposed methods were applied successfully for different pharmaceutical formulations analyses. These developed methods were simple and cost-effective compared with the manufacturer's RP-HPLC method.

A method for addressing differences in concentrations of fipronil and three degradates obtained by two different laboratory methods

USGS Publications Warehouse

Crawford, Charles G.; Martin, Jeffrey D.

2017-07-21

In October 2012, the U.S. Geological Survey (USGS) began measuring the concentration of the pesticide fipronil and three of its degradates (desulfinylfipronil, fipronil sulfide, and fipronil sulfone) by a new laboratory method using direct aqueous-injection liquid chromatography tandem mass spectrometry (DAI LC–MS/MS). This method replaced the previous method—in use since 2002—that used gas chromatography/mass spectrometry (GC/MS). The performance of the two methods is not comparable for fipronil and the three degradates. Concentrations of these four chemical compounds determined by the DAI LC–MS/MS method are substantially lower than the GC/MS method. A method was developed to correct for the difference in concentrations obtained by the two laboratory methods based on a methods comparison field study done in 2012. Environmental and field matrix spike samples to be analyzed by both methods from 48 stream sites from across the United States were sampled approximately three times each for this study. These data were used to develop a relation between the two laboratory methods for each compound using regression analysis. The relations were used to calibrate data obtained by the older method to the new method in order to remove any biases attributable to differences in the methods. The coefficients of the equations obtained from the regressions were used to calibrate over 16,600 observations of fipronil, as well as the three degradates determined by the GC/MS method retrieved from the USGS National Water Information System. The calibrated values were then compared to over 7,800 observations of fipronil and to the three degradates determined by the DAI LC–MS/MS method also retrieved from the National Water Information System. The original and calibrated values from the GC/MS method, along with measures of uncertainty in the calibrated values and the original values from the DAI LC–MS/MS method, are provided in an accompanying data release.

Research study on high energy radiation effect and environment solar cell degradation methods

NASA Technical Reports Server (NTRS)

Horne, W. E.; Wilkinson, M. C.

1974-01-01

The most detailed and comprehensively verified analytical model was used to evaluate the effects of simplifying assumptions on the accuracy of predictions made by the external damage coefficient method. It was found that the most serious discrepancies were present in heavily damaged cells, particularly proton damaged cells, in which a gradient in damage across the cell existed. In general, it was found that the current damage coefficient method tends to underestimate damage at high fluences. An exception to this rule was thick cover-slipped cells experiencing heavy degradation due to omnidirectional electrons. In such cases, the damage coefficient method overestimates the damage. Comparisons of degradation predictions made by the two methods and measured flight data confirmed the above findings.

Application of contact glow discharge electrolysis method for degradation of batik dye waste Remazol Red by the addition of Fe2+ ion

NASA Astrophysics Data System (ADS)

Saksono, Nelson; Puspita, Indah; Sukreni, Tulus

2017-03-01

Contact Glow Discharge Electrolysis (CGDE) has been shown to degrade much weight organic compounds such as dyes because the production of hydroxil radical (•OH) is excess. This research aims to degrade batik dye waste Remazol Red, using CGDE method with the addition of Fe2+ ion. The addition of iron salt compounds has proven to increase process efficiency. Dye degradation is known by measure its absorbances with Spectrophotometer UV-Vis. The result of study showed that percentage degradation was 99.92% in 20 minutes which obtained by using Na2SO4 0.01 M, with addition FeSO4 0,1 gram, applied voltage 860 volt, and 1 wolfram anode 5 mm depth.

Methods of degrading napalm B

DOEpatents

Tyndall, Richard L.; Vass, Arpad

1995-01-01

Methods of degrading napalm and/or trinitrotoluene involve contacting the waste with specific intra-amoebic isolates of ATCC 40908 and/or dispersants derived therefrom. Useful isolates include is deposited as ATCC 77529, NAP-1 deposited as ATCC 77526 and 13 deposited as ATCC 77527.

Observation method to predict meander migration and vertical degradation of rivers.

DOT National Transportation Integrated Search

2014-05-01

Meander migration and vertical degradation of river bed are processes that have been studied for years. : Different methods have been proposed to make predictions of the behavior of rivers with respect to these : processes. These two erosion controll...

Methods of degrading napalm B

DOEpatents

Tyndall, R.L.; Vass, A.

1995-09-12

Methods of degrading napalm and/or trinitrotoluene involve contacting the waste with specific intra-amoebic isolates of ATCC 40908 and/or dispersants derived therefrom. Useful isolates are deposited as ATCC 77529, NAP-1 deposited as ATCC 77526 and 13 deposited as ATCC 77527.

Blind estimation of blur in hyperspectral images

NASA Astrophysics Data System (ADS)

Zhang, Mo; Vozel, Benoit; Chehdi, Kacem; Uss, Mykhail; Abramov, Sergey; Lukin, Vladimir

2017-10-01

Hyperspectral images acquired by remote sensing systems are generally degraded by noise and can be sometimes more severely degraded by blur. When no knowledge is available about the degradations present on the original image, blind restoration methods can only be considered. By blind, we mean absolutely no knowledge neither of the blur point spread function (PSF) nor the original latent channel and the noise level. In this study, we address the blind restoration of the degraded channels component-wise, according to a sequential scheme. For each degraded channel, the sequential scheme estimates the blur point spread function (PSF) in a first stage and deconvolves the degraded channel in a second and final stage by means of using the PSF previously estimated. We propose a new component-wise blind method for estimating effectively and accurately the blur point spread function. This method follows recent approaches suggesting the detection, selection and use of sufficiently salient edges in the current processed channel for supporting the regularized blur PSF estimation. Several modifications are beneficially introduced in our work. A new selection of salient edges through thresholding adequately the cumulative distribution of their corresponding gradient magnitudes is introduced. Besides, quasi-automatic and spatially adaptive tuning of the involved regularization parameters is considered. To prove applicability and higher efficiency of the proposed method, we compare it against the method it originates from and four representative edge-sparsifying regularized methods of the literature already assessed in a previous work. Our attention is mainly paid to the objective analysis (via ݈l1-norm) of the blur PSF error estimation accuracy. The tests are performed on a synthetic hyperspectral image. This synthetic hyperspectral image has been built from various samples from classified areas of a real-life hyperspectral image, in order to benefit from realistic spatial distribution of reference spectral signatures to recover after synthetic degradation. The synthetic hyperspectral image has been successively degraded with eight real blurs taken from the literature, each of a different support size. Conclusions, practical recommendations and perspectives are drawn from the results experimentally obtained.

Quality by design approach for the separation of naproxcinod and its related substances by fused core particle technology column.

PubMed

Inugala, Ugandar Reddy; Pothuraju, Nageswara Rao; Vangala, Ranga Reddy

2013-01-01

This paper describes the development of a rapid, novel, stability-indicating gradient reversed-phase high-performance liquid chromatographic method and associated system suitability parameters for the analysis of naproxcinod in the presence of its related substances and degradents using a quality-by-design approach. All of the factors that affect the separation of naproxcinod and its impurities and their mutual interactions were investigated and robustness of the method was ensured. The method was developed using an Ascentis Express C8 150 × 4.6 mm, 2.7 µm column with a mobile phase containing a gradient mixture of two solvents. The eluted compounds were monitored at 230 nm, the run time was 20 min within which naproxcinod and its eight impurities were satisfactorily separated. Naproxcinod was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Naproxcinod was found to degrade significantly in acidic and basic conditions and to be stable in thermal, photolytic, oxidative and aqueous degradation conditions. The degradation products were satisfactorily resolved from the primary peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.

Gravimetric screening method for fungal decay of paper: inoculation with Trametes versicolor.

PubMed

Råberg, Ulrika; Hafrén, Jonas

2009-10-01

The European standard test EN 113 for fungal degradation of solid wood has been adapted for degradation of paper by white rot fungus (Trametes versicolor). Fungal degradation of paper sheets may potentially be used for screening different wood preservatives on paper instead of solid wood. The paper samples showed higher relative mass losses compared to wood, and samples pretreated with boric acid, copper sulfate and polymerized linseed oil were successfully tested for biodegradation using the paper sheet method. The results on paper degradation were compared with wood, both as wood blocks (according to standard test) and wood cut in sections forming layered structures mimicking paper layers.

Analytical method development of nifedipine and its degradants binary mixture using high performance liquid chromatography through a quality by design approach

NASA Astrophysics Data System (ADS)

Choiri, S.; Ainurofiq, A.; Ratri, R.; Zulmi, M. U.

2018-03-01

Nifedipin (NIF) is a photo-labile drug that easily degrades when it exposures a sunlight. This research aimed to develop of an analytical method using a high-performance liquid chromatography and implemented a quality by design approach to obtain effective, efficient, and validated analytical methods of NIF and its degradants. A 22 full factorial design approach with a curvature as a center point was applied to optimize of the analytical condition of NIF and its degradants. Mobile phase composition (MPC) and flow rate (FR) as factors determined on the system suitability parameters. The selected condition was validated by cross-validation using a leave one out technique. Alteration of MPC affected on time retention significantly. Furthermore, an increase of FR reduced the tailing factor. In addition, the interaction of both factors affected on an increase of the theoretical plates and resolution of NIF and its degradants. The selected analytical condition of NIF and its degradants has been validated at range 1 – 16 µg/mL that had good linearity, precision, accuration and efficient due to an analysis time within 10 min.

A New Platform for Profiling Degradation-Related Impurities Via Exploiting the Opportunities Offered by Ion-Selective Electrodes: Determination of Both Diatrizoate Sodium and Its Cytotoxic Degradation Product.

PubMed

Riad, Safaa M; Abd El-Rahman, Mohamed K; Fawaz, Esraa M; Shehata, Mostafa A

2018-05-01

Although the ultimate goal of administering active pharmaceutical ingredients (APIs) is to save countless lives, the presence of impurities and/or degradation products in APIs or formulations may cause harmful physiological effects. Today, impurity profiling (i.e., the identity as well as the quantity of impurity in a pharmaceutical) is receiving critical attention from regulatory authorities. Despite the predominant use of spectroscopic and chromatographic methods over electrochemical methods for impurity profiling of APIs, this work investigates the opportunities offered by electroanalytical methods, particularly, ion-selective electrodes (ISEs), for profiling degradation-related impurities (DRIs) compared with conventional spectroscopic and chromatographic methods. For a meaningful comparison, diatrizoate sodium (DTA) was chosen as the anionic X-ray contrast agent based on its susceptibility to deacetylation into its cytotoxic and mutagenic degradation product, 3,5-diamino-2,4,6 triiodobenzoic acid (DTB). This cationic diamino compound can be also detected as an impurity in the final product because it is used as a synthetic precursor for the synthesis of DTA. In this study, four novel sensitive and selective sensors for the determination of both DTA and its cytotoxic degradation products are presented. Sensors I and II were developed for the determination of the anionic drug, DTA, and sensors III and IV were developed for the determination of the cationic cytotoxic impurity. The use of these novel sensors not only provides a stability-indicating method for the selective determination of DTA in the presence of its degradation product, but also permits DRI profiling. Moreover, a great advantage of these proposed ISE systems is their higher sensitivity for the quantification of DTB relative to other spectroscopic and chromatographic methods, so it can measure trace amounts of DTB impurities in DTA bulk powder and pharmaceutical formulation without a need for preliminary separation.

DEVELOPMENT OF METHOD 535 FOR THE DETERMINATION OF CHLOROACETANILIDE AND OTHER ACETAMIDE HERBICIDE DEGRADATES IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

EPA Science Inventory

EPA Method 535 has been developed in order to provide a method for the analysis of "Alachlor ESA and other acetanilide degradation products" which are listed on U.S. EPA's 1998 Drinking Water Contaminant Candidate List. Method 535 uses solid phase extraction with a nonporous gr...

Photocatalytic degradation of methylene blue dye by zinc oxide nanoparticles obtained from precipitation and sol-gel methods.

PubMed

Balcha, Abebe; Yadav, Om Prakash; Dey, Tania

2016-12-01

Zinc oxide (ZnO) nanoparticles were synthesized by precipitation and sol-gel methods. The aim of this study was to understand how different synthetic methods can affect the photocatalytic activity of ZnO nanoparticles. As-synthesized ZnO nanoparticles were characterized by X-ray diffraction (XRD) and UV-Visible spectroscopic techniques. XRD patterns of ZnO powders synthesized by precipitation and sol-gel methods revealed their hexagonal wurtzite structure with crystallite sizes of 30 and 28 nm, respectively. Their photocatalytic activities were evaluated by photocatalytic degradation of methylene blue, a common water pollutant, under UV radiation. The effects of operational parameters such as photocatalyst load and initial concentration of the dye on photocatalytic degradation of methylene blue were investigated. While the degradation of dye decreased over the studied dye concentration range of 20 to 100 mg/L, an optimum photocatalyst load of 250 mg/L was needed to achieve dye degradation as high as 81 and 92.5 % for ZnO prepared by precipitation and sol-gel methods, respectively. Assuming pseudo first-order reaction kinetics, this corresponded to rate constants of 8.4 × 10 -3 and 12.4 × 10 -3  min -1 , respectively. Hence, sol-gel method is preferred over precipitation method in order to achieve higher photocatalytic activity of ZnO nanostructures. Photocatalytic activity is further augmented by better choice of capping ligand for colloidal stabilization, starch being more effective than polyethylene glycol (PEG).

Gradient RP-HPLC method for the determination of potential impurities in atazanavir sulfate.

PubMed

Chitturi, Sreenivasa Rao; Somannavar, Yallappa Somappa; Peruri, Badarinadh Gupta; Nallapati, Sreenivas; Sharma, Hemant Kumar; Budidet, Shankar Reddy; Handa, Vijay Kumar; Vurimindi, Hima Bindu

2011-04-28

This paper proposes a simple and selective RP-HPLC method for the determination of process impurities and degradation products (degradants) of atazanavir sulfate (ATV) drug substance. Chromatographic separation was achieved on Ascentis(®) Express C8, (150mm×4.6mm, 2.7μm) column thermostated at 30°C under gradient elution by a binary mixture of potassium dihydrogen phosphate (pH 3.5, 0.02M) and ACN at a flow rate of 1.0ml/min. A photodiode array (PDA) detector set at 250nm was used for detection. Stress testing (forced degradation) of ATV was carried out under acidic, alkaline, oxidative, photolytic, thermal and humidity conditions. In presence of alkali, ATV transformed into cyclized products and the order of degradation reaction is determined by the method of initial rates. The unknown process impurities and alkaline degradants are isolated by preparative LC and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectral data. The developed method is validated with respect to sensitivity (lod and loq), linearity, precision, accuracy and robustness and can be implemented for routine quality control analysis and stability testing of ATV. Copyright © 2011 Elsevier B.V. All rights reserved.

Method for determination of methyl tert-butyl ether and its degradation products in water

USGS Publications Warehouse

Church, C.D.; Isabelle, L.M.; Pankow, J.F.; Rose, D.L.; Tratnyek, P.G.

1997-01-01

An analytical method is described that can detect the major alkyl ether compounds that are used as gasoline oxygenates (methyl tert-butyl ether, MTBE; ethyl tert-butyl ether, ETBE; and tert-amyl methyl ether, TAME) and their most characteristic degradation products (tert-butyl alcohol, TBA; tert-butyl formate, TBF; and tert-amyl alcohol, TAA) in water at sub-ppb concentrations. The new method involves gas chromatography (GC) with direct aqueous injection (DAI) onto a polar column via a splitless injector, coupled with detection by mass spectrometry (MS). DAI-GC/MS gives excellent agreement with conventional purge-and-trap methods for MTBE over a wide range of environmentally relevant concentrations. The new method can also give simultaneous identification of polar compounds that might occur as degradation products of gasoline oxygenates, such as TBA, TBF, TAA, methyl acetate, and acetone. When the method was applied to effluent from a column microcosm prepared with core material from an urban site in New Jersey, conversion of MTBE to TBA was observed after a lag period of 35 days. However, to date, analyses of water samples from six field sites using the DAI-GC/MS method have not produced evidence for the expected products of in situ degradation of MTBE.An analytical method is described that can detect the major alkyl ether compounds that are used as gasoline oxygenates (methyl tert-butyl ether, MTBE; ethyl tert-butyl ether, ETBE; and tert-amyl methyl ether, TAME) and their most characteristic degradation products (tert-butyl alcohol, TBA; tert-butyl formate, TBF; and tert-amyl alcohol, TAA) in water at sub-ppb concentrations. The new method involves gas chromatography (GC) with direct aqueous injection (DAI) onto a polar column via a splitless injector, coupled with detection by mass spectrometry (MS). DAI-GC/MS gives excellent agreement with conventional purge-and-trap methods for MTBE over a wide range of environmentally relevant concentrations. The new method can also give simultaneous identification of polar compounds that might occur as degradation products of gasoline oxygenates, such as TBA, TBF, TAA, methyl acetate, and acetone. When the method was applied to effluent from a column microcosm prepared with core material from an urban site in New Jersey, conversion of MTBE to TBA was observed after a lag period of 35 days. However, to date, analyses of water samples from six field sites using the DAI-GC/MS method have not produced evidence for the expected products of in situ degradation of MTBE.

Short stack modeling of degradation in solid oxide fuel cells. Part II. Sensitivity and interaction analysis

NASA Astrophysics Data System (ADS)

Gazzarri, J. I.; Kesler, O.

In the first part of this two-paper series, we presented a numerical model of the impedance behaviour of a solid oxide fuel cell (SOFC) aimed at simulating the change in the impedance spectrum induced by contact degradation at the interconnect-electrode, and at the electrode-electrolyte interfaces. The purpose of that investigation was to develop a non-invasive diagnostic technique to identify degradation modes in situ. In the present paper, we appraise the predictive capabilities of the proposed method in terms of its robustness to uncertainties in the input parameters, many of which are very difficult to measure independently. We applied this technique to the degradation modes simulated in Part I, in addition to anode sulfur poisoning. Electrode delamination showed the highest robustness to input parameter variations, followed by interconnect oxidation and interconnect detachment. The most sensitive degradation mode was sulfur poisoning, due to strong parameter interactions. In addition, we simulate several simultaneous two-degradation-mode scenarios, assessing the method's capabilities and limitations for the prediction of electrochemical behaviour of SOFC's undergoing multiple simultaneous degradation modes.

40 CFR 265.314 - Special requirements for bulk and containerized liquids.

Code of Federal Regulations, 2012 CFR

2012-07-01

... (iii) Mixtures of these non-bio-degrad-a-ble materials. (2) Tests for non-bio-degrad-a-ble sorbents. (i) The sorbent material is determined to be non-bio-degrad-a-ble under ASTM Method G21-70 (1984a...

40 CFR 265.314 - Special requirements for bulk and containerized liquids.

Code of Federal Regulations, 2013 CFR

2013-07-01

... (iii) Mixtures of these non-bio-degrad-a-ble materials. (2) Tests for non-bio-degrad-a-ble sorbents. (i) The sorbent material is determined to be non-bio-degrad-a-ble under ASTM Method G21-70 (1984a...

40 CFR 265.314 - Special requirements for bulk and containerized liquids.

Code of Federal Regulations, 2014 CFR

2014-07-01

... (iii) Mixtures of these non-bio-degrad-a-ble materials. (2) Tests for non-bio-degrad-a-ble sorbents. (i) The sorbent material is determined to be non-bio-degrad-a-ble under ASTM Method G21-70 (1984a...

40 CFR 265.314 - Special requirements for bulk and containerized liquids.

Code of Federal Regulations, 2010 CFR

2010-07-01

... (iii) Mixtures of these non-bio-degrad-a-ble materials. (2) Tests for non-bio-degrad-a-ble sorbents. (i) The sorbent material is determined to be non-bio-degrad-a-ble under ASTM Method G21-70 (1984a...

Development and validation of a UV-spectrophotometric method for the determination of pheniramine maleate and its stability studies

NASA Astrophysics Data System (ADS)

Raghu, M. S.; Basavaiah, K.; Ramesh, P. J.; Abdulrahman, Sameer A. M.; Vinay, K. B.

2012-03-01

A sensitive, precise, and cost-effective UV-spectrophotometric method is described for the determination of pheniramine maleate (PAM) in bulk drug and tablets. The method is based on the measurement of absorbance of a PAM solution in 0.1 N HCl at 264 nm. As per the International Conference on Harmonization (ICH) guidelines, the method was validated for linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), and robustness and ruggedness. A linear relationship between absorbance and concentration of PAM in the range of 2-40 μg/ml with a correlation coefficient (r) of 0.9998 was obtained. The LOD and LOQ values were found to be 0.18 and 0.39 μg/ml PAM, respectively. The precision of the method was satisfactory: the value of relative standard deviation (RSD) did not exceed 3.47%. The proposed method was applied successfully to the determination of PAM in tablets with good accuracy and precision. Percentages of the label claims ranged from 101.8 to 102.01% with the standard deviation (SD) from 0.64 to 0.72%. The accuracy of the method was further ascertained by recovery studies via a standard addition procedure. In addition, the forced degradation of PAM was conducted in accordance with the ICH guidelines. Acidic and basic hydrolysis, thermal stress, peroxide, and photolytic degradation were used to assess the stability-indicating power of the method. A substantial degradation was observed during oxidative and alkaline degradations. No degradation was observed under other stress conditions.

Computer-aided diagnosis based on enhancement of degraded fundus photographs.

PubMed

Jin, Kai; Zhou, Mei; Wang, Shaoze; Lou, Lixia; Xu, Yufeng; Ye, Juan; Qian, Dahong

2018-05-01

Retinal imaging is an important and effective tool for detecting retinal diseases. However, degraded images caused by the aberrations of the eye can disguise lesions, so that a diseased eye can be mistakenly diagnosed as normal. In this work, we propose a new image enhancement method to improve the quality of degraded images. A new method is used to enhance degraded-quality fundus images. In this method, the image is converted from the input RGB colour space to LAB colour space and then each normalized component is enhanced using contrast-limited adaptive histogram equalization. Human visual system (HVS)-based fundus image quality assessment, combined with diagnosis by experts, is used to evaluate the enhancement. The study included 191 degraded-quality fundus photographs of 143 subjects with optic media opacity. Objective quality assessment of image enhancement (range: 0-1) indicated that our method improved colour retinal image quality from an average of 0.0773 (variance 0.0801) to an average of 0.3973 (variance 0.0756). Following enhancement, area under curves (AUC) were 0.996 for the glaucoma classifier, 0.989 for the diabetic retinopathy (DR) classifier, 0.975 for the age-related macular degeneration (AMD) classifier and 0.979 for the other retinal diseases classifier. The relatively simple method for enhancing degraded-quality fundus images achieves superior image enhancement, as demonstrated in a qualitative HVS-based image quality assessment. This retinal image enhancement may, therefore, be employed to assist ophthalmologists in more efficient screening of retinal diseases and the development of computer-aided diagnosis. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Determination of LEDs degradation with entropy generation rate

NASA Astrophysics Data System (ADS)

Cuadras, Angel; Yao, Jiaqiang; Quilez, Marcos

2017-10-01

We propose a method to assess the degradation and aging of light emitting diodes (LEDs) based on irreversible entropy generation rate. We degraded several LEDs and monitored their entropy generation rate ( S ˙ ) in accelerated tests. We compared the thermoelectrical results with the optical light emission evolution during degradation. We find a good relationship between aging and S ˙ (t), because S ˙ is both related to device parameters and optical performance. We propose a threshold of S ˙ (t) as a reliable damage indicator of LED end-of-life that can avoid the need to perform optical measurements to assess optical aging. The method lays beyond the typical statistical laws for lifetime prediction provided by manufacturers. We tested different LED colors and electrical stresses to validate the electrical LED model and we analyzed the degradation mechanisms of the devices.

Testing Method of Degrading Heavy Oil Pollution by Microorganisms

NASA Astrophysics Data System (ADS)

Wu, Qi; Zhao, Lin; Ma, Aijin

2018-01-01

With the development of human society, we are more and more relying on the petrochemical energy. The use of petrochemical energy not only brings us great convenience, but is also accompanied by a series of environmental pollution problems, especially oil pollution. Since it is impractical to restore all pollution problems, the proper use of some remedial measures, under the guidance of functional orientation, may be sufficient to minimize the risk of persistent and diffusing pollutants. In recent years, bioremediation technology has been gradually developed into a promising stage and has played a crucial role in the degradation of heavy oil pollution. Specially, microbes in the degradation of heavy oil have made a great contribution. This paper mainly summarizes the different kinds of microorganisms for degrading heavy oil and the detection method for degradation efficiency of heavy oil pollution.

Degradation of learned skills. Effectiveness of practice methods on simulated space flight skill retention

NASA Technical Reports Server (NTRS)

Sitterley, T. E.; Berge, W. A.

1972-01-01

Manual flight control and emergency procedure task skill degradation was evaluated after time intervals of from 1 to 6 months. The tasks were associated with a simulated launch through the orbit insertion flight phase of a space vehicle. The results showed that acceptable flight control performance was retained for 2 months, rapidly deteriorating thereafter by a factor of 1.7 to 3.1 depending on the performance measure used. Procedural task performance showed unacceptable degradation after only 1 month, and exceeded an order of magnitude after 4 months. The effectiveness of static rehearsal (checklists and briefings) and dynamic warmup (simulator practice) retraining methods were compared for the two tasks. Static rehearsal effectively countered procedural skill degradation, while some combination of dynamic warmup appeared necessary for flight control skill retention. It was apparent that these differences between methods were not solely a function of task type or retraining method, but were a function of the performance measures used for each task.

Validation of a headspace trap gas chromatography and mass spectrometry method for the quantitative analysis of volatile compounds from degraded rapeseed oil.

PubMed

Sghaier, Lilia; Cordella, Christophe B Y; Rutledge, Douglas N; Watiez, Mickaël; Breton, Sylvie; Sassiat, Patrick; Thiebaut, Didier; Vial, Jérôme

2016-05-01

Due to lipid oxidation, off-flavors, characterized by a fishy odor, are emitted during the heating of rapeseed oil in a fryer and affect the flavor of rapeseed oil even at low concentrations. Thus, there is a need for analytical methods to identify and quantify these products. To study the headspace composition of degraded rapeseed oil, and more specifically the compounds responsible for the fishy odor, a headspace trap gas chromatography with mass spectrometry method was developed and validated. Six volatile compounds formed during the degradation of rapeseed oil were quantified: 1-penten-3-one, (Z)-4-heptenal, hexanal, nonanal, (E,E)-heptadienal, and (E)-2-heptenal. Validation using accuracy profiles allowed us to determine the valid ranges of concentrations for each compound, with acceptance limits of 40% and tolerance limits of 80%. This method was then successfully applied to real samples of degraded oils. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An overview of state-of-the-art image restoration in electron microscopy.

PubMed

Roels, J; Aelterman, J; Luong, H Q; Lippens, S; Pižurica, A; Saeys, Y; Philips, W

2018-06-08

In Life Science research, electron microscopy (EM) is an essential tool for morphological analysis at the subcellular level as it allows for visualization at nanometer resolution. However, electron micrographs contain image degradations such as noise and blur caused by electromagnetic interference, electron counting errors, magnetic lens imperfections, electron diffraction, etc. These imperfections in raw image quality are inevitable and hamper subsequent image analysis and visualization. In an effort to mitigate these artefacts, many electron microscopy image restoration algorithms have been proposed in the last years. Most of these methods rely on generic assumptions on the image or degradations and are therefore outperformed by advanced methods that are based on more accurate models. Ideally, a method will accurately model the specific degradations that fit the physical acquisition settings. In this overview paper, we discuss different electron microscopy image degradation solutions and demonstrate that dedicated artefact regularisation results in higher quality restoration and is applicable through recently developed probabilistic methods. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Derivative spectrophotometry for the determination of faropenem in the presence of degradation products: an application for kinetic studies.

PubMed

Cielecka-Piontek, Judyta

2013-07-01

A simple and selective derivative spectrophotometric method was developed for the quantitative determination of faropenem in pure form and in pharmaceutical dosage. The method is based on the zero-crossing effect of first-derivative spectrophotometry (λ = 324 nm), which eliminates the overlapping effect caused by the excipients present in the pharmaceutical preparation, as well as degradation products, formed during hydrolysis, oxidation, photolysis, and thermolysis. The method was linear in the concentration range 2.5-300 μg/mL (r = 0.9989) at λ = 341 nm; the limits of detection and quantitation were 0.16 and 0.46 μg/mL, respectively. The method had good precision (relative standard deviation from 0.68 to 2.13%). Recovery of faropenem ranged from 97.9 to 101.3%. The first-order rate constants of the degradation of faropenem in pure form and in pharmaceutical dosage were determined by using first-derivative spectrophotometry. A statistical comparison of the validation results and the observed rate constants for faropenem degradation with these obtained with the high-performance liquid chromatography method demonstrated that both were compatible.

ANALYTICAL METHOD DEVELOPMENT FOR ALACHLOR ESA AND OTHER ACETANILIDE HERBICIDE DEGRADATION PRODUCTS

EPA Science Inventory

In 1998, USEPA published a Drinking Water Contaminant Candidate List (CCL) of 50 chemicals and 10 microorganisms. "Alachlor ESA and other acetanilide herbicide degradation products" is listed on the the 1998 CCL. Acetanilide degradation products are generally more water soluble...

[Mitigative effect of micribial degradation on autotoxicity of Panax ginseng].

PubMed

Li, Yong; Long, Qi-Liang; Ding, Wan-Long; Zhao, Dong-Yue

2014-08-01

Continuously cropping obstacle restricts ginseng production and rational use of land resource severely, and autotoxicity is one of the most important factors. In our previous work, ginseng autotoxin degrading bacteria were isolated, in the present re- search, plate culturing method and traditional physiological and biochemical method were used to analyze biological indices and protective enzyme activities, in order to elucidate the mitigative effect of autotoxin degrading bacteria on autotoxicity of P. ginseng. Results indicated that, except for palmitic acid, autotoxicity of benzonic acid, diisobutyl phthalate, diisobutyl succinate, and 2,2-bis (4- hydroxyphenyl) propane on the growth of ginseng seeds was significantly alleviated after autotoxins degrading bacteria was inoculated, and which have no evident difference with control. Except for benzoic acid, enzyme activity of SOD, POD and CAT in other autotoxin degrading treatments decreased significantly. The present research showed that, microbial degradation could alleviate the autotoxicity of autotoxins on ginseng seeds effectively, and which will be helpful for the resolution of ginseng continuously cropping obstacle problem.

Multistage degradation modeling for BLDC motor based on Wiener process

NASA Astrophysics Data System (ADS)

Yuan, Qingyang; Li, Xiaogang; Gao, Yuankai

2018-05-01

Brushless DC motors are widely used, and their working temperatures, regarding as degradation processes, are nonlinear and multistage. It is necessary to establish a nonlinear degradation model. In this research, our study was based on accelerated degradation data of motors, which are their working temperatures. A multistage Wiener model was established by using the transition function to modify linear model. The normal weighted average filter (Gauss filter) was used to improve the results of estimation for the model parameters. Then, to maximize likelihood function for parameter estimation, we used numerical optimization method- the simplex method for cycle calculation. Finally, the modeling results show that the degradation mechanism changes during the degradation of the motor with high speed. The effectiveness and rationality of model are verified by comparison of the life distribution with widely used nonlinear Wiener model, as well as a comparison of QQ plots for residual. Finally, predictions for motor life are gained by life distributions in different times calculated by multistage model.

Polymer scaffold degradation control via chemical control

DOEpatents

Hedberg-Dirk, Elizabeth L.; Dirk, Shawn; Cicotte, Kirsten

2016-01-05

A variety of polymers and copolymers suitable for use as biologically compatible constructs and, as a non-limiting specific example, in the formation of degradable tissue scaffolds as well methods for synthesizing these polymers and copolymers are described. The polymers and copolymers have degradation rates that are substantially faster than those of previously described polymers suitable for the same uses. Copolymers having a synthesis route which enables one to fine tune the degradation rate by selecting the specific stoichiometry of the monomers in the resulting copolymer are also described. The disclosure also provides a novel synthesis route for maleoyl chloride which yields monomers suitable for use in the copolymer synthesis methods described herein.

A thin film degradation study of a fluorinated polyether liquid lubricant using an HPLC method

NASA Technical Reports Server (NTRS)

Morales, W.

1986-01-01

A High Pressure Liquid Chromatography (HPLC) separation method was developed to study and analyze a fluorinated polyether fluid which is promising liquid lubricant for future applications. This HPLC separation method was used in a preliminary study investigating the catalytic effect of various metal, metal alloy, and ceramic engineering materials on the degradation of this fluid in a dry air atmosphere at 345 C. Using a 440 C stainless steel as a reference catalytic material it was found that a titanium alloy and a chromium plated material degraded the fluorinated polyether fluid substantially more than the reference material.

Analysis and detection of the herbicides dimethenamid and flufenacet and their sulfonic and oxanilic acid degradates in natural water

USGS Publications Warehouse

Zimmerman, L.R.; Schneider, R.J.; Thurman, E.M.

2002-01-01

Dimethenamid [2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1-methylethyl)acetamide] and flufenacet [N-(4-fluorophenyl)-N-(1-methylethyl)-2-(5-(trifluoromethyl)-1,3,4- thiadiazol-2-yl)oxy] were isolated by C-18 solid-phase extraction and separated from their ethanesulfonic acid (ESA) and oxanilic acid (OXA) degradates during their elution using ethyl acetate for the parent compound, followed by methanol for the polar degradates. The parent compounds were detected using gas chromatography-mass spectrometry in selected-ion mode. The ESA and OXA degradates were detected using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESPMS) in negative-ion mode. The method detection limits for a 123-mL sample ranged from 0.01 to 0.07 μg/L. These methods are compatible with existing methods and thus allow for analysis of 17 commonly used herbicides and 18 of their degradation compounds with one extraction. In a study of herbicide transport near the mouth of the Mississippi River during 1999 and 2000, dimethenamid and its ESA and OXA degradates were detected in surface water samples during the annual spring flushes. For flufenacet, the only detections at the study site were for the ESA degradates in samples collected at the peak of the herbicide spring flush in 2000. The low frequency of detections in surface water likely is due to dimethenamid and flufenacet being relatively new herbicides. In addition, detectable amounts of the stable degradates have not been detected in ground water.

Stability-indicating methods for the determination of piretanide in presence of the alkaline induced degradates.

PubMed

Youssef, Nadia F

2005-10-04

Stability-indicating high performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and first-derivative of ratio spectra (1DD) methods are developed for the determination of piretanide in presence of its alkaline induced degradates. HPLC method depends on separation of piretanide from its degradates on mu-Bondapak C18 column using methanol:water:acetic acid (70:30:1, v/v/v) as a mobile phase at flow rate 1.0 ml/min and UV detector at 275 nm. TLC densitometic method is based on the difference in Rf-values between the intact drug and its degradates on thin-layer silica gel. Iso-propanol:ammonia 33% (8:2, v/v) was used as a developing mobile phase and the chromatogram was scanned at 275 nm. The derivative of ratio spectra method (1DD) depends on the measurement of the absorbance at 288 nm in the first-derivative of ratio spectra for the determination of the cited drug in the presence of its degradates. Calibration graphs of the three suggested methods are linear in the concentration ranges 0.02-0.3 microg/20 microl, 0.5-10 microg/spot and 5-50 microg/ml, with mean percentage recovery 99.27+/-0.52, 99,17+/-1.01 and 99.65+/-1.01%, respectively. The three proposed methods were successfully applied for the determination of piretanide in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage form with good accuracy and precision. The results were statistically analyzed and compared with those obtained by the official method. Validation of the method was determined with favourable specificity, linearity, precision, and accuracy was assessed by applying the standard addition technique.

Ratio manipulating spectrophotometry versus chemometry as stability indicating methods for cefquinome sulfate determination.

PubMed

Yehia, Ali M; Arafa, Reham M; Abbas, Samah S; Amer, Sawsan M

2016-01-15

Spectral resolution of cefquinome sulfate (CFQ) in the presence of its degradation products was studied. Three selective, accurate and rapid spectrophotometric methods were performed for the determination of CFQ in the presence of either its hydrolytic, oxidative or photo-degradation products. The proposed ratio difference, derivative ratio and mean centering are ratio manipulating spectrophotometric methods that were satisfactorily applied for selective determination of CFQ within linear range of 5.0-40.0 μg mL(-1). Concentration Residuals Augmented Classical Least Squares was applied and evaluated for the determination of the cited drug in the presence of its all degradation products. Traditional Partial Least Squares regression was also applied and benchmarked against the proposed advanced multivariate calibration. Experimentally designed 25 synthetic mixtures of three factors at five levels were used to calibrate and validate the multivariate models. Advanced chemometrics succeeded in quantitative and qualitative analyses of CFQ along with its hydrolytic, oxidative and photo-degradation products. The proposed methods were applied successfully for different pharmaceutical formulations analyses. These developed methods were simple and cost-effective compared with the manufacturer's RP-HPLC method. Copyright © 2015 Elsevier B.V. All rights reserved.

Methods for locating ground faults and insulation degradation condition in energy conversion systems

DOEpatents

Agamy, Mohamed; Elasser, Ahmed; Galbraith, Anthony William; Harfman Todorovic, Maja

2015-08-11

Methods for determining a ground fault or insulation degradation condition within energy conversion systems are described. A method for determining a ground fault within an energy conversion system may include, in part, a comparison of baseline waveform of differential current to a waveform of differential current during operation for a plurality of DC current carrying conductors in an energy conversion system. A method for determining insulation degradation within an energy conversion system may include, in part, a comparison of baseline frequency spectra of differential current to a frequency spectra of differential current transient at start-up for a plurality of DC current carrying conductors in an energy conversion system. In one embodiment, the energy conversion system may be a photovoltaic system.

Development and application of a validated stability-indicating high-performance liquid chromatographic method using photodiode array detection for simultaneous determination of granisetron, methylparaben, propylparaben, sodium benzoate, and their main degradation products in oral pharmaceutical preparations.

PubMed

Hewala, Ismail; El-Fatatry, Hamed; Emam, Ehab; Mabrouk, Mokhtar

2011-01-01

A simple, rapid, and sensitive RP-HPLC method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron), sodium benzoate, methylparaben, propylparaben, and 4-hydroxybenzoic acid (the main degradation product of parabens) in granisetron oral drops and solutions. The separation of the compounds was achieved within 8 min on a SymmetryShield RP18 column (100 x 4.6 mm id, 3.5 microm particle size) using the mobile phase acetonitrile--0.05 M KH2PO4 buffered to pH 3 using H3PO4 (3+7, v/v). The photodiode array detector was used to test the purity of the peaks, and the chromatograms were extracted at 240 nm. The method was validated, and validation acceptance criteria were met in all cases. The robust method was successfully applied to the determination of granisetron and preservatives, as well as their degradation products in different batches of granisetron oral drops and solutions. The method proved to be sensitive for determination down to 0.04% (w/w) of granisetron degradation product relative to granisetron and 0.03% (w/w) 4-hydroxybenzoic acid relative to total parabens.

Solar/UV-induced photocatalytic degradation of three commercial textile dyes.

PubMed

Neppolian, B; Choi, H C; Sakthivel, S; Arabindoo, Banumathi; Murugesan, V

2002-01-28

The photocatalytic degradation of three commercial textile dyes with different structure has been investigated using TiO(2) (Degussa P25) photocatalyst in aqueous solution under solar irradiation. Experiments were conducted to optimise various parameters viz. amount of catalyst, concentration of dye, pH and solar light intensity. Degradation of all the dyes were examined by using chemical oxygen demand (COD) method. The degradation efficiency of the three dyes is as follows: Reactive Yellow 17(RY17) > Reactive Red 2(RR2) > Reactive Blue 4 (RB4), respectively. The experimental results indicate that TiO(2) (Degussa P25) is the best catalyst in comparison with other commercial photocatalysts such as, TiO(2) (Merck), ZnO, ZrO(2), WO(3) and CdS. Though the UV irradiation can efficiently degrade the dyes, naturally abundant solar irradiation is also very effective in the mineralisation of dyes. The comparison between thin-film coating and aqueous slurry method reveals that slurry method is more efficient than coating but the problems of leaching and the requirement of separation can be avoided by using coating technique. These observations indicate that all the three dyes could be degraded completely at different time intervals. Hence, it may be a viable technique for the safe disposal of textile wastewater into the water streams.

Isolation of Inositol Hexaphosphate (IHP)-Degrading Bacteria from Arbuscular Mycorrhizal Fungal Hyphal Compartments Using a Modified Baiting Method Involving Alginate Beads Containing IHP

PubMed Central

Hara, Shintaro; Saito, Masanori

2016-01-01

Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF. PMID:27383681

High-performance liquid chromatography/high-resolution multiple stage tandem mass spectrometry using negative-ion-mode hydroxide-doped electrospray ionization for the characterization of lignin degradation products.

PubMed

Owen, Benjamin C; Haupert, Laura J; Jarrell, Tiffany M; Marcum, Christopher L; Parsell, Trenton H; Abu-Omar, Mahdi M; Bozell, Joseph J; Black, Stuart K; Kenttämaa, Hilkka I

2012-07-17

In the search for a replacement for fossil fuel and the valuable chemicals currently obtained from crude oil, lignocellulosic biomass has become a promising candidate as an alternative biorenewable source for crude oil. Hence, many research efforts focus on the extraction, degradation, and catalytic transformation of lignin, hemicellulose, and cellulose. Unfortunately, these processes result in the production of very complex mixtures. Further, while methods have been developed for the analysis of mixtures of oligosaccharides, this is not true for the complex mixtures generated upon degradation of lignin. For example, high-performance liquid chromatography/multiple stage tandem mass spectrometry (HPLC/MS(n)), a tool proven to be invaluable in the analysis of complex mixtures derived from many other biopolymers, such as proteins and DNA, has not been implemented for lignin degradation products. In this study, we have developed an HPLC separation method for lignin degradation products that is amenable to negative-ion-mode electrospray ionization (ESI doped with NaOH), the best method identified thus far for ionization of lignin-related model compounds without fragmentation. The separated and ionized compounds are then analyzed by MS(3) experiments to obtain detailed structural information while simultaneously performing high-resolution measurements to determine their elemental compositions in the two parts of a commercial linear quadrupole ion trap/Fourier-transform ion cyclotron resonance mass spectrometer. A lignin degradation product mixture was analyzed using this method, and molecular structures were proposed for some components. This methodology significantly improves the ability to analyze complex product mixtures that result from degraded lignin.

The behavior of compression and degradation for municipal solid waste and combined settlement calculation method.

PubMed

Shi, Jianyong; Qian, Xuede; Liu, Xiaodong; Sun, Long; Liao, Zhiqiang

2016-09-01

The total compression of municipal solid waste (MSW) consists of primary, secondary, and decomposition compressions. It is usually difficult to distinguish between the three parts of compressions. In this study, the odeometer test was used to distinguish between the primary and secondary compressions to determine the primary and secondary compression coefficient. In addition, the ending time of the primary compressions were proposed based on municipal solid waste compression tests in a degradation-inhibited condition by adding vinegar. The amount of the secondary compression occurring in the primary compression stage has a relatively high percentage to either the total compression or the total secondary compression. The relationship between the degradation ratio and time was obtained from the tests independently. Furthermore, a combined compression calculation method of municipal solid waste for all three parts of compressions including considering organics degradation is proposed based on a one-dimensional compression method. The relationship between the methane generation potential L0 of LandGEM model and degradation compression index was also discussed in the paper. A special column compression apparatus system, which can be used to simulate the whole compression process of municipal solid waste in China, was designed. According to the results obtained from 197-day column compression test, the new combined calculation method for municipal solid waste compression was analyzed. The degradation compression is the main part of the compression of MSW in the medium test period. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Sensor-Based Method for Diagnostics of Machine Tool Linear Axes.

PubMed

Vogl, Gregory W; Weiss, Brian A; Donmez, M Alkan

2015-01-01

A linear axis is a vital subsystem of machine tools, which are vital systems within many manufacturing operations. When installed and operating within a manufacturing facility, a machine tool needs to stay in good condition for parts production. All machine tools degrade during operations, yet knowledge of that degradation is illusive; specifically, accurately detecting degradation of linear axes is a manual and time-consuming process. Thus, manufacturers need automated and efficient methods to diagnose the condition of their machine tool linear axes without disruptions to production. The Prognostics and Health Management for Smart Manufacturing Systems (PHM4SMS) project at the National Institute of Standards and Technology (NIST) developed a sensor-based method to quickly estimate the performance degradation of linear axes. The multi-sensor-based method uses data collected from a 'sensor box' to identify changes in linear and angular errors due to axis degradation; the sensor box contains inclinometers, accelerometers, and rate gyroscopes to capture this data. The sensors are expected to be cost effective with respect to savings in production losses and scrapped parts for a machine tool. Numerical simulations, based on sensor bandwidth and noise specifications, show that changes in straightness and angular errors could be known with acceptable test uncertainty ratios. If a sensor box resides on a machine tool and data is collected periodically, then the degradation of the linear axes can be determined and used for diagnostics and prognostics to help optimize maintenance, production schedules, and ultimately part quality.

A Sensor-Based Method for Diagnostics of Machine Tool Linear Axes

PubMed Central

Vogl, Gregory W.; Weiss, Brian A.; Donmez, M. Alkan

2017-01-01

A linear axis is a vital subsystem of machine tools, which are vital systems within many manufacturing operations. When installed and operating within a manufacturing facility, a machine tool needs to stay in good condition for parts production. All machine tools degrade during operations, yet knowledge of that degradation is illusive; specifically, accurately detecting degradation of linear axes is a manual and time-consuming process. Thus, manufacturers need automated and efficient methods to diagnose the condition of their machine tool linear axes without disruptions to production. The Prognostics and Health Management for Smart Manufacturing Systems (PHM4SMS) project at the National Institute of Standards and Technology (NIST) developed a sensor-based method to quickly estimate the performance degradation of linear axes. The multi-sensor-based method uses data collected from a ‘sensor box’ to identify changes in linear and angular errors due to axis degradation; the sensor box contains inclinometers, accelerometers, and rate gyroscopes to capture this data. The sensors are expected to be cost effective with respect to savings in production losses and scrapped parts for a machine tool. Numerical simulations, based on sensor bandwidth and noise specifications, show that changes in straightness and angular errors could be known with acceptable test uncertainty ratios. If a sensor box resides on a machine tool and data is collected periodically, then the degradation of the linear axes can be determined and used for diagnostics and prognostics to help optimize maintenance, production schedules, and ultimately part quality. PMID:28691039

Stable-isotope-based labeling of styrene-degrading microorganisms in biofilters.

PubMed

Alexandrino, M; Knief, C; Lipski, A

2001-10-01

Deuterated styrene ([(2)H(8)]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [(2)H(8)]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [(2)H(8)]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.

Co-addition of manure increases the dissipation rates of tylosin A and the numbers of resistance genes in laboratory incubation experiments.

PubMed

Li, Qian; Wang, Yan; Zou, Yong-De; Liao, Xin-Di; Liang, Juan-Boo; Xin, Wen; Wu, Yin-Bao

2015-09-15

The behavior of veterinary antibiotics in the soil is commonly studied using the following methods to add antibiotics to the soil: (A) adding manure collected from animals fed a diet that includes antibiotics; (B) adding antibiotic-free animal manure spiked with antibiotics; and (C) the direct addition of antibiotics. However, most studies have only used methods (B) and (C) in their research, and few studies have simultaneously compared the different antibiotic addition methods. This study used tylosin A (TYLA) as a model antibiotic to compare the effects of these three commonly used antibiotic addition methods on the dissipation rates of TYLA and the numbers of resistance genes in laboratory incubation experiments. The results showed that the three treatment methods produced similar TYLA degradation trends; however, there were significant differences (P<0.05) in the TYLA degradation half-life (t1/2) among the three methods. The half-life of TYLA degradation in treatments A, B and C was 2.44 ± 0.04, 1.21 ± 0.03 and 5.13 ± 0.11 days, respectively. The presence of manure resulted in a higher electrical conductivity (EC), higher relative abundance of Citrobacter amalonaticus, higher macrolide resistant gene (ermB, ermF and ermT) count and lower ecological toxicity in the soil, which could partially explain the higher TYLA degradation rate in the treatments containing manure. The higher degradation rate of TYLA in treatment B when compared to treatment A could be due to the lower concentrations of tylosin B (TYLB) and tylosin D (TYLD). The main route for veterinary antibiotics to enter the soil is via the manure of animals that have been administered antibiotics. Therefore, the more appropriate method to study the degradation and ecotoxicity of antibiotic residues in the soil is by using manure from animals fed/administered the particular antibiotic rather than by adding the antibiotic directly to the soil. Copyright © 2015. Published by Elsevier B.V.

Preparation of magnetic imprinted graphene oxide composite for catalytic degradation of Congo red under dark ambient conditions.

PubMed

Yang, Xiaochao; You, Xiaoxiao; Zhang, Bin; Guo, Chuigen; Yu, Chaosheng

2017-10-01

Magnetic imprinted N-doped P25/Fe 3 O 4 -graphene oxide (MIGNT) was prepared with methyl orange as the dummy template and pyrrole as functional monomer for catalytic degradation of Congo red (CR). Hummers method and the hydrothermal method were used to synthesize Fe 3 O 4 -GO and N-doped P25, respectively. The results of adsorption and degradation experiments showed that the adsorption capacity and catalytic degradation ability of the imprinted composite for CR were obviously higher than those of a non-imprinted one. Moreover, the effect factors on degradation efficiency of CR, such as the initial concentration of CR, catalysis time, pH of the solution and temperature, were investigated. The MIGNT was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, a physical property measurement system and a thermal gravimetric analyzer. The degradation products of CR were detected with high performance liquid chromatography and a mass spectrometer. The MIGNT was a brand-new imprinted composite and had high degradation efficiency for CR under dark ambient conditions. The MIGNT could be recycled conveniently, due to its magnetic property, and could be used as an effective, environmentally friendly and low-cost catalytic degradation material for the treatment of water contaminated by CR.

Disruption of cell walls for enhanced lipid recovery

DOEpatents

Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

2015-03-24

Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

Comparative efficacy of multimodal digital methods in assessing trail/resource degradation

Treesearch

Logan O. Park

2014-01-01

Outdoor recreation can cause both positive and negative impacts on associated forest ecosystems. Forest recreation trails localize negative impacts to a controlled spatial extent while providing recreation access beyond developed areas and transportation networks. Current methods for assessing extent and severity of trail and proximal resource degradation require...

An improved method for determination of fumigant degradation half-life in soil

USDA-ARS?s Scientific Manuscript database

Using the current approach, measurement of fumigant degradation half-lives under realistic soil conditions is problematic due to the large headspace that is necessary above the soil during incubation. This results in a poor degree of contact between the fumigant and the soil’s degrading surfaces; di...

Marine Oil-Degrading Microorganisms and Biodegradation Process of Petroleum Hydrocarbon in Marine Environments: A Review.

PubMed

Xue, Jianliang; Yu, Yang; Bai, Yu; Wang, Liping; Wu, Yanan

2015-08-01

Due to the toxicity of petroleum compounds, the increasing accidents of marine oil spills/leakages have had a significant impact on our environment. Recently, different remedial techniques for the treatment of marine petroleum pollution have been proposed, such as bioremediation, controlled burning, skimming, and solidifying. (Hedlund and Staley in Int J Syst Evol Microbiol 51:61-66, 2001). This review introduces an important remedial method for marine oil pollution treatment-bioremediation technique-which is considered as a reliable, efficient, cost-effective, and eco-friendly method. First, the necessity of bioremediation for marine oil pollution was discussed. Second, this paper discussed the species of oil-degrading microorganisms, degradation pathways and mechanisms, the degradation rate and reaction model, and the factors affecting the degradation. Last, several suggestions for the further research in the field of marine oil spill bioremediation were proposed.

Classifying Degraded Modern Polymeric Museum Artefacts by Their Smell.

PubMed

Curran, Katherine; Underhill, Mark; Grau-Bové, Josep; Fearn, Tom; Gibson, Lorraine T; Strlič, Matija

2018-02-05

The use of VOC analysis to diagnose degradation in modern polymeric museum artefacts is reported. Volatile organic compound (VOC) analysis is a successful method for diagnosing medical conditions but to date has found little application in museums. Modern polymers are increasingly found in museum collections but pose serious conservation difficulties owing to unstable and widely varying formulations. Solid-phase microextraction gas chromatography/mass spectrometry and linear discriminant analysis were used to classify samples according to the length of time they had been artificially degraded. Accuracies in classification of 50-83 % were obtained after validation with separate test sets. The method was applied to three artefacts from collections at Tate to detect evidence of degradation. This approach could be used for any material in heritage collections and more widely in the field of polymer degradation. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Existence of the sugar-bisulfite adducts and its inhibiting effect on degradation of monosaccharide in acid system.

PubMed

Shi, Yan

2014-02-01

Degradation of fermentable monosaccharides is one of the primary concerns for acid prehydrolysis of lignocellulosic biomass. Recently, in our research on degradation of pure monosaccharides in aqueous SO₂ solution by gas chromatography (GC) analysis, we found that detected yield was not actual yield of each monosaccharide due to the existence of sugar-bisulfite adducts, and a new method was developed by ourselves which led to accurate detection of recovery yield of each monosaccharide in aqueous SO₂ solution by GC analysis. By the use of this method, degradation of each monosaccharide in aqueous SO₂ was investigated and results showed that sugar-bisulfite adducts have different inhibiting effect on degradation of each monosaccharide in aqueous SO₂ because of their different stability. In addition, NMR testing also demonstrated possible existence of reaction between conjugated based HSO₃(-) and aldehyde group of sugars in acid system.

Bacterial degradation of chlorophenols and their derivatives

PubMed Central

2014-01-01

Chlorophenols (CPs) and their derivatives are persistent environmental pollutants which are used in the manufacture of dyes, drugs, pesticides and other industrial products. CPs, which include monochlorophenols, polychlorophenols, chloronitrophenols, chloroaminophenols and chloromethylphenols, are highly toxic to living beings due to their carcinogenic, mutagenic and cytotoxic properties. Several physico-chemical and biological methods have been used for removal of CPs from the environment. Bacterial degradation has been considered a cost-effective and eco-friendly method of removing CPs from the environment. Several bacteria that use CPs as their sole carbon and energy sources have been isolated and characterized. Additionally, the metabolic pathways for degradation of CPs have been studied in bacteria and the genes and enzymes involved in the degradation of various CPs have been identified and characterized. This review describes the biochemical and genetic basis of the degradation of CPs and their derivatives. PMID:24589366

Identification of land degradation evidences in an organic farm using probability maps (Croatia)

NASA Astrophysics Data System (ADS)

Pereira, Paulo; Bogunovic, Igor; Estebaranz, Ferran

2017-04-01

Land degradation is a biophysical process with important impacts on society, economy and policy. Areas affected by land degradation do not provide services in quality and with capacity to full-field the communities that depends on them (Amaya-Romero et al., 2015; Beyene, 2015; Lanckriet et al., 2015). Agricultural activities are one of the main causes of land degradation (Kraaijvanger and Veldkamp, 2015), especially when they decrease soil organic matter (SOM), a crucial element for soil fertility. In temperate areas, the critical level of SOM concentration in agricultural soils is 3.4%. Below this level there is a potential decrease of soil quality (Loveland and Weeb, 2003). However, no previous work was carried out in other environments, such as the Mediterranean. The spatial distribution of potential degraded land is important to be identified and mapped, in order to identify the areas that need restoration (Brevik et al., 2016; Pereira et al., 2017). The aim of this work is to assess the spatial distribution of areas with evidences of land degradation (SOM bellow 3.4%) using probability maps in an organic farm located in Croatia. In order to find the best method, we compared several probability methods, such as Ordinary Kriging (OK), Simple Kriging (SK), Universal Kriging (UK), Indicator Kriging (IK), Probability Kriging (PK) and Disjunctive Kriging (DK). The study area is located on the Istria peninsula (45°3' N; 14°2' E), with a total area of 182 ha. One hundred eighty-two soil samples (0-30 cm) were collected during July of 2015 and SOM was assessed using wet combustion procedure. The assessment of the best probability method was carried out using leave one out cross validation method. The probability method with the lowest Root Mean Squared Error (RMSE) was the most accurate. The results showed that the best method to predict the probability of potential land degradation was SK with an RMSE of 0.635, followed by DK (RMSE=0.636), UK (RMSE=0.660), OK (RMSE=0.660), IK (RMSE=0.722) and PK (RMSE=1.661). According to the most accurate method, it is observed that the majority of the area studied has a high probability to be degraded. Measures are needed to restore this area. References Amaya-Romero, M., Abd-Elmabod, S., Munoz-Rojas, M., Castellano, G., Ceacero, C., Alvarez, S., Mendez, M., De la Rosa, D. (2015) Evaluating soil threats under climate change scenarios in the Andalusia region, Southern Spain. Land Degradation and Development, 26, 441-449. Beyene, F. (2015) Incentives and challenges in community based rangeland management: Evidence from Eastern Ethiopia. Land Degradation and Development, 26, 502-509. Brevik, E., Calzolari, C., Miller, B., Pereira, P., Kabala, C., Baumgarten, A., Jordán, A. (2016) Historical perspectives and future needs in soil mapping, classification and pedological modelling, Geoderma, 264, Part B, 256-274. Kraaijvanger, R., Veldkamp, T. (2015) Grain productivity, fertilizer response and nutrient balance of farming systems in Tigray, Ethiopia: A Multiprespective view in relation do soil fertility degradation. Land Degradation and Development, 26, 701-710. Lanckriet, S., Derudder, B., Naudts, J., Bauer, H., Deckers, J., Haile, M., Nyssen, J. (2015) A political ecology perspective of land degradation in the North Ethiopian Highlands. Land Degradation and Development, 26, 521-530. Loveland, P., Weeb, J. (2003) Is there a critical level of organic matter in the agricultural soils of temperate regions: a review. Soil & Tillage Research, 70, 1-18. Pereira, P., Brevik, E., Munoz-Rojas, M., Miller, B., Smetanova, A., Depellegrin, D., Misiune, I., Novara, A., Cerda, A. Soil mapping and process modelling for sustainable land management. In: Pereira, P., Brevik, E., Munoz-Rojas, M., Miller, B. (Eds.) Soil mapping and process modelling for sustainable land use management (Elsevier Publishing House) ISBN: 9780128052006

Degradation data analysis based on a generalized Wiener process subject to measurement error

NASA Astrophysics Data System (ADS)

Li, Junxing; Wang, Zhihua; Zhang, Yongbo; Fu, Huimin; Liu, Chengrui; Krishnaswamy, Sridhar

2017-09-01

Wiener processes have received considerable attention in degradation modeling over the last two decades. In this paper, we propose a generalized Wiener process degradation model that takes unit-to-unit variation, time-correlated structure and measurement error into considerations simultaneously. The constructed methodology subsumes a series of models studied in the literature as limiting cases. A simple method is given to determine the transformed time scale forms of the Wiener process degradation model. Then model parameters can be estimated based on a maximum likelihood estimation (MLE) method. The cumulative distribution function (CDF) and the probability distribution function (PDF) of the Wiener process with measurement errors are given based on the concept of the first hitting time (FHT). The percentiles of performance degradation (PD) and failure time distribution (FTD) are also obtained. Finally, a comprehensive simulation study is accomplished to demonstrate the necessity of incorporating measurement errors in the degradation model and the efficiency of the proposed model. Two illustrative real applications involving the degradation of carbon-film resistors and the wear of sliding metal are given. The comparative results show that the constructed approach can derive a reasonable result and an enhanced inference precision.

Application of excitation and emission matrix fluorescence (EEM) and UV-vis absorption to monitor the characteristics of Alizarin Red S (ARS) during electro-Fenton degradation process.

PubMed

Lai, Bo; Zhou, Yuexi; Wang, Juling; Yang, Zhishan; Chen, Zhiqiang

2013-11-01

Oxidative degradation of Alizarin Red S (ARS) in aqueous solutions by using electro-Fenton was studied. At first, effect of operating parameters such as current density, aeration rate and initial pH on the degradation of ARS were studied by using UV-vis spectrum, respectively. Then, under the optimal operating conditions (current density: 10.0mAcm(-2), aeration rate: 1000mLmin(-1), initial pH: 2.8), the identification of degradation products of ARS was carried out by using GC-MS and HPLC, meanwhile its degradation pathway was proposed according to the intermediates. Considering the location, intensity and intensity ratio of fluorescence center peak of the ARS in aqueous solution, a convenient and quick monitoring method by using excitation-emission matrix fluorescence spectrum technology was developed to monitor the degradation degree of ARS through electro-Fenton process. Furthermore, it is suggested that the developed method would be promising for the quick analysis and evaluation of the degradation degree of the pollutants with π-conjugated system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of a validated liquid chromatographic method for quantification of sorafenib tosylate in the presence of stress-induced degradation products and in biological matrix employing analytical quality by design approach.

PubMed

Sharma, Teenu; Khurana, Rajneet Kaur; Jain, Atul; Katare, O P; Singh, Bhupinder

2018-05-01

The current research work envisages an analytical quality by design-enabled development of a simple, rapid, sensitive, specific, robust and cost-effective stability-indicating reversed-phase high-performance liquid chromatographic method for determining stress-induced forced-degradation products of sorafenib tosylate (SFN). An Ishikawa fishbone diagram was constructed to embark upon analytical target profile and critical analytical attributes, i.e. peak area, theoretical plates, retention time and peak tailing. Factor screening using Taguchi orthogonal arrays and quality risk assessment studies carried out using failure mode effect analysis aided the selection of critical method parameters, i.e. mobile phase ratio and flow rate potentially affecting the chosen critical analytical attributes. Systematic optimization using response surface methodology of the chosen critical method parameters was carried out employing a two-factor-three-level-13-run, face-centered cubic design. A method operable design region was earmarked providing optimum method performance using numerical and graphical optimization. The optimum method employed a mobile phase composition consisting of acetonitrile and water (containing orthophosphoric acid, pH 4.1) at 65:35 v/v at a flow rate of 0.8 mL/min with UV detection at 265 nm using a C 18 column. Response surface methodology validation studies confirmed good efficiency and sensitivity of the developed method for analysis of SFN in mobile phase as well as in human plasma matrix. The forced degradation studies were conducted under different recommended stress conditions as per ICH Q1A (R2). Mass spectroscopy studies showed that SFN degrades in strongly acidic, alkaline and oxidative hydrolytic conditions at elevated temperature, while the drug was per se found to be photostable. Oxidative hydrolysis using 30% H 2 O 2 showed maximum degradation with products at retention times of 3.35, 3.65, 4.20 and 5.67 min. The absence of any significant change in the retention time of SFN and degradation products, formed under different stress conditions, ratified selectivity and specificity of the systematically developed method. Copyright © 2017 John Wiley & Sons, Ltd.

Development and application of a validated stability-indicating HPLC method for simultaneous determination of granisetron hydrochloride, benzyl alcohol and their main degradation products in parenteral dosage forms.

PubMed

Hewala, Ismail; El-Fatatre, Hamed; Emam, Ehab; Mubrouk, Mokhtar

2010-06-30

A simple, rapid and sensitive reversed phase high performance liquid chromatographic method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, benzyl alcohol, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron) and benzaldehyde (the main degradation product of benzyl alcohol) in granisetron injections. The separation was achieved on Hypersil BDS C8 (250 mm x 4.6 mm i.d., 5 microm particle diameter) column using a mobile phase consisted of acetonitrile:0.05 M KH(2)PO(4):triethylamine (22:100:0.15) adjusted to pH 4.8. The column was maintained at 25 degrees C and 20 microL of solutions was injected. Photodiode array detector was used to test the peak purity and the chromatograms were extracted at 210 nm. Naphazoline hydrochloride was used as internal standard. The method was validated with respect to specificity, linearity, accuracy, precision, limit of quantitation and limit of detection. The validation acceptance criteria were met in all cases. Identification of the pure peaks was carried out using library match programmer and wavelengths of derivative optima of the spectrograms of the peaks. The method was successfully applied to the determination of the investigated drugs and their degradation products in different batches of granisetron injections. The method was proved to be sensitive for the determination down to 0.03 and 0.01% of granisetron degradation product and benzaldehyde, respectively, which are far below the compendia limits for testing these degradation products in their corresponding intact drugs. Copyright 2010 Elsevier B.V. All rights reserved.

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

NASA Astrophysics Data System (ADS)

Petre, Brînduşa-Alina; Ulrich, Martina; Stumbaum, Mihaela; Bernevic, Bogdan; Moise, Adrian; Döring, Gerd; Przybylski, Michael

2012-11-01

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts.

PubMed

Taveira, Gabriel B; Mathias, Luciana S; da Motta, Olney V; Machado, Olga L T; Rodrigues, Rosana; Carvalho, André O; Teixeira-Ferreira, André; Perales, Jonas; Vasconcelos, Ilka M; Gomes, Valdirene M

2014-01-01

Plants defend themselves against pathogens with production of antimicrobial peptides (AMPs). Herein we describe the discovery of a new antifungal and antibacterial peptide from fruits of Capsicum annuum that showed similarity to an already well characterized family of plant AMPs, thionins. Other fraction composed of two peptides, in which the major peptide also showed similarity to thionins. Among the obtained fractions, fraction 1, which is composed of a single peptide of 7 kDa, was sequenced by Edman method and its comparative sequence analysis in database (nr) showed similarity to thionin-like peptides. Tests against microorganisms, fraction 1 presented inhibitory activity to the cells of yeast Saccharomyces cerevisiae, Candida albicans, and Candida tropicalis and caused growth reduction to the bacteria species Escherichia coli and Pseudomonas aeruginosa. Fraction 3 caused inhibitory activity only for C. albicans and C. tropicalis. This fraction was composed of two peptides of ∼7 and 10 kDa, and the main protein band correspondent to the 7 kDa peptide, also showed similarity to thionins. This plasma membrane permeabilization assay demonstrates that the peptides present in the fractions 1 and 3 induced changes in the membranes of all yeast strains, leading to their permeabilization. Fraction 1 was capable of inhibiting acidification of the medium of glucose-induced S. cerevisiae cells 78% after an incubation time of 30 min, and opposite result was obtained for C. albicans. Experiments demonstrate that the fraction 1 and 3 were toxic and induced changes in the membranes of all yeast strains, leading to their permeabilization. Copyright © 2013 Wiley Periodicals, Inc.

Identification and immunologic characterization of an allergen, alliin lyase, from garlic (Allium sativum).

PubMed

Kao, Shao-Hsuan; Hsu, Ching-Hsian; Su, Song-Nan; Hor, Wei-Ting; Chang T, Wen-Hong; Chow, Lu-Ping

2004-01-01

Garlic (Allium sativum) is one of the most common relishes used in cooking worldwide. Very few garlic allergens have been reported, and garlic allergy has been rarely studied. The aim of the study was to identify allergenic proteins in garlic and to investigate their importance in allergies to other Allium species (leek, shallot, and onion). A crude extract of garlic proteins was separated by SDS-PAGE and 2-dimensional electrophoresis; immunoblotting was then performed with the use of individual and pooled sera from patients with garlic allergy, and the major IgE-binding proteins were analyzed by amino acid sequencing and mass spectrometry. The putative allergens were further purified by chromatography; the antigenicity, allergenicity, and IgE-binding cross-reactivity of the purified protein were then studied by immunoblotting, periodate oxidation, skin tests, and IgE-binding inhibition assays. A major allergen, alliin lyase, was identified by mass spectrometry and Edman sequencing and purified to homogeneity through the use of a simple 2-step chromatographic method. Skin tests showed that the purified protein elicited IgE-mediated hypersensitive responses in patients with garlic allergy. Periodate oxidation showed that carbohydrate groups were involved in the antigenicity, allergenicity, and cross-reactivity. Garlic alliin lyase showed strong cross-reactivity with alliin lyases from other Allium species, namely leek, shallot, and onion. Alliin lyase was found to be a major garlic allergen in a garlic-allergic group of patients in Taiwan. The wide distribution of alliin lyase in Allium suggests it may be a new cross-reactive allergen.

Modeling of ultrasonic degradation of non-volatile organic compounds by Langmuir-type kinetics.

PubMed

Chiha, Mahdi; Merouani, Slimane; Hamdaoui, Oualid; Baup, Stéphane; Gondrexon, Nicolas; Pétrier, Christian

2010-06-01

Sonochemical degradation of phenol (Ph), 4-isopropylphenol (4-IPP) and Rhodamine B (RhB) in aqueous solutions was investigated for a large range of initial concentrations in order to analyze the reaction kinetics. The initial rates of substrate degradation and H(2)O(2) formation as a function of initial concentrations were determined. The obtained results show that the degradation rate increases with increasing initial substrate concentration up to a plateau and that the sonolytic destruction occurs mainly through reactions with hydroxyl radicals in the interfacial region of cavitation bubbles. The rate of H(2)O(2) formation decreases with increasing substrate concentration and reaches a minimum, followed by almost constant production rate for higher substrate concentrations. Sonolytic degradation data were analyzed by the models of Okitsu et al. [K. Okitsu, K. Iwasaki, Y. Yobiko, H. Bandow, R. Nishimura, Y. Maeda, Sonochemical degradation of azo dyes in aqueous solution: a new heterogeneous kinetics model taking into account the local concentration OH radicals and azo dyes, Ultrason. Sonochem. 12 (2005) 255-262.] and Seprone et al. [N. Serpone, R. Terzian, H. Hidaka, E. Pelizzetti, Ultrasonic induced dehalogenation and oxidation of 2-, 3-, and 4-chlorophenol in air-equilibrated aqueous media. Similarities with irradiated semiconductor particulates, J. Phys. Chem. 98 (1994) 2634-2640.] developed on the basis of a Langmuir-type mechanism. The five linearized forms of the Okitsu et al.'s equation as well as the non-linear curve fitting analysis method were discussed. Results show that it is not appropriate to use the coefficient of determination of the linear regression method for comparing the best-fitting. Among the five linear expressions of the Okitsu et al.'s kinetic model, form-2 expression very well represent the degradation data for Ph and 4-IPP. Non-linear curve fitting analysis method was found to be the more appropriate method to determine the model parameters. An excellent representation of the experimental results of sonolytic destruction of RhB was obtained using the Serpone et al.'s model. The Serpone et al.'s model gives a worse fit for the sonolytic degradation data of Ph and 4-IPP. These results indicate that Ph and 4-IPP undergo degradation predominantly at the bubble/solution interface, whereas RhB undergoes degradation at both bubble/solution interface and in the bulk solution. (c) 2010 Elsevier B.V. All rights reserved.

Determination of degradation products and process related impurities of asenapine maleate in asenapine sublingual tablets by UPLC

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Sangeetha, D.; Kalyanraman, L.

2017-11-01

For determination of process related impurities and degradation products of asenapine maleate in asenapine sublingual Tablets, a reversed phase, stability indicating UPLC method was developed. Acetonitrile, methanol and potassium dihydrogen phosphate buffer with tetra-n- butyl ammonium hydrogen sulphate as ion pair (pH 2.2; 0.01 M) at flow rate of 0.2 ml/min were used in gradient elution mode. Separation was achieved by using acquity BEH Shield RP18 column (1.7 μm, 2.1 mm×100 mm) at 35 ºC. UV detection was performed at 228 nm. Subsequently the liquid chromatography method was validated as per ICH. The drug product was exposed to the stress conditions of acid hydrolysis, base hydrolysis, water hydrolysis, oxidative, thermal, and photolytic. In oxidative stress and thermal stress significant degradation was observed. All the degradation products were well separated from analyte peak and its impurities. Stability indicating nature of the method was proved by demonstrating the peak purity of Asenapine peak in all the stressed samples. The mass balance was found >95% for all the stress conditions. Based on method validation, the method was found specific, linear, accurate, precise, rugged and robust.

Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product

PubMed Central

Hassan, Mostafa A.; Zaghary, Wafaa A.

2018-01-01

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm–260.4 nm, amplitudes at 260.4 nm–274.0 nm, and mean-centered values at 287.6 nm–257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5–50 μg/mL and 2.5–25 μg/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD. PMID:29629213

Different Spectrophotometric Methods for Simultaneous Determination of Trelagliptin and Its Acid Degradation Product.

PubMed

Mowaka, Shereen; Ayoub, Bassam M; Hassan, Mostafa A; Zaghary, Wafaa A

2018-01-01

New spectrophotometric and chemometric methods were carried out for the simultaneous assay of trelagliptin (TRG) and its acid degradation product (TAD) and applied successfully as a stability indicating assay to recently approved Zafatek® tablets. TAD was monitored using TLC to ensure complete degradation. Furthermore, HPLC was used to confirm dealing with one major acid degradation product. The proposed methods were developed by manipulating zero-order, first-derivative, and ratio spectra of TRG and TAD using simultaneous equation, first-derivative, and mean-centering methods, respectively. Using Spectra Manager II and Minitab v.14 software, the absorbance at 274 nm-260.4 nm, amplitudes at 260.4 nm-274.0 nm, and mean-centered values at 287.6 nm-257.2 nm were measured against methanol as a blank for TRG and TAD, respectively. Linearity and the other validation parameters were acceptable at concentration ranges of 5-50  μ g/mL and 2.5-25  μ g/mL for TRG and TAD, respectively. Using one-way analysis of variance (ANOVA), the optimized methods were compared and proved to be accurate for the simultaneous assay of TRG and TAD.

Methods and reagents. Degraded DNA and gel tornados.

PubMed

Hengen, P N

1997-04-01

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a case of inexplicable DNA degradation and tornados seen in agarose gels. For details on how to partake in the newsgroup, see the accompanying box.

Aircraft Flight Envelope Determination using Upset Detection and Physical Modeling Methods

NASA Technical Reports Server (NTRS)

Keller, Jeffrey D.; McKillip, Robert M. Jr.; Kim, Singwan

2009-01-01

The development of flight control systems to enhance aircraft safety during periods of vehicle impairment or degraded operations has been the focus of extensive work in recent years. Conditions adversely affecting aircraft flight operations and safety may result from a number of causes, including environmental disturbances, degraded flight operations, and aerodynamic upsets. To enhance the effectiveness of adaptive and envelope limiting controls systems, it is desirable to examine methods for identifying the occurrence of anomalous conditions and for assessing the impact of these conditions on the aircraft operational limits. This paper describes initial work performed toward this end, examining the use of fault detection methods applied to the aircraft for aerodynamic performance degradation identification and model-based methods for envelope prediction. Results are presented in which a model-based fault detection filter is applied to the identification of aircraft control surface and stall departure failures/upsets. This application is supported by a distributed loading aerodynamics formulation for the flight dynamics system reference model. Extensions for estimating the flight envelope due to generalized aerodynamic performance degradation are also described.

Stability, Antioxidant Capacity and Degradation Kinetics of Pelargonidin-3-glucoside Exposed to Ultrasound Power at Low Temperature.

PubMed

Sun, Jianxia; Mei, Zhouxiong; Tang, Yajuan; Ding, Lijun; Jiang, Guichuan; Zhang, Chi; Sun, Aidong; Bai, Weibin

2016-08-24

As an alternative preservation method to thermal treatment, ultrasound is a novel non-thermal processing technology that can significantly avoid undesirable nutritional changes. However, recently literature indicated that anthocyanin degradation occurred when high amplitude ultrasound was applied to juice. This work mainly studied the effect of ultrasound on the stability and antioxidant capacity of pelargonidin-3-glucoside (Pg-3-glu) and the correlation between anthocyanin degradation and •OH generation in a simulated system. Results indicated that the spectral intensities of Pg-3-glu decreased with increasing ultrasound power (200-500 W) and treatment time (0-60 min). The degradation trend was consistent with first-order reaction kinetics (R² > 0.9100). Further study showed that there was a good linear correlation between Pg-3-glu degradation and •OH production (R² = 0.8790), which indicated the important role of •OH in the degradation of anthocyanin during ultrasound exposure. Moreover, a decrease in the antioxidant activity of solution(s) containing Pg-3-glu as evaluated by the DPPH and FRAP methods was observed after ultrasound treatment.

Selenylation Modification of Degraded Polysaccharide from Enteromorpha prolifera and Its Biological Activities

NASA Astrophysics Data System (ADS)

Lv, Haitao; Duan, Ke; Shan, Hu

2018-04-01

Polysaccharide extracted from Enteromorpha prolifera possessed excellent biological activities, but its molecular weight was greatly high which influenced the activity. Organic Se had higher biological activities and was safer than inorganic Se species. In the present study, Enteromorpha polysaccharide was degraded to low molecular weight by free-radical degradation method of H2O2 and ascorbic acid. By single factor and orthogonal experiments, the optimal degradation conditions were reaction time of 2 h, reaction temperature of 50°C, H2O2/ascorbic acid (n/n=1:1) concentration of 15 mmol L-1, and solid-liquid ratio of 1:50 (g mL-1). Then, the degraded polysaccharide was chemically modified to obtain its selenide derivatives by nitric acid-sodium selenite method. The selenium content was 1137.29 μg g-1, while the content of sulfate radical had no change. IR spectra indicated that the selenite ester group was formed. Degraded polysaccharide selenide was characterized and evaluated for antioxidant, antifungal and antibacterial activities. The results showed that degraded polysaccharide selenide had strong capacity of scavenging DPPH and ·OH free radical. It had significant antibacterial properties for Escherichia coli, Bacillus subtilis and Salmonella spp., and it also had significant antifungal properties for Apple anthrax. The result ascertained degradation and selenylation modification did not change the main structure of polysaccharides. It was possible that free-radical degradation was an effective way for enhancing antioxidant activity to decrease molecular weight of polysaccharides.

25 years monitoring of PAHs and petroleum hydrocarbons biodegradation in soil.

PubMed

Harmsen, Joop; Rietra, René P J J

2018-05-10

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) and total petroleum hydrocarbons (TPH) in sediment and soil has been monitored on seven experimental fields during periods up to 25 years. With this unique dataset, we investigated long-term very slow biodegradation under field conditions. . The data show that three biodegradation rates can be distinguished for PAHs: 1) rapid degradation during the first year, 2) slow degradation during the following 6 years and 3), subject of this paper, a very slow degradation after 7 years until at least 25 years. Beside 2-, 3- and 4-ring PAHs, also 5- and 6-ring PAHs (aromatic rings) were degraded, all at the same rate during very slow degradation. In the period of very slow degradation, 6% yr -1 of the PAHs present were removed in five fields and 2% yr -1 in two other fields, while in the same period no very slow degradation of TPH could be observed. The remaining petroleum hydrocarbons were high boiling and non-toxic. Using the calculated degradation rates and the independently measured bioavailability of the PAHs (Tenax-method), the PAHs degradation curves of all seven monitored fields could be modelled. Applying the model and data obtained with the Tenax-method for fresh contaminated material, results of long-term biodegradation can be predicted, which can support the use of bioremediation in order to obtain a legally acceptable residual concentration. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Color transplant for reverse ageing of faded artworks

NASA Astrophysics Data System (ADS)

Del Mastio, A.; Piva, A.; Barni, M.; Cappellini, V.; Stefanini, L.

2008-02-01

Nowadays, photographs are one of the most used media for communication. Images are used for the representation of documents, Cultural goods, and so on: they are used to pass on a wedge of historical memory of the society. Since its origin, the photographic technique has got several improvements; nevertheless, photos are liable to several damages, both concerning the physical support and concerning the colors and figures which are depicted in it: for example, think about scratches or rips happened to a photo, or think about the fading or red (or yellow) toning concerning the colors of a photo. In this paper, we propose a novel method which is able to assess the original beauty of digital reproductions of aged photos, as well as digital reproductions of faded goods. The method is based on the comparison of the degraded image with a not-degraded one showing similar contents; thus, the colors of the not-degraded image can be transplanted in the degraded one. The key idea is a dualism between the analytical mechanics and the color theory: for each of the degraded and not-degraded images we compute first a scatter plot of the x and y normalized coordinates of their colors; these scatter diagrams can be regarded as a system of point masses, thus provided of inertia axes and an inertia ellipsoid. Moving the scatter diagram of the degraded image over the one belonging to the not-degraded image, the colors of the degraded image can be restored.

Identification and Structure Elucidation of Forced Degradation Products of the Novel Propionic acid Derivative Loxoprofen: Development of Stability-Indicating Chromatographic Methods Validated as per ICH Guidelines.

PubMed

Eissa, Maya S; Abd El-Sattar, Osama I

2017-04-01

Loxoprofen sodium (LOX) is a recently developed novel propionic acid derivative. Owing to its instability under both hydrolytic and oxidative conditions, the development of simple, rapid and sensitive methods for its determination in the presence of its possible forced degradation products becomes essential. Two simple chromatographic methods, high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC), were developed associated with ultraviolet (UV) detection. In HPTLC-densitometric method, the separation of LOX from its degradation products was achieved using silica gel F254 plates and toluene:acetone:acetic acid (1.8:1.0:0.1, v/v/v) as the developing system followed by densitometric scanning at 220 nm. In the HPLC-UV method, the separation was performed using isocratic elution system with acetonitrile: 0.15% triethylamine (pH 2.2) (50:50, v/v) on C18 analytical column. The flow rate was optimized at 1.0 mL·min-1 and UV detection was achieved at 220 nm. Validation was performed in accordance with the International Conference on Harmonization guidelines and the method was perfectly applied for determination of LOX in its pharmaceutical preparation. The results obtained were statistically compared to those obtained after application of the official HPLC method, where no significant difference was found incompliance with precision and accuracy. Identification and characterization of the possible hydrolytic degradation product under alkaline conditions and that produced during oxidative degradation using hydrogen peroxide were structurally elucidated using infrared and mass spectrometry analyses. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Research on degradation of omethoate with Y2O3:Er3+ and TiO2

NASA Astrophysics Data System (ADS)

Liu, Zhiping; Mai, Yanling; Yan, Aiguo; Fan, Hailu; Yuan, Taidou

2018-06-01

Application of visible light excited photocatalytic degradation reagent of pesticide residues is not only suitable for the farmers, can also be used for city residents for daily use. Up conversion material Y2O3:Er3+ was prepared by sol gel method, then mixed with anatase TiO2 sol solution, to carry out the research of omethoate degradation under visible light. In order to get the higher degradability, it's important to study the technological parameters. Among so many parameters, four parameters were selected. They were vegetable surface omethoate concentration, photocatalytic degradation reagent dosage, pH value and degradation time. Utilizing orthogonal experimental design program, all parameters were optimized. The results showed that: the degradation rate was the largest concerned with the vegetable surface omethoate concentration, and then the degradation time.

Degradation of atrazine by microwave-assisted electrodeless discharge mercury lamp in aqueous solution.

PubMed

Ta, Na; Hong, Jun; Liu, Tingfeng; Sun, Cheng

2006-11-02

The present study investigates the degradation of atrazine (2-chloro-4-(ethyl amino)-6-isopropyl amino-s-triazine) in aqueous solution by a developed new method, namely by means of a microwave-assisted electrodeless discharge mercury lamp (MW-EDML). An experimental design was conducted to assess the influence of various parameters: pH value, initial concentration, amount of EDML, initial volume and coexisted solvent. Atrazine was degraded completely by EDML in a relatively short time (i.e. t(1/2)=1.2 min for 10 mg/l). Additionally, the identification of main degradation products during atrazine degradation process was conducted by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). This study proposes the degradation mechanism including four possible pathways for atrazine degradation according to the degradation products.

[Effect of enzymolysis after acid and alkali pretreatment on extraction of tanshinones from Salviae Miltiorrhizae Radix et Rhizoma residues].

PubMed

Dai, Xin-Xin; Shen, Fei; Su, Shu-Lan; Zhang, Sen; Guo, Sheng; Jiang, Shu; Qian, Da-Wei; Duan, Jin-Ao

2016-09-01

Salviae Miltiorrhizae Radix et Rhizoma residues were pre-treated with acid and alkali, degraded by using cellulose, and the effects of different processing methods on the extraction rate of tanshinones were compared to provide scientific basis for development and utilization of tanshinones from Salviae Miltiorrhizae Radix et Rhizoma residues. The results showed that in the Salviae Miltiorrhizae Radix et Rhizoma residues without pre-treatment, enzymatic hydrolysis time of 4.5 d could make most of the cellulose degraded when the concentration of substrate enzyme concentration was 6 U•mL-1, and the highest glucose concentration was 59.74 mg•g⁻¹. It was found that the best effect was achieved after alkali pre-treatment-cellulose C degradation among the different pre-treatment methods, and the glucose content reached 119.50 mg•g⁻¹, followed by the same concentration of acid pre-treatment-cellulose C degradation. The extraction amount of tanshinone ⅡA was increased by 82.54% after enzyme degradation, with a mass fraction of 2.451 mg•g⁻¹; extraction amount of tanshinone I was increased by 81.82% after enzyme degradation, with a mass fraction of 2.373 mg•g⁻¹; extraction amount of cryptotanshinone was increased by 64.4% after enzyme degradation, with a mass fraction of 1.080 mg•g⁻¹; extraction amount of dihydrotanshinone I was increased by 61.3% after enzyme degradation, with a mass fraction of 0.601 2 mg•g⁻¹. Acid and alkali pre-treatment combined with cellulose degradation could effectively improve the extraction rate of tanshinones from Salviae Miltiorrhizae Radix et Rhizoma residues. This method is operable and practical, and it is beneficial for improving the utilization efficiency of tanshinones (resource based chemicals) from Salviae Miltiorrhizae Radix et Rhizoma residues. Copyright© by the Chinese Pharmaceutical Association.

Structure elucidation and degradation kinetic study of Ofloxacin using surface enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

El-Zahry, Marwa R.; Lendl, Bernhard

2018-03-01

A simple, fast and sensitive surface enhanced Raman spectroscopy (SERS) method for quantitative determination of fluoroquinolone antibiotic Ofloxacin (OFX) is presented. Also the stability behavior of OFX was investigated by monitoring the SERS spectra of OFX after various degradation processes. Acidic, basic and oxidative force degradation processes were applied at different time intervals. The forced degradation conditions were conducted and followed using SERS method utilizing silver nanoparticles (Ag NPs) as a SERS substrate. The Ag NPs colloids were prepared by reduction of silver nitrate using polyethyelene glycol (PEG) as a reducing and stabilizing agent. Validation tests were done in accordance with International Conference on Harmonization (ICH) guidelines. The calibration curve with a correlation coefficient (R = 0.9992) was constructed as a relationship between the concentration range of OFX (100-500 ng/ml) and SERS intensity at 1394 cm- 1 band. LOD and LOQ values were calculated and found to be 23.5 ng/ml and 72.6 ng/ml, respectively. The developed method was applied successfully for quantitation of OFX in different pharmaceutical dosage forms. Kinetic parameters were calculated including rate constant of the degradation of the studied antibiotic.

Degradation of bisphenol A in water by the heterogeneous photo-Fenton.

PubMed

Jiang, Chuanrui; Xu, Zhencheng; Guo, Qingwei; Zhuo, Qiongfang

2014-01-01

Bisphenol A (BPA) is a kind of a controversial endocrine disruptor, and is ubiquitous in environment. The degradation of BPA with the heterogeneous photo-Fenton system was demonstrated in this study. The Fe-Y molecular sieve catalyst was prepared with the ion exchange method, and it was characterized by X-ray radiation diffraction (XRD). The effects ofpH, initial concentration of H2O2, initial BPA concentration, and irradiation intensity on the degradation of BPA were investigated. The service life and iron solubility of catalyst were also tested. XRD test shows that the major phase of the Fe-Y catalyst was Fe2O3. The method of heterogeneous photo-Fenton with Fe-Y catalyst was superior to photolysis, photo-oxidation with only hydrogen, heterogeneous Fenton, and homogeneous photo-Fenton approaches. pH value had no obvious effects on BPA degradation over the range of 2.2-7.2. The initial concentration of H2O2 had an optimal value of 20 x 10(-4) mol/L. The decrease in initial concentration of BPA was favourable for degradation. The intensity of ultraviolet irradiation has no obvious effect on the BPA removal. The stability tests indicated that the Fe-Y catalyst can be reused and iron solubility concentration ranged from NA to 0.0062 mg/L. Based on the results, the heterogeneous photo-Fenton treatment is the available method for the degradation of BPA.

Modeling the degradation kinetics of ascorbic acid.

PubMed

Peleg, Micha; Normand, Mark D; Dixon, William R; Goulette, Timothy R

2018-06-13

Most published reports on ascorbic acid (AA) degradation during food storage and heat preservation suggest that it follows first-order kinetics. Deviations from this pattern include Weibullian decay, and exponential drop approaching finite nonzero retention. Almost invariably, the degradation rate constant's temperature-dependence followed the Arrhenius equation, and hence the simpler exponential model too. A formula and freely downloadable interactive Wolfram Demonstration to convert the Arrhenius model's energy of activation, E a , to the exponential model's c parameter, or vice versa, are provided. The AA's isothermal and non-isothermal degradation can be simulated with freely downloadable interactive Wolfram Demonstrations in which the model's parameters can be entered and modified by moving sliders on the screen. Where the degradation is known a priori to follow first or other fixed order kinetics, one can use the endpoints method, and in principle the successive points method too, to estimate the reaction's kinetic parameters from considerably fewer AA concentration determinations than in the traditional manner. Freeware to do the calculations by either method has been recently made available on the Internet. Once obtained in this way, the kinetic parameters can be used to reconstruct the entire degradation curves and predict those at different temperature profiles, isothermal or dynamic. Comparison of the predicted concentration ratios with experimental ones offers a way to validate or refute the kinetic model and the assumptions on which it is based.

Multi-omic Mitoprotease Profiling Defines a Role for Oct1p in Coenzyme Q Production.

PubMed

Veling, Mike T; Reidenbach, Andrew G; Freiberger, Elyse C; Kwiecien, Nicholas W; Hutchins, Paul D; Drahnak, Michael J; Jochem, Adam; Ulbrich, Arne; Rush, Matthew J P; Russell, Jason D; Coon, Joshua J; Pagliarini, David J

2017-12-07

Mitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis-a pathway essential for mitochondrial respiration. Through Edman sequencing and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Additionally, our data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations. Copyright © 2017 Elsevier Inc. All rights reserved.

Visual Reading Method for Detection of Bacterial Tannase

PubMed Central

Osawa, R.; Walsh, T. P.

1993-01-01

Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free gallic acid and (ii) the green to brown coloration of gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes. PMID:16348918

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

PubMed

Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

1992-02-01

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs. The presence in shark liver of an FABP which differs substantially in primary structure from mammalian liver FABP, while being closely related to the FABP expressed in mammalian heart muscle, peripheral nerve myelin and adipocytes, opens a further dimension regarding the question of the existence of structure-dependent and tissue-specific specialization of FABP function in lipid metabolism.

Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl(3H)adenosine at 65 degrees C

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

Bullough, D.A.; Yoshida, M.; Allison, W.S.

1986-02-01

Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 whichmore » was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with (3H)FSBA by about 90%, greater than 20 mol of (3H)SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM (3H)FSBA at 65 degrees C and pH 6.4, about 10 mol of (3H)SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985)), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.« less

Elucidating PID Degradation Mechanisms and In Situ Dark I–V Monitoring for Modeling Degradation Rate in CdTe Thin-Film Modules

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

Hacke, Peter; Spataru, Sergiu; Johnston, Steve

A progression of potential-induced degradation (PID) mechanisms are observed in CdTe modules, including shunting/junction degradation and two different manifestations of series resistance depending on the stress level and water ingress. The dark I-V method for in-situ characterization of Pmax based on superposition was adapted for the thin-film modules undergoing PID in view of the degradation mechanisms observed. An exponential model based on module temperature and relative humidity was fit to the PID rate for multiple stress levels in chamber tests and validated by predicting the observed degradation of the module type in the field.

Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate.

PubMed

Wallenius, Kaisa; Lappi, Kaisa; Mikkonen, Anu; Wickström, Annika; Vaalama, Anu; Lehtinen, Taru; Suominen, Leena

2012-02-01

We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.

Selective determination of ertapenem in the presence of its degradation product

NASA Astrophysics Data System (ADS)

Hassan, Nagiba Y.; Abdel-Moety, Ezzat M.; Elragehy, Nariman A.; Rezk, Mamdouh R.

2009-06-01

Stability-indicative determination of ertapenem (ERTM) in the presence of its β-lactam open-ring degradation product, which is also the metabolite, is investigated. The degradation product has been isolated, via acid-degradation, characterized and elucidated. Selective quantification of ERTM, singly in bulk form, pharmaceutical formulations and/or in the presence of its major degradant is demonstrated. The indication of stability has been undertaken under conditions likely to be expected at normal storage conditions. Among the spectrophotometric methods adopted for quantification are first derivative ( 1D), first derivative of ratio spectra ( 1DD) and bivariate analysis.

Microbial degradation of an organophosphate pesticide, malathion.

PubMed

Singh, Baljinder; Kaur, Jagdeep; Singh, Kashmir

2014-05-01

Organophosphorus pesticide, malathion, is used in public health, residential, and agricultural settings worldwide to control the pest population. It is proven that exposure to malathion produce toxic effects in humans and other mammals. Due to high toxicity, studies are going on to design effective methods for removal of malathion and its associated compounds from the environment. Among various techniques available, degradation of malathion by microbes proves to be an effective and environment friendly method. Recently, research activities in this area have shown that a diverse range of microorganisms are capable of degrading malathion. Therefore, we aimed at providing an overview of research accomplishments on this subject and discussed the toxicity of malathion and its metabolites, various microorganisms involved in its biodegradation and effect of various environmental parameters on its degradation.

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

Pezeshki, Alan M.; Sacci, Robert L.; Veith, Gabriel M.

Here, we demonstrate a novel method to accelerate electrode degradation in redox flow batteries and apply this method to the all-vanadium chemistry. Electrode performance degradation occurred seven times faster than in a typical cycling experiment, enabling rapid evaluation of materials. This method also enables the steady-state study of electrodes. In this manner, it is possible to delineate whether specific operating conditions induce performance degradation; we found that both aggressively charging and discharging result in performance loss. Post-mortem x-ray photoelectron spectroscopy of the degraded electrodes was used to resolve the effects of state of charge (SoC) and current on the electrodemore » surface chemistry. For the electrode material tested in this work, we found evidence that a loss of oxygen content on the negative electrode cannot explain decreased cell performance. Furthermore, the effects of decreased electrode and membrane performance on capacity fade in a typical cycling battery were decoupled from crossover; electrode and membrane performance decay were responsible for a 22% fade in capacity, while crossover caused a 12% fade.« less

Minimizing thermal degradation in gas chromatographic quantitation of pentaerythritol tetranitrate.

PubMed

Lubrano, Adam L; Field, Christopher R; Newsome, G Asher; Rogers, Duane A; Giordano, Braden C; Johnson, Kevin J

2015-05-15

An analytical method for establishing calibration curves for the quantitation of pentaerythriol tetranitrate (PETN) from sorbent-filled thermal desorption tubes by gas chromatography with electron capture detection (TDS-GC-ECD) was developed. As PETN has been demonstrated to thermally degrade under typical GC instrument conditions, peaks corresponding to both PETN degradants and molecular PETN are observed. The retention time corresponding to intact PETN was verified by high-resolution mass spectrometry with a flowing atmospheric pressure afterglow (FAPA) ionization source, which enabled soft ionization of intact PETN eluting the GC and subsequent accurate-mass identification. The GC separation parameters were transferred to a conventional GC-ECD instrument where analytical method-induced PETN degradation was further characterized and minimized. A method calibration curve was established by direct liquid deposition of PETN standard solutions onto the glass frit at the head of sorbent-filled thermal desorption tubes. Two local, linear relationships between detector response and PETN concentration were observed, with a total dynamic range of 0.25-25ng. Published by Elsevier B.V.

Methods for degrading lignocellulosic materials

DOEpatents

Vlasenko, Elena [Davis, CA; Cherry, Joel [Davis, CA; Xu, Feng [Davis, CA

2008-04-08

The present invention relates to methods for degrading a lignocellulosic material, comprising: treating the lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying a lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant; (b) fermenting the saccharified lignocellulosic material of step (a) with one or more fermentating microoganisms; and (c) recovering the organic substance from the fermentation.

Methods for degrading lignocellulosic materials

DOEpatents

Vlasenko, Elena [Davis, CA; Cherry, Joel [Davis, CA; Xu, Feng [Davis, CA

2011-05-17

The present invention relates to methods for degrading a lignocellulosic material, comprising: treating the lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying a lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant; (b) fermenting the saccharified lignocellulosic material of step (a) with one or more fermenting microorganisms; and (c) recovering the organic substance from the fermentation.

Evaluating paper degradation progress. Cross-linking between chromatographic, spectroscopic and chemical results

NASA Astrophysics Data System (ADS)

Łojewski, Tomasz; Zięba, Katarzyna; Knapik, Arkadiusz; Bagniuk, Jacek; Lubańska, Anna; Łojewska, Joanna

2010-09-01

The study presents an overview of the chromatographic (SEC), spectroscopic (FTIR, UV/VIS), viscometric (DP) and chemical methods (titration, pH) used for the evaluation of the degradation progress of various kinds of paper under various conditions. The methods were chosen to follow different routes of paper degradation. Model paper samples represented boundary paper types from pure cellulose cotton paper, through softwood to low quality acidic, sized groundwood paper The accelerated ageing conditions were adjusted to achieve maximum effect (climatic chamber RH 59%, 90oC) and also to mimic the environment inside books (closed vials). The results were settled on the literature data on the degradation mechanisms and compared in terms of the paper types and ageing conditions. The estimators of coupled de-polymerisation and oxidation have been proposed based on the correlation between SEC, UV/VIS and titrative coppper number determination. The overall oxidation index derived from FTIR results was shown to correlate with the summary -CHO and -COOH concentration determined by titrative methods.

Methods for degrading lignocellulosic materials

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

Vlasenko, Elena; Cherry, Joel; Xu, Feng

2008-04-08

The present invention relates to methods for degrading a lignocellulosic material, comprising: treating the lignocellulosic material with an effective amount of one or more cellulolytic enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying a lignocellulosic material with an effective amount of one or more cellulolyticmore » enzymes in the presence of at least one surfactant selected from the group consisting of a secondary alcohol ethoxylate, fatty alcohol ethoxylate, nonylphenol ethoxylate, tridecyl ethoxylate, and polyoxyethylene ether, wherein the presence of the surfactant increases the degradation of lignocellulosic material compared to the absence of the surfactant; (b) fermenting the saccharified lignocellulosic material of step (a) with one or more fermentating microoganisms; and (c) recovering the organic substance from the fermentation.« less

A validated stability-indicating LC method for the separation of enantiomer and potential impurities of Linezolid using polar organic mode.

PubMed

Satyanarayana Raju, T; Vishweshwari Kutty, O; Ganesh, V; Yadagiri Swamy, P

2012-08-01

Although a number of methods are available for evaluating Linezolid and its possible impurities, a common method for separation if its potential impurities, degradants and enantiomer in a single method with good efficiency remain unavailable. With the objective of developing an advanced method with shorter runtimes, a simple, precise, accurate stability-indicating LC method was developed for the determination of purity of Linezolid drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. This method is capable of separating all the related substances of Linezolid along with the chiral impurity. This method can also be used for the estimation of assay of Linezolid in drug substance as well as in drug product. The method was developed using Chiralpak IA (250 mm×4.6 mm, 5 μm) column. A mixture of acetonitrile, ethanol, n-butyl amine and trifluoro acetic acid in 96:4:0.10:0.16 (v/v/v/v) ratio was used as a mobile phase. The eluted compounds were monitored at 254 nm. Linezolid was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness and system suitability.

Measurement of Pyrethroids and Their Environmental Degradates in Fruits and Vegetables using a Modification of the Quick Easy Cheap Effective Rugged Safe (QuEChERS) Method

EPA Science Inventory

Pyrethroid insecticides are used extensively in agriculture and they, as well as their environmental degradates, may remain as residues on food products such as fruits and vegetables. Since pyrethroid degradates can be identical to the urinary markers used in human biomonitoring ...

Rumen-degradable protein in roll conditioned or macerated legume hays and silages estimated by in situ kinetics vs. alternative methods

USDA-ARS?s Scientific Manuscript database

Alternatives to the in situ method for estimating rumen-degradable protein (RDP) in diverse forage legumes should be validated. In this study, RDP in roll conditioned or macerated silages and hays of Medicago, Lotus, and Trifolium species with differing polyphenol compositions were estimated from in...

Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems.

PubMed

McDowell, Gary S; Philpott, Anna

2018-05-16

The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts. © 2019 Cold Spring Harbor Laboratory Press.

Identification and in vitro cytotoxicity of ochratoxin A degradation products formed during coffee roasting.

PubMed

Cramer, Benedikt; Königs, Maika; Humpf, Hans-Ulrich

2008-07-23

The mycotoxin ochratoxin A is degraded by up to 90% during coffee roasting. In order to investigate this degradation, model heating experiments with ochratoxin A were carried out, and the reaction products were analyzed by HPLC-DAD and HPLC-MS/MS. Two ochratoxin A degradation products were identified, and their structure and absolute configuration were determined. As degradation reactions, the isomerization to 14-(R)-ochratoxin A and the decarboxylation to 14-decarboxy-ochratoxin A were identified. Subsequently, an analytical method for the determination of these compounds in roasted coffee was developed. Quantification was carried out by HPLC-MS/MS and the use of stable isotope dilution analysis. By using this method for the analysis of 15 coffee samples from the German market, it could be shown that, during coffee roasting, the ochratoxin A diastereomer 14-(R)-ochratoxin A was formed in amounts of up to 25.6% relative to ochratoxin A. The decarboxylation product was formed only in traces. For toxicity evaluations, first preliminary cell culture assays were performed with the two new substances. Both degradation products exhibited higher IC50 values and caused apoptotic effects with higher concentrations than ochratoxin A in cultured human kidney epithelial cells. Thus, these cell culture data suggest that the degradation products are less cytotoxic than ochratoxin A.

Degradation of learned skills. Static practice effectiveness for visual approach and landing skill retention

NASA Technical Reports Server (NTRS)

Sitterley, T. E.

1974-01-01

The effectivess of an improved static retraining method was evaluated for a simulated space vehicle approach and landing under instrument and visual flight conditions. Experienced pilots were trained and then tested after 4 months without flying to compare their performance using the improved method with three methods previously evaluated. Use of the improved static retraining method resulted in no practical or significant skill degradation and was found to be even more effective than methods using a dynamic presentation of visual cues. The results suggested that properly structured open loop methods of flight control task retraining are feasible.

Chemotactic selection of pollutant degrading soil bacteria

DOEpatents

Hazen, Terry C.

1994-01-01

A method for identifying soil microbial strains which may be bacterial degraders of pollutants comprising the steps of placing a concentration of a pollutant in a substantially closed container, placing the container in a sample of soil for a period of time ranging from one minute to several hours, retrieving the container, collecting the contents of the container, and microscopically determining the identity of the bacteria present. Different concentrations of the pollutant can be used to determine which bacteria respond to each concentration. The method can be used for characterizing a polluted site or for looking for naturally occurring biological degraders of the pollutant. Then bacteria identified as degraders of the pollutant and as chemotactically attracted to the pollutant are used to inoculate contaminated soil. To enhance the effect of the bacteria on the pollutant, nutrients are cyclicly provided to the bacteria then withheld to alternately build up the size of the bacterial colony or community and then allow it to degrade the pollutant.

Biodegradation of oil refinery wastes under OPA and CERCLA

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

Gamblin, W.W.; Banipal, B.S.; Myers, J.M.

1995-12-31

Land treatment of oil refinery wastes has been used as a disposal method for decades. More recently, numerous laboratory studies have been performed attempting to quantify degradation rates of more toxic polycyclic aromatic hydrocarbon compounds (PAHs). This paper discusses the results of the fullscale aerobic biodegradation operations using land treatment at the Macmillan Ring-Free Oil refining facility. The tiered feasibility approach of evaluating biodegradation as a treatment method to achieve site-specific cleanup criteria, including pilot biodegradation operations, is discussed in an earlier paper. Analytical results of biodegradation indicate that degradation rates observed in the laboratory can be met and exceededmore » under field conditions and that site-specific cleanup criteria can be attained within a proposed project time. Also prevented are degradation rates and half-lives for PAHs for which cleanup criteria have been established. PAH degradation rates and half-life values are determined and compared with the laboratory degradation rates and half-life values which used similar oil refinery wastes by other in investigators (API 1987).« less

Degradation of phenol and Cr (VI) wastewater with contact glow discharge electrolysis method and the addition of Fe2+

NASA Astrophysics Data System (ADS)

Kurniawan, Raden Ridzki Aditya; Saksono, Nelson

2017-11-01

Phenol and Cr (VI) are an organic waste and dangerous heavy metals which generated from a wide variety of industrial processes such as textiles, paints, dyes, and others. For that reason, we need effective waste treatment technologies, one of them is Contact Glow Discharge Electrolysis (CGDE). This method produce reactive species such as radical hidroxyl so as to be able to degradate phenol and Cr(VI) wastewater effectively. This research aims to obtain the effect of Fe 2+ and air bubbles in degradation of phenol and Cr (VI) waste simultaneously. Waste degradation is measured its absorbance with UV-Vis spectrophotometer. In the conditions of 600 Volt voltage, Na2SO4 0.02 M, anode depth of 1.5 cm, the addition of Fe2+ 40 ppm and the addition of air bubbles for 30 minutes was obtained a percentage degradation of phenol 99.47%, Cr (VI) 76.75% and specific energy of 344.473 kJ / mmol.

Effects of rotor model degradation on the accuracy of rotorcraft real time simulation

NASA Technical Reports Server (NTRS)

Houck, J. A.; Bowles, R. L.

1976-01-01

The effects are studied of degrading a rotating blade element rotor mathematical model to meet various real-time simulation requirements of rotorcraft. Three methods of degradation were studied: reduction of number of blades, reduction of number of blade segments, and increasing the integration interval, which has the corresponding effect of increasing blade azimuthal advance angle. The three degradation methods were studied through static trim comparisons, total rotor force and moment comparisons, single blade force and moment comparisons over one complete revolution, and total vehicle dynamic response comparisons. Recommendations are made concerning model degradation which should serve as a guide for future users of this mathematical model, and in general, they are in order of minimum impact on model validity: (1) reduction of number of blade segments, (2) reduction of number of blades, and (3) increase of integration interval and azimuthal advance angle. Extreme limits are specified beyond which the rotating blade element rotor mathematical model should not be used.

On-Orbit Performance Degradation of the International Space Station P6 Photovoltaic Arrays

NASA Technical Reports Server (NTRS)

Kerslake, Thomas W.; Gustafson, Eric D.

2003-01-01

This paper discusses the on-orbit performance and performance degradation of the International Space Station P6 solar array wings (SAWs) from the period of December 2000 through February 2003. Data selection considerations and data reduction methods are reviewed along with the approach for calculating array performance degradation based on measured string shunt current levels. Measured degradation rates are compared with those predicted by the computational tool SPACE and prior degradation rates measured with the same SAW technology on the Mir space station. Initial results show that the measured SAW short-circuit current is degrading 0.2 to 0.5 percent per year. This degradation rate is below the predicted rate of 0.8 percent per year and is well within the 3 percent estimated uncertainty in measured SAW current levels. General contributors to SAW degradation are briefly discussed.

Assessment of bioavailability of pesticides in soils and identification of pesticide degradation drivers using the in-situ Mass Distribution Quotient (iMDQ)

NASA Astrophysics Data System (ADS)

Folberth, Christian

2010-05-01

The in-situ Mass Distribution Quotient (iMDQ) has recently been shown to reliably describe the bioavailability and mineralization of the widely applied pesticide isoproturon in agricultural soils. It is determined by pore water extraction from previously incubated soil samples and subsequent assessment of the mass distribution between solid and liquid phase. The method was verified by comparing the bioavailability with co-metabolic mineralization in soils under optimum microbial soil conditions (water tension -15 kPa and bulk density 1.3 g cm-3). A comparison of the results with the chemical partitioning assessed by the Kd method has shown a higher accuracy of the new method. By combining the iMDQ/pore water extraction method with mineralization of the pesticide under optimum microbial conditions in the soils, further information about mineralization and degradation processes could be obtained or confirmed: a) Metabolically outstanding soils could be identified due to inconsistency between bioavailability and mineralization when compared to the co-metabolic soils. In a metabolically hampered soil, the mineralization was very low compared to the bioavailability and in a soil with metabolically IPU degrading microorganisms the mineralization was extremely high despite low bioavailability. b) Analysis of metabolite patterns in soil water fractions of a degradation experiment allowed for an additional identification of the metabolic status of the soil. In co-metabolic soils, the diversity of metabolites increased proportionally with the degree of mineralization of the parent compound, whereas in a metabolically hampered soil the metabolite pattern was very diverse despite low mineralization. c) A quite stable fractioning between total mineralization of the parent compound to CO2 and build-up of non-extractable bound residues was found. This is a hint that also during co-metabolic degradation that can up to now not be attributed to a certain group of microorganisms, very similar processes take place in different soils. d) It could be shown that soil parameters governing the bioavailability of the compound differ between soils. Although TOC and pH could again be identified as the most important factors for the sorption strength of soils towards isoproturon, the bioavailability itself was driven by a combination of water content and sorption strength that was unique for each soil sample. f) The partitioning of parent compound and primary metabolites remained quite stable during the degradation and mineralization. Further investigations focusing on the microbial side of co-metabolic degradation are in progress. In the future, the method could be used to investigate more compounds, the effectiveness of methods to increase bioavailability in-situ without the need for degradation experiments, and the identification and analysis of degradation pathways in-situ. Other processes that are important for risk assessment, like leaching, have already been investigated with similar methods.

[Photocatalytic Degradation of Perfluorooctanoic Acid by Pd-TiO2 Photocatalyst].

PubMed

Liu, Qing; Yu, Ze-bin; Zhang, Rui-han; Li, Ming-jie; Chen, Ying; Wang, Li; Kuang, Yu; Zhang, Bo; Zhu, You-hui

2015-06-01

Perfluorooctanoic acid (PFOA) is a new persistent organic pollutant which has got global concern for its wide distribution, high bioaccumulation and strong biological toxicity. In present study, the photocatalytic degradation of PFOA using palladium doped TiO2 (Pd-TiO2) prepared by chemical reduction method was investigated. The photocatalysts were characterized by XRD, FESEM and UV-vis DRS and were used for PFOA degradation under 365 nm UV irradiation. The results indicated that the grain size of TiO2 was smaller while the specific surface area increased and the absorption of ultraviolet light also enhanced after using chemical reduction method, but all these changes had no influence on PFOA degradation. However, the degradation was significantly enhanced because of the deposition of Pd, the fluoride concentration of PFOA was 6.62 mg x L(-1) after 7 h irradiation which was 7.3 times higher than that of TiO2 (P25). Experiments with the addition of trapping agent and nitrogen indicated that *OH played an important role in PFOA degradation while the presence of O2 accelerated the degradation. The main intermediate products of photocatalytic degradation of PFOA were authenticated by an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry systems (UPLC-QTOF-MS). The probable photocatalytic degradation mechanism involves h+ attacking the carboxyl of PFOA and resulting in decarboxylation. The produced *CnF(2n +1) was oxidized by *OH underwent defluorinetion to form shorter-chain perfluorinated carboxylic acids. The significant enhancement of PFOA degradation can be ascribed to the palladium deposits, acting as electron traps on the Pd-TiO2 surface, which facilitated the transfer of photogenerated electrons and retarded the accumulation of electrons.

Enhanced degradation of perfluorooctanoic acid by a genome shuffling-modified Pseudomonas parafulva YAB-1.

PubMed

Yi, Langbo; Peng, Qingzhong; Liu, Deming; Zhou, Lulu; Tang, Chongjian; Zhou, Yaoyu; Chai, Liyuan

2018-05-02

Perfluorooctanoic acid (PFOA) as an emerging persistent organic pollutant is hard to be degraded by conventional methods because of its stable physical and chemical properties. Microbial transformation is an attractive remediation approach to prevent and clean up PFOA contamination. To date, several strains of wild microbes have been reported to have limited capacity to degrade PFOA, selection of superior strains degrading PFOA become urgently necessary. Here, we report the application of genome shuffling to improve the PFOA-degrading bacterium Pseudomonas Parafulva YAB-1. The initial mutant populations of strain YAB1 were generated by nitrosoguanidine and ultraviolet irradiation mutagenesis respectively, resulting in mutants YM-9 and YM-19 with slightly improved PFOA-degrading ability. YM-9 and YM-19 were used as the starting strains for three rounds of recursive protoplast fusion. The positive mutants were screened on inorganic salt medium plates containing different concentrations of PFOA and selected based on their PFOA degradability in shake-flask fermentation test. The best performing recombinant F3-52 was isolated after three rounds of genome shuffling. In batch fermentation, the PFOA degradation rate of mutant F3-52 was up to 58.6%, which was 1.8-fold higher than that of the parent strain YAB1, and 1.6-fold higher than the initial mutants YM-9 and YM-19. Pass-generation test indicated that the heredity character of F3-52 was stable. The results demonstrated that genome shuffling was an efficient method for improving PFOA degradation of Pseudomonas Parafulva YAB1. The bred mutant F3-52 with 58.6% PFOA-degrading rate could be used for the environmental control of PFOA pollutant.

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning.

PubMed

Sivakumaran, Daryl; Bakaic, Emilia; Campbell, Scott B; Xu, Fei; Mueller, Eva; Hoare, Todd

2018-04-16

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Tension degradation of anterior cruciate ligament grafts with dynamic flexion-extension loading: a biomechanical model in porcine knees.

PubMed

Dargel, Jens; Koebke, Jürgen; Brüggemann, Gert-Peter; Pennig, Dietmar; Schmidt-Wiethoff, Rüdiger

2009-10-01

This study investigates the influence of various femoral anterior cruciate ligament graft fixation methods on the amount of tension degradation and the initial fixation strength after cyclic flexion-extension loading in a porcine knee model. One hundred twenty porcine digital extensor tendons, used as 4-stranded free tendon grafts, were fixated within porcine femoral bone tunnels by use of extracortical button, cross-pin, or interference screw fixation. One hundred twenty porcine patellar tendon-bone grafts were fixated by use of cross-pin, interference screw, or press-fit fixation. Each femur-graft complex was submitted to cyclic flexion-extension loading for 1,000 cycles throughout different loading ranges, and the total loss of tension was determined. After cyclic testing, the grafts were loaded to failure, and the data were compared with a pullout series without cyclic loading. Tension degradation after 1,000 cycles of flexion-extension loading averaged 62.6% +/- 10.0% in free tendon grafts and 48.9% +/- 13.35% in patellar tendon-bone grafts. There was no influence of the loading range on the total amount of tension degradation. The total amount of tension degradation was the highest with interference screw fixation of free tendon and patellar tendon-bone grafts. Despite excessive loss of tension, the initial fixation strength of the femur-graft complex was not reduced. The method of femoral graft fixation significantly influenced tension degradation during dynamic flexion-extension loading. Femoral graft fixation methods that secure the graft close to the tunnel entrance and that displace the graft substance from the center of the bone tunnel show the largest amount of tension degradation during cyclic flexion-extension loading. The graft substance, not the fixation site, was the weakest link of the graft complex within this investigation. We believe that the graft fixation method should be considered when aiming to improve the precision of femoral graft placement in anterior cruciate ligament reconstruction.

Results of analyses of the fungicide Chlorothalonil, its degradation products, and other selected pesticides at 22 surface-water sites in five Southern states, 2003-04

USGS Publications Warehouse

Scribner, Elisabeth A.; Orlando, James L.; Battaglin, William A.; Sandstrom, Mark; Kuivila, Kathryn; Meyer, Michael T.

2006-01-01

In accordance with the mission of the U.S. Geological Survey (USGS) Toxic Substances Hydrology Program, a pesticide study was conducted during 2003-04 to determine the occurrence of the fungicide chlorothalonil and its degradation products at 22 surface-water sites in five Southern States. Water-quality samples were collected during the peanut-growing season (June-September) in 2003. During the peanut-growing season in 2004, samples were collected after large storms. An analytical method was developed at the USGS Organic Geochemistry Research Laboratory in Lawrence, Kansas, to measure chlorothalonil and its degradation products by liquid chromatography/mass spectrometry (LC/MS). Chlorothalonil was detected in 4 of the 113 surface-water samples. The primary degradation product of chlorothalonil, 4-hydroxy-chlorothalonil, was detected in 26 of the 113 samples with concentrations ranging from 0.002 to 0.930 microgram per liter. The chlorothalonil degradation products, 1-amide-4-hydroxy-chorothalonil and 1,3-diamide-chlorothalonil, were detected in one water sample each at 0.020 and 0.161 microgram per liter, respectively. The USGS Methods and Research Development Group, Lakewood, Colorado, developed a custom method for chlorothalonil using gas chromatography/mass spectrometry (GC/MS) in an effort to achieve a lower laboratory reporting level (LRL) than the USGS National Water-Quality Laboratory (NWQL) schedule 2060, which analyzes the compound chlorothalonil at a LRL of 0.035 ?g/L. The group succeeded in achieving a lower GC/MS reporting level of 0.01 ?g/L. Chlorothalonil was detected in 5 of 68 water samples analyzed using the custom GC/MS method, whereas chlorothalonil was detected in 2 of 21 water samples analyzed using NWQL schedule 2060. In addition to analysis of chlorothalonil and its degradation products, samples were analyzed using the USGS NWQL schedules 2001 and 2060 for about 114 pesticides and their degradation products. Samples also were analyzed for dissolved organic carbon, suspended sediment, and percentage of silt- and clay-sized particles. Overall, it was found that chlorothalonil was detected only infrequently and at relatively low concentrations. Chlorothalonil's major degradation product, 4-hydroxy-chlorothalonil, was detected most frequently, occurred generally at higher concentrations in water samples than did the parent fungicide, and the data from this study reaffirmed that it is the dominant degradation product of chlorothalonil in the peanut-growing environment.

Photocatalytic treatment of RDX wastewater with nano-sized titanium dioxide.

PubMed

Liu, Zongkuan; He, Yanling; Li, Feng; Liu, Yonghong

2006-09-01

The polynitramines, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), are important military explosives and regulated toxic hazardous compounds. Production, testing and use of the compounds has resulted in numerous acres of contaminated soils and groundwater near many munitions facilities. Economical and efficient methods for treatment of wastewater and cleanup of soils or groundwater containing RDX and HMX are needed. This study focuses on the photocatalytic treatment of RDX wastewater with nano-sized titanium dioxide (nano-TiO2) under simulated sunlight, whose intensity and wavelength are similar to that of the real sunlight in Xi'an at noon. The objective is to determine the potential for RDX destruction with nano-TiO2 in aqueous solution. An activated carbon fiber (ACF) cloth-loaded with nano-TiO2 was put into the RDX containing solution, and the concentration of RDX was measured (by HPLC-UV) at regular time intervals under simulated sunlight. The RDX degradation percentage of the photocatalytic process is higher than that of Fenton oxidation before 80 min, equivalent after 80 min, and it reaches 95% or above after 120 min. The nano-TiO2 catalyst can be used repeatedly. The photocatalytic degradation kinetics of RDX under simulated sunlight can be described by a first-order reaction kinetics equation. The possible degradation mechanism of RDX was presented and the degradation performance was compared with that of biological method. It was demonstrated that the degradation of RDX wastewater is very effective with nano-TiO2 as the photocatalytic catalyst under simulated sunlight. The efficiency of the nano-TiO2 catalyst for RDX degradation under simulated sunlight is nearly identical to that of Fenton oxidation. To date, a number of catalysts show poor absorption and utilization of sunlight, and still need ultraviolet light irradiation during wastewater degradation. The nano-TiO2 used in the described experiments features very good degradation of RDX under simulated sunlight, and the manufacturing costs are rather low (around 10 Euro/m2). Moreover, the degradation efficiency is higher compared to that of the biological method. This method exhibits great potential for practical applications owing to its easiness and low cost. If it can be applied extensively, the efficiency of wastewater treatment will be enhanced greatly.

Methods of analysis by the U.S. Geological Survey Organic Geochemistry Research Group : determination of triazine and phenylurea herbicides and their degradation products in water using solid-phase extraction and liquid chromatography/mass spectrometry

USGS Publications Warehouse

Lee, Edward Alan; Strahan, Alex P.; Thurman, Earl Michael

2002-01-01

An analytical method for the determination of 7 triazine and phenylurea herbicides and 12 of their degradation products in natural water samples using solid-phase extraction and liquid chromatography/mass spectrometry is presented in this report. Special consideration was given during the development of the method to prevent the formation of degradation products during the analysis. Filtered water samples were analyzed using 0.5 gram graphitized carbon as the solid-phase extraction media followed by liquid chromatography/mass spectrometry. Three different water-sample matrices?ground-water, surface-water, and reagent-water samples?spiked at 0.2 and 2.0 micrograms per liter were analyzed. Method detection limits ranged from 0.013 to 0.168 microgram per liter for the parent triazine herbicides and the triazine degradation products. Method detection limits ranged from 0.042 to 0.141 microgram per liter for the parent phenylurea herbicides and their degradation products. Mean recoveries for the triazine compounds in the ground- and surface-water samples generally ranged from 72.6 to 117.5 percent, but deethyl-cyanazine amide was recovered at 140.5 percent. Mean recoveries from the ground- and surface-water samples for the phenylurea compounds spiked at the 2.0-micrograms-per-liter level ranged from 82.1 to 114.4 percent. The mean recoveries for the phenylureas spiked at 0.2-microgram per liter were less consistent, ranging from 87.0 to 136.0 percent. Mean recoveries from reagent-water samples ranged from 87.0 to 109.5 percent for all compounds. The triazine compounds and their degradation products are reported in concentrations ranging from 0.05 to 2.0 micrograms per liter, with the exception of deethylcyanazine and deethylcyanazine amide which are reported at 0.20 to 2.0 micrograms per liter. The phenylurea compounds and their degradation products are reported in concentrations ranging from 0.20 to 2.0 micrograms per liter. The upper concentration limit was 2.0 micrograms per liter for all compounds without dilution.

Development of a pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray ionization tandem mass spectrometry method for the quantitative determination of benzoxazolinones and their degradation products in agricultural soil.

PubMed

Villagrasa, M; Guillamón, M; Navarro, A; Eljarrat, E; Barceló, D

2008-02-01

A new analytical method for the quantitative determination of benzoxazolinones and their degradation products in agricultural soils based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) and then instrumental determination using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation and quantitative detection of a mixture of two benzoxazolinones, benzoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-2-one (MBOA) and their degradation products, 2-aminophenol (APH), N-(2-hydroxyphenyl)malonamic acid (HMPMA), 2-amino-3-H-phenoxazin-3-one (APO), 9-methoxy-2-amino-3-H-phenoxazin-3-one (AMPO), 2-acetylamino-3-H-phenoxazin-3-one (AAPO) and 2-acetylamino-9-methoxy-2-amino-3-H-phenoxazin-3-one (AAMPO) was achieved. The complete LC-ESI-MS-MS precursor-product ion fragmentation pathways for the degradation products of benzoxazolinones are described for the first time. Quantitative analysis was done in the multiple reaction mode using two specific combinations of precursor-product ion transitions for each compound. The optimized method was quality assessed by the measure of parameter as recovery, linearity, sensitivity, repeatability and reproducibility. Recoveries of the analytes ranged from 53 to 123%. The developed method offered improvements to the sensitivity as compared with our previously LC-MS method, with detection limits down to 2.4-21 ng/g of dry weight. This achievement allows us to identify and quantify for the first time degradation products of benzoxazolinones in real agricultural soil samples. Analytes were found in the range of 20.6-149 ng/g dry weight.

Stability-indicating assay of repaglinide in bulk and optimized nanoemulsion by validated high performance thin layer chromatography technique.

PubMed

Akhtar, Juber; Fareed, Sheeba; Aqil, Mohd

2013-07-01

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for analysis of repaglinide both as a bulk drug and in nanoemulsion formulation was developed and validated. The method employed TLC aluminum plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of chloroform/methanol/ammonia/glacial acetic acid (7.5:1.5:0.9:0.1, v/v/v/v). This system was found to give compact spots for repaglinide (R f value of 0.38 ± 0.02). Repaglinide was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also, the degraded products were well separated from the pure drug. Densitometric analysis of repaglinide was carried out in the absorbance mode at 240 nm. The linear regression data for the calibration plots showed good linear relationship with r (2)= 0.998 ± 0.032 in the concentration range of 50-800 ng. The method was validated for precision, accuracy as recovery, robustness and specificity. The limits of detection and quantitation were 0.023 and 0.069 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of the degraded product were resolved from the standard drug with significantly different R f values. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the degradation kinetics in 1M NaOH.

Bio-Augmentation of Cupriavidus sp. CY-1 into 2,4-D Contaminated Soil: Microbial Community Analysis by Culture Dependent and Independent Techniques

PubMed Central

Chang, Young-Cheol; Reddy, M. Venkateswar; Umemoto, Honoka; Sato, Yuki; Kang, Mi-Hye; Yajima, Yuka; Kikuchi, Shintaro

2015-01-01

In the present study, a 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterial strain CY-1 was isolated from the forest soil. Based on physiological, biochemical and 16S rRNA gene sequence analysis it was identified as Cupriavidus sp. CY-1. Further 2,4-D degradation experiments at different concentrations (200 to 800 mg l-1) were carried out using CY-1. Effect of NaCl and KNO3 on 2,4-D degradation was also evaluated. Degradation of 2,4-D and the metabolites produced during degradation process were analyzed using high pressure liquid chromatography (HPLC) and GC-MS respectively. The amount of chloride ions produced during the 2,4-D degradation were analyzed by Ion chromatography (IC) and it is stoichiometric with 2,4-D dechlorination. Furthermore two different types of soils collected from two different sources were used for 2,4-D degradation studies. The isolated strain CY-1 was bio-augmented into 2,4-D contaminated soils to analyze its degradation ability. Culture independent methods like denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), and culture dependent methods like colony forming units (CFU) and most probable number (MPN) were used to analyze the survivability of strain CY-1 in contaminated soil. Results of T-RFLP were coincident with the DGGE analysis. From the DGGE, T-RFLP, MPN and HPLC results it was concluded that strain CY-1 effectively degraded 2,4-D without disturbing the ecosystem of soil indigenous microorganisms. PMID:26710231

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC.

PubMed

Lau, Hollis; Pace, Danielle; Yan, Boxu; McGrath, Theresa; Smallwood, Scott; Patel, Ketaki; Park, Jihea; Park, Sungae S; Latypov, Ramil F

2010-04-01

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab-Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs. 2010 Elsevier B.V. All rights reserved.

A validated stability indicating RP-HPLC method for estimation of Armodafinil in pharmaceutical dosage forms and characterization of its base hydrolytic product.

PubMed

Venkateswarlu, Kambham; Rangareddy, Ardhgeri; Narasimhaiah, Kanaka; Sharma, Hemraj; Bandi, Naga Mallikarjuna Raja

2017-01-01

The main objective of present study was to develop a RP-HPLC method for estimation of Armodafinil in pharmaceutical dosage forms and characterization of its base hydrolytic product. The method was developed for Armodafinil estimation and base hydrolytic products were characterized. The separation was carried out on C18 column by using mobile phase as mixture of water and methanol (45:55%v/v). Eluents were detected at 220nm at 1ml/min. Stress studies were performed with milder conditions followed by stronger conditions so as to get sufficient degradation around 20%. A total of five degradation products were detected and separated from analyte. The linearity of the proposed method was investigated in the range of 20-120µg/ml for Armodafinil. The detection limit and quantification limit was found to be 0.01183μg/ml and 0.035µg/ml respectively. The precision % RSD was found to be less than 2% and the recovery was between 98-102%. Armodafinil was found to be more sensitive to the base hydrolysis and yielded its carboxylic acid as degradant. The developed method was stability indicating assay, suitable to quantify Armodafinil in presence of possible degradants. The drug was sensitive to acid, base &photolytic stress and resistant to thermal &oxidation.

Development and validation of a stability-indicating RP-HPLC method for determination of atomoxetine hydrochloride in tablets.

PubMed

Patel, Sejal K; Patel, Natvarlal J

2010-01-01

This paper describes the development of a stability-indicating RP-HPLC method for the determination of atomoxetine hydrochloride (ATX) in the presence of its degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of acid, base, oxidation, wet heat, dry heat, and photodegradation. In stability tests, the drug was susceptible to acid, base, oxidation, and dry and wet heat degradation. It was found to be stable under the photolytic conditions tested. The drug was successfully separated from the degradation products formed under stress conditions on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) by using acetonitrile-methanol-0.032 M ammonium acetate (55 + 05 + 40, v/v/v) as the mobile phase at 1.0 mL/min and 40 degrees C. Photodiode array detection at 275 nm was used for quantitation after RP-HPLC over the concentration range of 0.5-5 microg/mL with a mean recovery of 100.8 +/- 0.4% for ATX. Statistical analysis demonstrated that the method is repeatable, specific, and accurate for the estimation of ATX. Because the method effectively separates the drug from its degradation products, it can be used as a stability-indicating method.

Spectrofluorometry, thin layer chromatography, and column high-performance liquid chromatography determination of rabeprazole sodium in the presence of its acidic and oxidized degradation products.

PubMed

Osman, Afaf Osman; Osman, Afaf; Osman, Mohamed

2009-01-01

The objective of this study is to develop validated stability-indicating spectrofluorometric, TLC-densitometric, and HPLC methods for the determination of rabeprazole sodium and its degradation products. The first method was based on measuring the fluorescence intensity of the drug at 416 and 311 nm for the emission and at 320 and 274 nm for the excitation for acid and oxidized solutions, respectively. The second method was based on the separation of the drug from its acidic and oxidized degradation products followed by densitometric measurement of the intact drug spot at 284 nm. The separation was carried out on Fluka TLC sheets of silica gel 60 F254 using isopropyl alcohol--30% ammonia (80 + 2, v/v) mobile phase. The third method was based on HPLC separation of rabeprazole sodium from its acidic and oxidized degradation products on a reversed-phase Waters Nova-Pak C18 column using 0.05 M potassium dihydrogen phosphate-methanol-acetonitrile (5 + 3 + 2, v/v/v) pH 7 +/- 0.2 mobile phase. The proposed procedures were successfully applied for the determination of rabeprazole sodium in pure form, laboratory-prepared mixtures, tablet, and expired batch. The obtained results were statistically compared with those of a reported method and validated according to United States Pharmacopeia guidelines. Two main acidic degradation products of the drug were separated and subjected to IR spectrometry and MS to confirm their structures, and the schemes for their formation were elucidated.

Chemical degradation of drinking water disinfection byproducts by millimeter-sized particles of iron-silicon and magnesium-aluminum alloys.

PubMed

Li, Tianyu; Chen, Yongmei; Wan, Pingyu; Fan, Maohong; Yang, X Jin

2010-03-03

The candidature of Fe-Si and Mg-Al alloys at millimeter-scale particle sizes for chemical degradation of disinfection byproducts (DBPs) in drinking water systems was substantiated by their enhanced corrosion resistance and catalytic effect on the degradation. The Mg-Al particles supplied electrons for reductive degradation, and the Fe-Si particles acted as a catalyst and provided the sites for the reaction. The alloy particles are obtained by mechanical milling and stable under ambient conditions. The proposed method for chemical degradation of DBPs possesses the advantages of relatively constant degradation performance, long-term durability, no secondary contamination, and ease of handling, storage and maintenance in comparison with nanoparticle systems.

Proteomic researches for lignocellulose-degrading enzymes: A mini-review.

PubMed

Guo, Hongliang; Wang, Xiao-Dong; Lee, Duu-Jong

2018-05-31

Protective action of lignin/hemicellulose networks and crystalline structures of embedded cellulose render lignocellulose material resistant to external enzymatic attack. To eliminate this bottleneck, research has been conducted in which advanced proteomic techniques are applied to identify effective commercial hydrolytic enzymes. This mini-review summarizes researches on lignocellulose-degrading enzymes, the mechanisms of the responses of various lignocellulose-degrading strains and microbial communities to various carbon sources and various biomass substrates, post-translational modifications of lignocellulose-degrading enzymes, new lignocellulose-degrading strains, new lignocellulose-degrading enzymes and a new method of secretome analysis. The challenges in the practical use of enzymatic hydrolysis process to realize lignocellulose biorefineries are discussed, along with the prospects for the same. Copyright © 2018 Elsevier Ltd. All rights reserved.

Poly[(ethylene oxide)-co-(methylene ethylene oxide)]: A hydrolytically-degradable poly(ethylene oxide) platform.

PubMed

Lundberg, Pontus; Lee, Bongjae F; van den Berg, Sebastiaan A; Pressly, Eric D; Lee, Annabelle; Hawker, Craig J; Lynd, Nathaniel A

2012-11-20

A facile method for imparting hydrolytic degradability to poly(ethylene oxide) (PEO), compatible with current PEGylation strategies, is presented. By incorporating methylene ethylene oxide (MEO) units into the parent PEO backbone, complete degradation was defined by the molar incorporation of MEO, and the structure of the degradation byproducts was consistent with an acid-catalyzed vinyl-ether hydrolysis mechanism. The hydrolytic degradation of poly[(ethylene oxide)-co-(methylene ethylene oxide)] was pH-sensitive, with degradation at pH 5 being significantly faster than at pH 7.4 at 37 °C in PBS buffer while long-term stability could be obtained in either the solid-state or at pH 7.4 at 6 °C.

Measurement of pyrethroids and their environmental degradation products in fresh fruits and vegetables using a modification of the quick easy cheap effective rugged safe (QuEChERS) method

EPA Science Inventory

Pyrethroid insecticides are used extensively in agriculture, and they, as well as their environmental degradates, may remain as residues on foods such as fruits and vegetables. Since pyrethroid degradates can be identical to the urinary markers used in human biomonitoring, it is ...

Method and apparatus to predict the remaining service life of an operating system

DOEpatents

Greitzer, Frank L.; Kangas, Lars J.; Terrones, Kristine M.; Maynard, Melody A.; Pawlowski, Ronald A. , Ferryman; Thomas A.; Skorpik, James R.; Wilson, Bary W.

2008-11-25

A method and computer-based apparatus for monitoring the degradation of, predicting the remaining service life of, and/or planning maintenance for, an operating system are disclosed. Diagnostic information on degradation of the operating system is obtained through measurement of one or more performance characteristics by one or more sensors onboard and/or proximate the operating system. Though not required, it is preferred that the sensor data are validated to improve the accuracy and reliability of the service life predictions. The condition or degree of degradation of the operating system is presented to a user by way of one or more calculated, numeric degradation figures of merit that are trended against one or more independent variables using one or more mathematical techniques. Furthermore, more than one trendline and uncertainty interval may be generated for a given degradation figure of merit/independent variable data set. The trendline(s) and uncertainty interval(s) are subsequently compared to one or more degradation figure of merit thresholds to predict the remaining service life of the operating system. The present invention enables multiple mathematical approaches in determining which trendline(s) to use to provide the best estimate of the remaining service life.

A Review of Molecular-Level Mechanism of Membrane Degradation in the Polymer Electrolyte Fuel Cell

PubMed Central

Ishimoto, Takayoshi; Koyama, Michihisa

2012-01-01

Chemical degradation of perfluorosulfonic acid (PFSA) membrane is one of the most serious problems for stable and long-term operations of the polymer electrolyte fuel cell (PEFC). The chemical degradation is caused by the chemical reaction between the PFSA membrane and chemical species such as free radicals. Although chemical degradation of the PFSA membrane has been studied by various experimental techniques, the mechanism of chemical degradation relies much on speculations from ex-situ observations. Recent activities applying theoretical methods such as density functional theory, in situ experimental observation, and mechanistic study by using simplified model compound systems have led to gradual clarification of the atomistic details of the chemical degradation mechanism. In this review paper, we summarize recent reports on the chemical degradation mechanism of the PFSA membrane from an atomistic point of view. PMID:24958288

Effect of preparation methods on the activity of titanium dioxide-carbon nitride composites for photocatalytic degradation of salicylic acid

NASA Astrophysics Data System (ADS)

Yuliati, L.; Salleh, A. M.; Hatta, M. H. M.; Lintang, H. O.

2018-04-01

In this study, titanium dioxide-carbon nitride (TiO2-CN) composites were prepared by three methods, which were one pot oxidation, impregnation, and physical mixing. Each series of the photocatalysts was prepared with different ratios of titanium to carbon (Ti/C), i.e., 1, 5, 10, 20, and 50 mol%. All samples were characterized by X-ray diffraction (XRD) and diffuse reflectance ultraviolet-visible (DR UV-Vis) spectroscopies. The characterization results confirmed the successful preparation of TiO2, CN, and the TiO2-CN composites. Photocatalytic activity tests were carried out for degradation of salicylic acid at room temperature for 6 h under UV and visible light irradiations. It was confirmed that all the prepared TiO2-CN composites showed better photocatalytic activities than the bare TiO2 and the bare CN. Under UV light irradiation, 90.6% of salicylic acid degradation was achieved on the best composite prepared by one pot oxidation with 5 mol% of titanium to carbon (Ti/C) ratio. On the other hand, the highest degradation under visible light irradiation was 94.3%, observed on the composite that was prepared also by one pot oxidation method with the Ti/C ratio of 10 mol%. Therefore, among the investigated methods, the best method to prepare the titanium dioxide-carbon nitride composites with high photocatalytic activity was one pot oxidation method.

Adaptive hidden Markov model-based online learning framework for bearing faulty detection and performance degradation monitoring

NASA Astrophysics Data System (ADS)

Yu, Jianbo

2017-01-01

This study proposes an adaptive-learning-based method for machine faulty detection and health degradation monitoring. The kernel of the proposed method is an "evolving" model that uses an unsupervised online learning scheme, in which an adaptive hidden Markov model (AHMM) is used for online learning the dynamic health changes of machines in their full life. A statistical index is developed for recognizing the new health states in the machines. Those new health states are then described online by adding of new hidden states in AHMM. Furthermore, the health degradations in machines are quantified online by an AHMM-based health index (HI) that measures the similarity between two density distributions that describe the historic and current health states, respectively. When necessary, the proposed method characterizes the distinct operating modes of the machine and can learn online both abrupt as well as gradual health changes. Our method overcomes some drawbacks of the HIs (e.g., relatively low comprehensibility and applicability) based on fixed monitoring models constructed in the offline phase. Results from its application in a bearing life test reveal that the proposed method is effective in online detection and adaptive assessment of machine health degradation. This study provides a useful guide for developing a condition-based maintenance (CBM) system that uses an online learning method without considerable human intervention.

A Rapid Novel HPLC Method for Estimation of Eight Related Compounds in Azilsartan Kamedoxomil and Identification of Degradation Compounds by Using LC-MS.

PubMed

Sreenivasulu, J; Venkata Ramana, P; Sampath Kumar Reddy, G; Nagaraju, Ch V S; Thirumalai Rajan, S; Eswaraiah, S

2015-10-01

A novel, rapid, specific and stability-indicating reverse-phase high-performance liquid chromatography method was developed for the quantitative determination of related compounds, obtained from two different synthetic routes and degradation products of Azilsartan kamedoxomil (AZL). The method was developed by using a YMC-Pack pro C18 (150 × 4.6 mm, 3 µm) column with a mobile phase containing a gradient mobile phase combination. The eluted compounds were measured at wavelength 220 nm. The developed method run time was 25 min, within which AZL and its eight impurities were well separated with minimum 3.0 resolution. The drug substance was subjected to stress conditions of hydrolysis (acid, base and water), oxidation, photolysis, sunlight, 75% relative humidity and thermal degradation as per International Conference on Harmonization (ICH) prescribed stress conditions to ascertain the stability-indicating power of the method. Significant degradation was observed during acid, base, peroxide, water hydrolysis and 75% relative humidity studies. The mass balance of AZL was close to 100% in all the stress condition. The developed method was validated as per the ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Stress Degradation Studies on Varenicline Tartrate and Development of a Validated Stability-Indicating HPLC Method

PubMed Central

Pujeri, Sudhakar S.; Khader, Addagadde M. A.; Seetharamappa, Jaldappagari

2012-01-01

A simple, rapid and stability-indicating reversed-phase liquid chromatographic method was developed for the assay of varenicline tartrate (VRT) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 Inertsil column (250 mm × 4.6 mm i.d. particle size is 5 μm) employing a mobile phase consisting of ammonium acetate buffer containing trifluoroacetic acid (0.02M; pH 4) and acetonitrile in gradient program mode with a flow rate of 1.0 mL min−1. The UV detector was operated at 237 nm while column temperature was maintained at 40 °C. The developed method was validated as per ICH guidelines with respect to specificity, linearity, precision, accuracy, robustness and limit of quantification. The method was found to be simple, specific, precise and accurate. Selectivity of the proposed method was validated by subjecting the stock solution of VRT to acidic, basic, photolysis, oxidative and thermal degradation. The calibration curve was found to be linear in the concentration range of 0.1–192 μg mL−1 (R2 = 0.9994). The peaks of degradation products did not interfere with that of pure VRT. The utility of the developed method was examined by analyzing the tablets containing VRT. The results of analysis were subjected to statistical analysis. PMID:22396908

Efficient and selective degradation of polyethylenes into liquid fuels and waxes under mild conditions.

PubMed

Jia, Xiangqing; Qin, Chuan; Friedberger, Tobias; Guan, Zhibin; Huang, Zheng

2016-06-01

Polyethylene (PE) is the largest-volume synthetic polymer, and its chemical inertness makes its degradation by low-energy processes a challenging problem. We report a tandem catalytic cross alkane metathesis method for highly efficient degradation of polyethylenes under mild conditions. With the use of widely available, low-value, short alkanes (for example, petroleum ethers) as cross metathesis partners, different types of polyethylenes with various molecular weights undergo complete conversion into useful liquid fuels and waxes. This method shows excellent selectivity for linear alkane formation, and the degradation product distribution (liquid fuels versus waxes) can be controlled by the catalyst structure and reaction time. In addition, the catalysts are compatible with various polyolefin additives; therefore, common plastic wastes, such as postconsumer polyethylene bottles, bags, and films could be converted into valuable chemical feedstocks without any pretreatment.

Method and compositions for the degradation of tributyl phosphate in chemical waste mixtures

DOEpatents

Stoner, Daphne L.; Tien, Albert J.

1995-01-01

A method and process for the degradation of tributyl phosphate in an organic waste mixture and a biologically pure, novel bacteria culture for accomplishing the same. A newly-discovered bacteria (a strain of Acinetobacter sp. ATCC 55587) is provided which is combined in a reactor vessel with a liquid waste mixture containing tributyl phosphate and one or more organic waste compounds capable of functioning as growth substrates for the bacteria. The bacteria is thereafter allowed to incubate within the waste mixture. As a result, the tributyl phosphate and organic compounds within the waste mixture are metabolized (degraded) by the bacteria, thereby eliminating such materials which are environmentally hazardous. In addition, the bacteria is capable of degrading waste mixtures containing high quantities of tributyl phosphate (e.g. up to about 1.0% by weight tributyl phosphate).

Efficient and selective degradation of polyethylenes into liquid fuels and waxes under mild conditions

PubMed Central

Jia, Xiangqing; Qin, Chuan; Friedberger, Tobias; Guan, Zhibin; Huang, Zheng

2016-01-01

Polyethylene (PE) is the largest-volume synthetic polymer, and its chemical inertness makes its degradation by low-energy processes a challenging problem. We report a tandem catalytic cross alkane metathesis method for highly efficient degradation of polyethylenes under mild conditions. With the use of widely available, low-value, short alkanes (for example, petroleum ethers) as cross metathesis partners, different types of polyethylenes with various molecular weights undergo complete conversion into useful liquid fuels and waxes. This method shows excellent selectivity for linear alkane formation, and the degradation product distribution (liquid fuels versus waxes) can be controlled by the catalyst structure and reaction time. In addition, the catalysts are compatible with various polyolefin additives; therefore, common plastic wastes, such as postconsumer polyethylene bottles, bags, and films could be converted into valuable chemical feedstocks without any pretreatment. PMID:27386559

Semi-empirical study of ortho-cresol photo degradation in manganese-doped zinc oxide nanoparticles suspensions

PubMed Central

2012-01-01

The optimization processes of photo degradation are complicated and expensive when it is performed with traditional methods such as one variable at a time. In this research, the condition of ortho-cresol (o-cresol) photo degradation was optimized by using a semi empirical method. First of all, the experiments were designed with four effective factors including irradiation time, pH, photo catalyst’s amount, o-cresol concentration and photo degradation % as response by response surface methodology (RSM). The RSM used central composite design (CCD) method consists of 30 runs to obtain the actual responses. The actual responses were fitted with the second order algebraic polynomial equation to select a model (suggested model). The suggested model was validated by a few numbers of excellent statistical evidences in analysis of variance (ANOVA). The used evidences include high F-value (143.12), very low P-value (<0.0001), non-significant lack of fit, the determination coefficient (R2 = 0.99) and the adequate precision (47.067). To visualize the optimum, the validated model simulated the condition of variables and response (photo degradation %) be using a few number of three dimensional plots (3D). To confirm the model, the optimums were performed in laboratory. The results of performed experiments were quite close to the predicted values. In conclusion, the study indicated that the model is successful to simulate the optimum condition of o-cresol photo degradation under visible-light irradiation by manganese doped ZnO nanoparticles. PMID:22909072

Monitoring of Gasoline-ethanol Degradation In Undisturbed Soil

NASA Astrophysics Data System (ADS)

Österreicher-Cunha, P.; Nunes, C. M. F.; Vargas, E. A.; Guimarães, J. R. D.; Costa, A.

Environmental contamination problems are greatly emphasised nowadays because of the direct threat they represent for human health. Traditional remediation methods fre- quently present low efficiency and high costs; therefore, biological treatment is being considered as an accessible and efficient alternative for soil and water remediation. Bioventing, commonly used to remediate petroleum hydrocarbon spills, stimulates the degradation capacity of indigenous microorganisms by providing better subsur- face oxygenation. In Brazil, gasoline and ethanol are mixed (78:22 v/v); some authors indicate that despite gasoline high degradability, its degradation in subsurface is hin- dered by the presence of much more rapidly degrading ethanol. Contaminant distribu- tion and degradation in the subsurface can be monitored by several physical, chemical and microbiological methodologies. This study aims to evaluate and follow the degra- dation of a gasoline-ethanol mixture in a residual undisturbed tropical soil from Rio de Janeiro. Bioventing was used to enhance microbial degradation. Shifts in bacte- rial culturable populations due to contamination and treatment effects were followed by conventional microbiology methods. Ground Penetrating Radar (GPR) measure- ments, which consist of the emission of electro-magnetic waves into the soil, yield a visualisation of contaminant degradation because of changes in soil conductivity due to microbial action on the pollutants. Chemical analyses will measure contaminant residue in soil. Our results disclosed contamination impact as well as bioventing stim- ulation on soil culturable heterotrophic bacterial populations. This multidisciplinary approach allows for a wider evaluation of processes occurring in soil.

Methods of saccharification of polysaccharides in plants

DOEpatents

Howard, John; Fake, Gina

2014-04-29

Saccharification of polysaccharides of plants is provided, where release of fermentable sugars from cellulose is obtained by adding plant tissue composition. Production of glucose is obtained without the need to add additional .beta.-glucosidase. Adding plant tissue composition to a process using a cellulose degrading composition to degrade cellulose results in an increase in the production of fermentable sugars compared to a process in which plant tissue composition is not added. Using plant tissue composition in a process using a cellulose degrading enzyme composition to degrade cellulose results in decrease in the amount of cellulose degrading enzyme composition or exogenously applied cellulase required to produce fermentable sugars.

[Assessment and early warning of land ecological security in rapidly urbanizing coastal area: A case study of Caofeidian new district, Hebei, China].

PubMed

Zhang, Li; Chen, Ying; Wang, Shu-tao; Men, Ming-xin; Xu, Hao

2015-08-01

Assessment and early warning of land ecological security (LES) in rapidly urbanizing coastal area is an important issue to ensure sustainable land use and effective maintenance of land ecological security. In this study, an index system for the land ecological security of Caofeidian new district was established based on the Pressure-State-Response (P-S-R) model. Initial assessment units of 1 km x 1 km created with the remote sensing data and GIS methods were spatially interpolated to a fine pixel size of 30 m x 30 m, which were combined with the early warning method (using classification tree method) to evaluate the land ecological security of Caofeidian in 2005 and 2013. The early warning level was classed into four categories: security with degradation potential, sub-security with slow degradation, sub-security with rapid degradation, and insecurity. Result indicated that, from 2005 to 2013, the average LES of Caofeidian dropped from 0.55 to 0.52, indicating a degradation of land ecological security from medium security level to medium-low security level. The areas at the levels of insecurity with rapid degradation were mainly located in the rapid urbanization areas, illustrating that rapid expansion of urban construction land was the key factor to the deterioration of the regional land ecological security. Industrial District, Shilihai town and Nanpu saltern, in which the lands at the levels of insecurity and sub-security with rapid degradation or slow degradation accounted for 58.3%, 98.9% and 81.2% of their respective districts, were at the stage of high early warning. Thus, land ecological security regulation for these districts should be strengthened in near future. The study could provide a reference for land use planning and ecological protection of Caofeidian new district.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

PubMed

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Sliding Window Analyses for Optimal Selection of Mini-Barcodes, and Application to 454-Pyrosequencing for Specimen Identification from Degraded DNA

PubMed Central

Boyer, Stephane; Brown, Samuel D. J.; Collins, Rupert A.; Cruickshank, Robert H.; Lefort, Marie-Caroline; Malumbres-Olarte, Jagoba; Wratten, Stephen D.

2012-01-01

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential ‘mini-barcodes’ for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation. PMID:22666489

Evaluation of HDPE and LDPE degradation by fungus, implemented by statistical optimization

NASA Astrophysics Data System (ADS)

Ojha, Nupur; Pradhan, Neha; Singh, Surjit; Barla, Anil; Shrivastava, Anamika; Khatua, Pradip; Rai, Vivek; Bose, Sutapa

2017-01-01

Plastic in any form is a nuisance to the well-being of the environment. The ‘pestilence’ caused by it is mainly due to its non-degradable nature. With the industrial boom and the population explosion, the usage of plastic products has increased. A steady increase has been observed in the use of plastic products, and this has accelerated the pollution. Several attempts have been made to curb the problem at large by resorting to both chemical and biological methods. Chemical methods have only resulted in furthering the pollution by releasing toxic gases into the atmosphere; whereas; biological methods have been found to be eco-friendly however they are not cost effective. This paves the way for the current study where fungal isolates have been used to degrade polyethylene sheets (HDPE, LDPE). Two potential fungal strains, namely, Penicillium oxalicum NS4 (KU559906) and Penicillium chrysogenum NS10 (KU559907) had been isolated and identified to have plastic degrading abilities. Further, the growth medium for the strains was optimized with the help of RSM. The plastic sheets were subjected to treatment with microbial culture for 90 days. The extent of degradation was analyzed by, FE-SEM, AFM and FTIR. Morphological changes in the plastic sheet were determined.

Evaluation of HDPE and LDPE degradation by fungus, implemented by statistical optimization.

PubMed

Ojha, Nupur; Pradhan, Neha; Singh, Surjit; Barla, Anil; Shrivastava, Anamika; Khatua, Pradip; Rai, Vivek; Bose, Sutapa

2017-01-04

Plastic in any form is a nuisance to the well-being of the environment. The 'pestilence' caused by it is mainly due to its non-degradable nature. With the industrial boom and the population explosion, the usage of plastic products has increased. A steady increase has been observed in the use of plastic products, and this has accelerated the pollution. Several attempts have been made to curb the problem at large by resorting to both chemical and biological methods. Chemical methods have only resulted in furthering the pollution by releasing toxic gases into the atmosphere; whereas; biological methods have been found to be eco-friendly however they are not cost effective. This paves the way for the current study where fungal isolates have been used to degrade polyethylene sheets (HDPE, LDPE). Two potential fungal strains, namely, Penicillium oxalicum NS4 (KU559906) and Penicillium chrysogenum NS10 (KU559907) had been isolated and identified to have plastic degrading abilities. Further, the growth medium for the strains was optimized with the help of RSM. The plastic sheets were subjected to treatment with microbial culture for 90 days. The extent of degradation was analyzed by, FE-SEM, AFM and FTIR. Morphological changes in the plastic sheet were determined.

Derivatization of organophosphorus nerve agent degradation products for gas chromatography with ICPMS and TOF-MS detection.

PubMed

Richardson, Douglas D; Caruso, Joseph A

2007-06-01

Separation and detection of seven V-type (venomous) and G-type (German) organophosphorus nerve agent degradation products by gas chromatography with inductively coupled plasma mass spectrometry (GC-ICPMS) is described. The nonvolatile alkyl phosphonic acid degradation products of interest included ethyl methylphosphonic acid (EMPA, VX acid), isopropyl methylphosphonic acid (IMPA, GB acid), ethyl hydrogen dimethylamidophosphate sodium salt (EDPA, GA acid), isobutyl hydrogen methylphosphonate (IBMPA, RVX acid), as well as pinacolyl methylphosphonic acid (PMPA), methylphosphonic acid (MPA), and cyclohexyl methylphosphonic acid (CMPA, GF acid). N-(tert-Butyldimethylsilyl)-N-methyltrifluroacetamide with 1% TBDMSCl was utilized to form the volatile TBDMS derivatives of the nerve agent degradation products for separation by GC. Exact mass confirmation of the formation of six of the TBDMS derivatives was obtained by GC-time of flight mass spectrometry (TOF-MS). The method developed here allowed for the separation and detection of all seven TBDMS derivatives as well as phosphate in less than ten minutes. Detection limits for the developed method were less than 5 pg with retention times and peak area precisions of less than 0.01 and 6%, respectively. This method was successfully applied to river water and soil matrices. To date this is the first work describing the analysis of chemical warfare agent (CWA) degradation products by GC-ICPMS.

High temperature polymer degradation: Rapid IR flow-through method for volatile quantification

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

Giron, Nicholas H.; Celina, Mathew C.

Accelerated aging of polymers at elevated temperatures often involves the generation of volatiles. These can be formed as the products of oxidative degradation reactions or intrinsic pyrolytic decomposition as part of polymer scission reactions. A simple analytical method for the quantification of water, CO 2, and CO as fundamental signatures of degradation kinetics is required. Here, we describe an analytical framework and develops a rapid mid-IR based gas analysis methodology to quantify volatiles that are contained in small ampoules after aging exposures. The approach requires identification of unique spectral signatures, systematic calibration with known concentrations of volatiles, and a rapidmore » acquisition FTIR spectrometer for time resolved successive spectra. Furthermore, the volatiles are flushed out from the ampoule with dry N2 carrier gas and are then quantified through spectral and time integration. This method is sufficiently sensitive to determine absolute yields of ~50 μg water or CO 2, which relates to probing mass losses of less than 0.01% for a 1 g sample, i.e. the early stages in the degradation process. Such quantitative gas analysis is not easily achieved with other approaches. Our approach opens up the possibility of quantitative monitoring of volatile evolution as an avenue to explore polymer degradation kinetics and its dependence on time and temperature.« less

High temperature polymer degradation: Rapid IR flow-through method for volatile quantification

DOE PAGES

Giron, Nicholas H.; Celina, Mathew C.

2017-05-19

Accelerated aging of polymers at elevated temperatures often involves the generation of volatiles. These can be formed as the products of oxidative degradation reactions or intrinsic pyrolytic decomposition as part of polymer scission reactions. A simple analytical method for the quantification of water, CO 2, and CO as fundamental signatures of degradation kinetics is required. Here, we describe an analytical framework and develops a rapid mid-IR based gas analysis methodology to quantify volatiles that are contained in small ampoules after aging exposures. The approach requires identification of unique spectral signatures, systematic calibration with known concentrations of volatiles, and a rapidmore » acquisition FTIR spectrometer for time resolved successive spectra. Furthermore, the volatiles are flushed out from the ampoule with dry N2 carrier gas and are then quantified through spectral and time integration. This method is sufficiently sensitive to determine absolute yields of ~50 μg water or CO 2, which relates to probing mass losses of less than 0.01% for a 1 g sample, i.e. the early stages in the degradation process. Such quantitative gas analysis is not easily achieved with other approaches. Our approach opens up the possibility of quantitative monitoring of volatile evolution as an avenue to explore polymer degradation kinetics and its dependence on time and temperature.« less

Evaluation of HDPE and LDPE degradation by fungus, implemented by statistical optimization

PubMed Central

Ojha, Nupur; Pradhan, Neha; Singh, Surjit; Barla, Anil; Shrivastava, Anamika; Khatua, Pradip; Rai, Vivek; Bose, Sutapa

2017-01-01

Plastic in any form is a nuisance to the well-being of the environment. The ‘pestilence’ caused by it is mainly due to its non-degradable nature. With the industrial boom and the population explosion, the usage of plastic products has increased. A steady increase has been observed in the use of plastic products, and this has accelerated the pollution. Several attempts have been made to curb the problem at large by resorting to both chemical and biological methods. Chemical methods have only resulted in furthering the pollution by releasing toxic gases into the atmosphere; whereas; biological methods have been found to be eco-friendly however they are not cost effective. This paves the way for the current study where fungal isolates have been used to degrade polyethylene sheets (HDPE, LDPE). Two potential fungal strains, namely, Penicillium oxalicum NS4 (KU559906) and Penicillium chrysogenum NS10 (KU559907) had been isolated and identified to have plastic degrading abilities. Further, the growth medium for the strains was optimized with the help of RSM. The plastic sheets were subjected to treatment with microbial culture for 90 days. The extent of degradation was analyzed by, FE-SEM, AFM and FTIR. Morphological changes in the plastic sheet were determined. PMID:28051105

Influence of engine coolant composition on the electrochemical degradation behavior of EPDM radiator hoses

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

Vroomen, G.L.M.; Lievens, S.S.; Maes, J.P.

1999-08-01

EPDM (ethylene-propylene rubber) has been used for more than 25 years as the main elastomer in radiator hoses because it offers a well-balanced price/performance ratio in this field of application. Some years ago the automotive and rubber industry became aware of a problem called electrochemical degradation and cracking. Cooling systems broke down due to a typical cracking failure of some radiator hoses. Different test methods were developed to simulate and solve the problem on laboratory scale. The influence of different variables with respect to the electrochemical degradation and cracking. Cooling systems broke down due to a typical cracking failure ofmore » some radiator hoses. Different test methods were developed to simulate and solve the problem on laboratory scale. The influence of different variables with respect to the electrochemical degradation process has been investigated, but until recently the influence of the engine coolant was ignored. Using a test method developed by DSM elastomers, the influence of the composition of the engine coolant as well as of the EPDM composition has now been evaluated. This paper gives an overview of test results with different coolant technologies and offers a plausible explanation of the degradation mechanisms as a function of the elastomer composition.« less

A data-driven multiplicative fault diagnosis approach for automation processes.

PubMed

Hao, Haiyang; Zhang, Kai; Ding, Steven X; Chen, Zhiwen; Lei, Yaguo

2014-09-01

This paper presents a new data-driven method for diagnosing multiplicative key performance degradation in automation processes. Different from the well-established additive fault diagnosis approaches, the proposed method aims at identifying those low-level components which increase the variability of process variables and cause performance degradation. Based on process data, features of multiplicative fault are extracted. To identify the root cause, the impact of fault on each process variable is evaluated in the sense of contribution to performance degradation. Then, a numerical example is used to illustrate the functionalities of the method and Monte-Carlo simulation is performed to demonstrate the effectiveness from the statistical viewpoint. Finally, to show the practical applicability, a case study on the Tennessee Eastman process is presented. Copyright © 2013. Published by Elsevier Ltd.

Validation of a Chiral Liquid Chromatographic Method for the Degradation Behavior of Flumequine Enantiomers in Mariculture Pond Water.

PubMed

Wang, Yan-Fei; Gao, Xiao-Feng; Jin, Huo-Xi; Wang, Yang-Guang; Wu, Wei-Jian; Ouyang, Xiao-Kun

2016-09-01

In this work, flumequine (FLU) enantiomers were separated using a Chiralpak OD-H column, with n-hexane-ethanol (20:80, v/v) as the mobile phase at a flow rate of 0.6 mL/min. Solid phase extraction (SPE) was used for cleanup and enrichment. The limit of detection, limit of quantitation, linearity, precision, and intra/interday variation of the chiral high-performance liquid chromatography (HPLC) method were determined. The developed method was then applied to investigate the degradation behavior of FLU enantiomers in mariculture pond water samples. The results showed that the degradation of FLU enantiomers under natural, sterile, or dark conditions was not enantioselective. Chirality 28:649-655, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Retrieve polarization aberration from image degradation: a new measurement method in DUV lithography

NASA Astrophysics Data System (ADS)

Xiang, Zhongbo; Li, Yanqiu

2017-10-01

Detailed knowledge of polarization aberration (PA) of projection lens in higher-NA DUV lithographic imaging is necessary due to its impact to imaging degradations, and precise measurement of PA is conductive to computational lithography techniques such as RET and OPC. Current in situ measurement method of PA thorough the detection of degradations of aerial images need to do linear approximation and apply the assumption of 3-beam/2-beam interference condition. The former approximation neglects the coupling effect of the PA coefficients, which would significantly influence the accuracy of PA retrieving. The latter assumption restricts the feasible pitch of test masks in higher-NA system, conflicts with the Kirhhoff diffraction model of test mask used in retrieving model, and introduces 3D mask effect as a source of retrieving error. In this paper, a new in situ measurement method of PA is proposed. It establishes the analytical quadratic relation between the PA coefficients and the degradations of aerial images of one-dimensional dense lines in coherent illumination through vector aerial imaging, which does not rely on the assumption of 3-beam/2- beam interference and linear approximation. In this case, the retrieval of PA from image degradation can be convert from the nonlinear system of m-quadratic equations to a multi-objective quadratic optimization problem, and finally be solved by nonlinear least square method. Some preliminary simulation results are given to demonstrate the correctness and accuracy of the new PA retrieving model.

Simultaneous sound velocity and thickness measurement by the ultrasonic pitch-catch method for corrosion-layer-forming polymeric materials.

PubMed

Kusano, Masahiro; Takizawa, Shota; Sakai, Tetsuya; Arao, Yoshihiko; Kubouchi, Masatoshi

2018-01-01

Since thermosetting resins have excellent resistance to chemicals, fiber reinforced plastics composed of such resins and reinforcement fibers are widely used as construction materials for equipment in chemical plants. Such equipment is usually used for several decades under severe corrosive conditions so that failure due to degradation may result. One of the degradation behaviors in thermosetting resins under chemical solutions is "corrosion-layer-forming" degradation. In this type of degradation, surface resins in contact with a solution corrode, and some of them remain asa corrosion layer on the pristine part. It is difficult to precisely measure the thickness of the pristine part of such degradation type materials by conventional pulse-echo ultrasonic testing, because the sound velocity depends on the degree of corrosion of the polymeric material. In addition, the ultrasonic reflection interface between the pristine part and the corrosion layer is obscure. Thus, we propose a pitch-catch method using a pair of normal and angle probes to measure four parameters: the thicknesses of the pristine part and the corrosion layer, and their respective sound velocities. The validity of the proposed method was confirmed by measuring a two-layer sample and a sample including corroded parts. The results demonstrate that the pitch-catch method can successfully measure the four parameters and evaluate the residual thickness of the pristine part in the corrosion-layer-forming sample. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel strategy to evaluate the degradation of quantum dots: identification and quantification of CdTe quantum dots and corresponding ionic species by CZE-ICP-MS.

PubMed

Meng, Peijun; Xiong, Yamin; Wu, Yingting; Hu, Yue; Wang, Hui; Pang, Yuanfeng; Jiang, Shuqing; Han, Sihai; Huang, Peili

2018-05-09

In view of the significance and urgency of the speciation analysis of quantum dots (QDs) and their degradation products for clarifying their degradation rules and toxicity mechanisms, a method for the identification and quantification of CdTe QDs and corresponding ionic species in complex matrices was developed using capillary zone electrophoresis (CZE) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). The quality assessment of commercial CdTe QDs and serum pharmacokinetics of synthesized CdTe QDs in rats were successfully undertaken using the developed CZE-ICP-MS method.

Effects of time-shifted data on flight determined stability and control derivatives

NASA Technical Reports Server (NTRS)

Steers, S. T.; Iliff, K. W.

1975-01-01

Flight data were shifted in time by various increments to assess the effects of time shifts on estimates of stability and control derivatives produced by a maximum likelihood estimation method. Derivatives could be extracted from flight data with the maximum likelihood estimation method even if there was a considerable time shift in the data. Time shifts degraded the estimates of the derivatives, but the degradation was in a consistent rather than a random pattern. Time shifts in the control variables caused the most degradation, and the lateral-directional rotary derivatives were affected the most by time shifts in any variable.

Determination of the kinetics of degradation of 13-cis-retinoic acid and all-trans-retinoic acid in solution.

PubMed

Tan, X; Meltzer, N; Lindenbaum, S

1993-09-01

The degradations of 13-cis-retinoic acid and all-trans-retinoic acid in an organic solvent were determined with an HPLC assay. The degradation curves at 70, 50 and 37 degrees C all showed autocatalytic characteristics for both isomers. For this kind of complex reaction, the usual method cannot be used to estimate the shelf-lives and half-lives at room temperature. In this work a new method was developed to directly calculate the shelf-lives and half-lives. From this equation the activation energy was found to change as the multiple step reaction progressed.

Simple Pixel Structure Using Video Data Correction Method for Nonuniform Electrical Characteristics of Polycrystalline Silicon Thin-Film Transistors and Differential Aging Phenomenon of Organic Light-Emitting Diodes

NASA Astrophysics Data System (ADS)

Hai-Jung In,; Oh-Kyong Kwon,

2010-03-01

A simple pixel structure using a video data correction method is proposed to compensate for electrical characteristic variations of driving thin-film transistors (TFTs) and the degradation of organic light-emitting diodes (OLEDs) in active-matrix OLED (AMOLED) displays. The proposed method senses the electrical characteristic variations of TFTs and OLEDs and stores them in external memory. The nonuniform emission current of TFTs and the aging of OLEDs are corrected by modulating video data using the stored data. Experimental results show that the emission current error due to electrical characteristic variation of driving TFTs is in the range from -63.1 to 61.4% without compensation, but is decreased to the range from -1.9 to 1.9% with the proposed correction method. The luminance error due to the degradation of an OLED is less than 1.8% when the proposed correction method is used for a 50% degraded OLED.

Prognostics of slurry pumps based on a moving-average wear degradation index and a general sequential Monte Carlo method

NASA Astrophysics Data System (ADS)

Wang, Dong; Tse, Peter W.

2015-05-01

Slurry pumps are commonly used in oil-sand mining for pumping mixtures of abrasive liquids and solids. These operations cause constant wear of slurry pump impellers, which results in the breakdown of the slurry pumps. This paper develops a prognostic method for estimating remaining useful life of slurry pump impellers. First, a moving-average wear degradation index is proposed to assess the performance degradation of the slurry pump impeller. Secondly, the state space model of the proposed health index is constructed. A general sequential Monte Carlo method is employed to derive the parameters of the state space model. The remaining useful life of the slurry pump impeller is estimated by extrapolating the established state space model to a specified alert threshold. Data collected from an industrial oil sand pump were used to validate the developed method. The results show that the accuracy of the developed method improves as more data become available.

Single underwater image enhancement based on color cast removal and visibility restoration

NASA Astrophysics Data System (ADS)

Li, Chongyi; Guo, Jichang; Wang, Bo; Cong, Runmin; Zhang, Yan; Wang, Jian

2016-05-01

Images taken under underwater condition usually have color cast and serious loss of contrast and visibility. Degraded underwater images are inconvenient for observation and analysis. In order to address these problems, an underwater image-enhancement method is proposed. A simple yet effective underwater image color cast removal algorithm is first presented based on the optimization theory. Then, based on the minimum information loss principle and inherent relationship of medium transmission maps of three color channels in an underwater image, an effective visibility restoration algorithm is proposed to recover visibility, contrast, and natural appearance of degraded underwater images. To evaluate the performance of the proposed method, qualitative comparison, quantitative comparison, and color accuracy test are conducted. Experimental results demonstrate that the proposed method can effectively remove color cast, improve contrast and visibility, and recover natural appearance of degraded underwater images. Additionally, the proposed method is comparable to and even better than several state-of-the-art methods.

Monitoring in Situ Anaerobic Alkylbenzene Biodegradation Based on Mass Spectrometric Detection of Unique Metabolites or Real-Time PCR Detection of a Catabolic Gene

NASA Astrophysics Data System (ADS)

Beller, H. R.; Kane, S. R.

2002-12-01

Monitored natural attenuation (MNA) can be a cost-effective and viable approach for remediation of hydrocarbon-contaminated groundwater. However, regulatory acceptance of the approach is often contingent on monitoring that can convincingly demonstrate the role of microbial degradation. Recent advances in anaerobic hydrocarbon biochemistry, analytical chemistry, and molecular biology have fostered the development of powerful new techniques that can be applied to MNA of BTEX (benzene, toluene, ethylbenzene, and xylenes). Here we report two independent methods that have been developed to monitor in situ, anaerobic biodegradation of toluene and xylenes. A method has been developed for rapid, sensitive, and highly selective detection of distinctive indicators of anaerobic alkylbenzene metabolism. The target metabolites, benzylsuccinic acid (BS) and methylbenzylsuccinic acid (MeBS) isomers, have no known sources other than anaerobic toluene or xylene degradation; thus, their mere presence in groundwater provides definitive evidence of in situ metabolism. The method, which involves small sample size (<1 mL) and no extraction/concentration steps, relies on isotope dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) with selected reaction monitoring. Detection limits for benzylsuccinates were determined to be ca. 0.3 μg/L and accuracy and precision were favorable in a groundwater matrix. The LC/MS/MS method was used to characterize geographic and temporal distributions of benzylsuccinates in an anaerobic, hydrocarbon-contaminated aquifer. BS was never detected and MeBS isomers were detected in the three wells with the highest concentrations of BTEX; MeBS concentrations ranged from <0.3 to 205 μg/L. A strong linear correlation was found between concentrations of total MeBS isomers and their parent compounds, xylenes. A monitoring method based on real-time Polymerase Chain Reaction (PCR) analysis has been developed to specifically quantify populations of anaerobic methylbenzene-degrading bacteria in aquifer sediment. The method targets a catabolic gene (bssA) associated with the first step of anaerobic toluene and xylene degradation. The method proved to be sensitive (detection limit ca. 5 gene copies) and had a linear range of > 7 orders of magnitude. In microcosm experiments involving toluene degradation under denitrifying conditions, population trends were generally consistent with observed toluene degradation activity. In the microcosms with the most rapid toluene degradation, numbers of bssA copies increased 100- to 1000-fold over the first four days of incubation, during which time most of the toluene had been consumed. These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons.

Combination of different methods to assess the fate of lignin in decomposing needle and leave litter

NASA Astrophysics Data System (ADS)

Klotzbücher, Thimo; Filley, Timothy; Kaiser, Klaus; Kalbitz, Karsten

2010-05-01

Lignin is a major component of plant litter. However, its fate during litter decay is still poorly understood. One reason is the difficult analysis. Commonly used methods utilize different methodological approaches and focus on different aspects, e.g., content of lignin and/or of lignin-derived phenols and the degree of oxidation. The comparability and feasibility of the methods has not been tested so far. Our aims were: (1) to compare different methods with respect to track lignin degradation during plant litter decay and (2) to evaluate possible advantages of combining the different results. We assessed lignin degradation in decaying litter by 13C-TMAH thermochemolysis and CuO oxidation (each combined with GC/MS) and by determination of acid-detergent lignin (ADL) combined with near infrared spectroscopy. Furthermore, water-extractable organic matter produced during litter decay was examined for indicators of lignin-derived compounds by UV absorbance at 280 nm, fluorescence spectroscopy, and 13C-TMAH GC/MS. The study included litter samples from 5 different tree species (acer, ash, beech, pine, spruce), exposed in litterbags to degradation in a spruce stand for 27 months. First results suggested stronger lignin degradation in coniferous than in deciduous litter. This was indicated by complementary results from various methods: Conifer litter showed a more pronounced decrease in ADL content and a stronger increase in oxidation degree of side chains (Ac/Al ratios of CuO oxidation and 13C-TMAH products). Furthermore water extracted organic matter from needles showed a higher aromaticity and molecule complexity. Thus properties of water extractable organic matter seemed to reflect the extents of lignin degradation in solid litter samples. Contents of lignin-derived phenols determined with the CuO method (VSC content) hardly changed during decay of needles and leaves. These results thus not matched the trends found with the ADL method. Our results suggested that water-soluble phenolic acids that are included in the CuO oxidation products, accumulated during decay of litter with less stable lignin and then contributed to VSC contents and to the pool of water- extractable organic matter. By combining results from different methods we gained a better understanding about the differences in lignin degradation between the litter species.

Methods of Analysis by the U.S. Geological Survey Organic Geochemistry Research Group?Determination of acetamide herbicides and their degradation products in water using online solid-phase extraction and liquid chromatography/mass spectrometry

USGS Publications Warehouse

Lee, E.A.; Strahan, A.P.

2003-01-01

An analytical method for the determination of 6 acetamide herbicides (acetochlor, alachlor, dimethenamid, flufenacet, metolachlor, and propachlor) and 16 of their degradation products in natural water samples using solid-phase extraction and liquid chromatography/mass spectrometry is described in this report. Special consideration was given during the development of the method to prevent the formation of degradation products during the analysis. Filtered water samples were analyzed using octadecylsilane as the solid-phase extraction media on online automated equipment followed by liquid chromatography/mass spectrometry. The method uses only 10 milliliters of sample per injection. Three different water-sample matrices, a reagent-water, a ground-water, and a surface-water sample spiked at 0.10 and 1.0 microgram per liter, were analyzed to determine method performance. Method detection limits ranged from 0.004 to 0.051 microgram per liter for the parent acetamide herbicides and their degradation products. Mean recoveries for the acetamide compounds in the ground- and surface-water samples ranged from 62.3 to 117.4 percent. The secondary amide of acetochlor/metolachlor ethanesulfonic acid (ESA) was recovered at an average rate of 43.5 percent. The mean recoveries for propachlor and propachlor oxanilic acid (OXA) were next lowest, ranging from 62.3 to 95.5 percent. Mean recoveries from reagent-water samples ranged from 90.3 to 118.3 percent for all compounds. Overall the mean of the mean recoveries of all compounds in the three matrices spiked at 0.10 and 1.0 microgram per liter ranged from 89.9 to 100.7 percent, including the secondary amide of acetochlor/metolachlor ESA and the propachlor compounds. The acetamide herbicides and their degradation products are reported in concentrations ranging from 0.05 to 2.0 micrograms per liter. The upper concentration limit is 2.0 micrograms per liter for all compounds without dilution. With the exception of the secondary amide of acetochlor/metolachlor ESA, good precision and accuracy for the chloroacetanalide herbicides and their degradation compounds were demonstrated for the method in buffered reagent water, ground water, and surface water. The extraction method as used did not optimize the recovery of the secondary amide of acetochlor/metolachlor ESA.

Difficulties in Interpreting Ballast Degradation Level Estimates from Synthetic Ground-Penetrating Radar Data

NASA Astrophysics Data System (ADS)

Scanlan, K. M.; Hendry, M. T.; Martin, C. D.; Schmitt, D. R.

2016-12-01

As fine-grained particles accumulate within railway ballast, it becomes more susceptible to differential deformations, which leads to the loss of proper track alignment and an increased risk for car derailment. Methods for estimating the ballast degradation level from low-frequency (<1 GHz) ground-penetrating radar (GPR) measurements exist, but their applicability in a wide range of track foundation conditions has yet to be evaluated. This analysis, based on simulated GPR data, evaluates the sensitivity of these methods to changing ballast moisture contents, ballast thicknesses and subballast material types. The results highlight that small changes to the track foundation, indepedent of the concentration of degraded ballast, significantly alter the attenuation and reflectivity characteristics of the simualted GPR measurements. As such, ballast degraded to a certain level will manifest with different attenuation and reflectivity characteristics, limiting the ability to accurately and reliably detect these changes using GPR. Radar propagation velocities within the ballast are less influenced by changes in ballast depth and subballast material type. However, ambiguous propagation velocities are observed for certain ballast moisture contents and degradation levels; furthermore, velocities can only be calculated when the ballast thickness is known. These results suggest that while under certain circumstances, with additional information on the track foundation conditons, ballast degradation levels can be estimated from GPR data; in general, the complicated nature of GPR signals makes the quantificaion of ballast degradation levels difficult.

Isolation and Characterization of Oil-Degrading Bacteria from One of South Sumatera’s Oilfield

NASA Astrophysics Data System (ADS)

Purwasena, I. A.; Astuti, D. I.; Fatmawati, R.; Afinanisa, Q.

2018-01-01

Microbial Enhanced Oil Recovery (MEOR) is a tertiary oil recovery method that utilizes microbes to enhance oil production. This research was focused on the isolation and characterization of indigenous bacteria from a South Sumatra’s oilfield which were able to degrade heavy crude oil and decrease oil viscosity. The total of 33 colonies were successfully isolated based on sequential isolation method and screened based on oil degradation activity and SARA analysis. Isolate G3, G7, and N6 were choosen as the best candidate as they were able to reduce oil viscosity up to 22,67%; 23,14%; and 24,36% respectively. Based on 16S rRNA analysis, isolate G3 which was able to degrade aromatic fraction (38,27%) and resin (29,26%) was identified as Pseudoxhantomonas taiwanensis. Isolate G7 which degraded aromatic fraction (61,14%) was identified as Brevibacillus agri while N6 which degraded asphaltene fraction (51.76%) was identified as Bacillus subtilis. In addition, the change in nalkana fraction (C11 - C28) abundance relative to phytan showed that all of the bacterial isolates were able to change those fractions of crude oil. This study showed that three bacterial species isolated from South Sumatran Oilfield were able to degrade heavier fraction of crude oil and reduce its viscosity. This result suggests that those bacteria are highly potential to be applied for MEOR technology.

Assessment of potential soil degradation on agricultural land in the czech republic.

PubMed

Šarapatka, Bořivoj; Bednář, Marek

2015-01-01

Many attempts have been made worldwide to develop methods to identify the areas most threatened by soil degradation. Some soils in afflicted areas may be irreversibly degraded and thus have very little resilience (the ability to restore themselves). For the purpose of assessing the current state of soil degradation in the Czech Republic (CZ) we have developed an overall indicator of land vulnerability to the threat of soil degradation on the basis of individual factors that contribute to soil degradation and are monitored on a long-term basis in various research worksites in the CZ. Individual degradation factors were divided into two groups: chemical and physical degradation. On the basis of principal component analysis, individual degradation factors were assigned a specific weight of influence. With the use of a GIS, the input factors of degradation were combined to create maps of chemical and physical soil degradation, and consequently a map of overall degradation-threatened soils for the CZ, along with a map of areas differentiated according to the prevailing type of degradation. Results showed that, at present, the most important degradation factor in the CZ is water erosion, followed by loss of organic matter. Statistical analysis showed that approximately 51% of agricultural land is moderately threatened in the CZ. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Survey of microbial degradation of asphalts with notes on relationship to nuclear waste management

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

ZoBell, C.E.; Molecke, M.A.

1978-12-01

A survey has been made of the microbial degradation of asphalts. Topics covered include chemical and physical properties of asphalts, their chemical stability, methods of demonstrating their microbial degradation, and environmental extremes for microbial activity based on existing literature. Specific concerns for the use of asphalt in nuclear waste management, plus potential effects and consequences thereof are discussed. 82 references.

Photocatalytic degradation of diethyl phthalate using TiO{sub 2} nanoparticles

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

Singla, Pooja, E-mail: pooja.singla@thapar.edu; Pandey, O. P., E-mail: pooja.singla@thapar.edu; Singh, K., E-mail: pooja.singla@thapar.edu

2014-04-24

TiO{sub 2} nanoparticles predominantly in rutile phase are synthesized by ultrasonication assisted sol-gel method. TiO{sub 2} powder is characterized using X-ray powder diffraction and UV-vis diffuse reflectance. TiO{sub 2} is used as catalyst in photocatalytic degradation of Diethyl Phthalate. TiO{sub 2} exhibits good photocatalytic activity for the degradation of diethyl phthalate.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

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

Thompson, David N.; Apel, William A.; Thompson, Vicki S.

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

2015-06-02

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2013-10-15

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Apel, William A [Jackson, WY; Thompson, Vicki S [Idaho Falls, ID; Reed, David W [Idaho Falls, ID; Lacey, Jeffrey A [Idaho Falls, ID; Henriksen, Emily D [Idaho Falls, ID

2012-06-19

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

2013-04-23

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

2010-12-28

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

2013-07-30

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

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

Thompson, David N; Apel, William A; Thompson, Vicki S

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Methods and compositions for simultaneous saccharification and fermentation

DOEpatents

Ingram, Lonnie O'Neal; Zhou, Shengde

2006-04-11

The invention provides compositions and methods for the synergistic degradation of oligosaccharides by endoglucanases. The invention further provides recombinant host cells containing one or more genes encoding endoglucanses which are capable of the synergistic degradation of oligosaccharides. Preferred host cells of the invention are ethanologenic and capable of carrying out simultaneous saccharification and fermentation resulting in the production of ethanol from complex cellulose substrates.

Development and validation of a novel stability-indicating HPLC method for the quantitative determination of eleven related substances in ezetimibe drug substance and drug product.

PubMed

Luo, Zhiqiang; Deng, Zhongqing; Liu, Yang; Wang, Guopeng; Yang, Wenning; Hou, Chengbo; Tang, Minming; Yang, Ruirui; Zhou, Huaming

2015-07-01

Ezetimibe is a novel lipid-lowering agent that inhibits intestinal absorption of dietary and biliary cholesterol. In the present work, a simple, sensitive and reproducible gradient reverse phase high performance liquid chromatographic (RP-HPLC) method for separation and determination of the related substances of ezetimibe was developed and validated. Eleven potential process-related impurities (starting materials, (3S,4S,3'S)-isomer, degradants and byproducts) were identified in the crude samples. Tentative structures for all the impurities were assigned primarily based on comparison of their retention time and mass spectrometric data with that of available standards and references. This method can be applied to routine analysis in quality control of both bulk drugs and commercial tablets. Separation of all these compounds was performed on a Phenomenex Luna Phenyl-Hexyl (100mm×4.6mm, 5μm) analytical column. The mobile phase-A consists of acetonitrile-water (pH adjusted to 4.0 with phosphoric acid)-methanol at 15:75:10 (v/v/v), and mobile phase-B contains acetonitrile. The eluted compounds were monitored at 210nm. Ezetimibe was subjected to hydrolytic, acid, base, oxidative, photolytic and thermal stress conditions as per ICH serves to generate degradation products that can be used as a worst case to assess the analytical method performance. The drug showed extensive degradation in thermal, acid, oxidative, base and hydrolytic stress conditions, while it was stable to photolytic degradation conditions. The main degradation product formed under thermal, acid, oxidative, base and hydrolytic stress conditions corresponding to (2R,3R,6S)-N, 6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-oxane-3-carboxamide (Ezetimibe tetrahydropyran impurity) was characterized by LC-MS/MS analysis. The degradation products were well resolved from the main peak and its impurities, thus proved the stability-indicating power of the method. The developed method was validated as per international conference on harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness. Copyright © 2015 Elsevier B.V. All rights reserved.

[On-Orbit Multispectral Sensor Characterization Based on Spectral Tarps].

PubMed

Li, Xin; Zhang, Li-ming; Chen, Hong-yao; Xu, Wei-wei

2016-03-01

The multispectral remote sensing technology has been a primary means in the research of biomass monitoring, climate change, disaster prediction and etc. The spectral sensitivity is essential in the quantitative analysis of remote sensing data. When the sensor is running in the space, it will be influenced by cosmic radiation, severe change of temperature, chemical molecular contamination, cosmic dust and etc. As a result, the spectral sensitivity will degrade by time, which has great implication on the accuracy and consistency of the physical measurements. This paper presents a characterization method of the degradation based on man-made spectral targets. Firstly, a degradation model is established in the paper. Then, combined with equivalent reflectance of spectral targets measured and inverted from image, the degradation characterization can be achieved. The simulation and on orbit experiment results showed that, using the proposed method, the change of center wavelength and band width can be monotored. The method proposed in the paper has great significance for improving the accuracy of long time series remote sensing data product and comprehensive utilization level of multi sensor data products.

Biocarrier composition for and method of degrading pollutants

DOEpatents

Fliermans, C.B.

1994-01-01

The present invention relates to biocarrier compositions that attract and bond pollutant-degrading antigens that will degrade the pollutants. Biocarriers are known generally as a variety of inert or semi-inert compounds or structures having the ability to sequester (attract), hold and biomagnify (enhance) specific microorganisms within their structure. Glass or polystyrene beads are the most well known biocarriers. The biocarrier, which is preferably in the form of glass microspheres, is coated with an antibody or group of antibodies that attract and react specifically with certain pollutant-degrading antigens. The antibody, once bonded to the biocarrier, is used by the composition to attract and bond those pollutant-degrading antigens. Each antibody is specific for an antigen that is specific for a given pollutant. The resulting composition is subsequently exposed to an environment contaminated with pollutants for degradation. In the preferred use, the degrading composition is formed and then injected directly into or near a plume or source of contamination.

Bacteria-mediated bisphenol A degradation.

PubMed

Zhang, Weiwei; Yin, Kun; Chen, Lingxin

2013-07-01

Bisphenol A (BPA) is an important monomer in the manufacture of polycarbonate plastics, food cans, and other daily used chemicals. Daily and worldwide usage of BPA and BPA-contained products led to its ubiquitous distribution in water, sediment/soil, and atmosphere. Moreover, BPA has been identified as an environmental endocrine disruptor for its estrogenic and genotoxic activity. Thus, BPA contamination in the environment is an increasingly worldwide concern, and methods to efficiently remove BPA from the environment are urgently recommended. Although many factors affect the fate of BPA in the environment, BPA degradation is mainly depended on the metabolism of bacteria. Many BPA-degrading bacteria have been identified from water, sediment/soil, and wastewater treatment plants. Metabolic pathways of BPA degradation in specific bacterial strains were proposed, based on the metabolic intermediates detected during the degradation process. In this review, the BPA-degrading bacteria were summarized, and the (proposed) BPA degradation pathway mediated by bacteria were referred.

Methods of Analysis by the U.S. Geological Survey Organic Geochemistry Research Group-Update and Additions to the Determination of Chloroacetanilide Herbicide Degradation Compounds in Water Using High-Performance Liquid Chromatography/Mass Spectrometry

USGS Publications Warehouse

Lee, E.A.; Kish, J.L.; Zimmerman, L.R.; Thurman, E.

2001-01-01

An analytical method using high-performance liquid chromatography/mass spectrometry (HPLC/MS) was developed by the U.S. Geological Survey in 1999 for the analysis of selected chloroacetanilide herbicide degradation compounds in water. These compounds were acetochlor ethane sulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. The HPLC/MS method was updated in 2000, and the method detection limits were modified accordingly. Four other degradation compounds also were added to the list of compounds that can be analyzed using HPLC/MS; these compounds were dimethenamid ESA, dimethenamid OXA, flufenacet ESA, and flufenacet OXA. Except for flufenacet OXA, good precision and accuracy were demonstrated for the updated HPLC/MS method in buffered reagent water, surface water, and ground water. The mean HPLC/MS recoveries of the degradation compounds from water samples spiked at 0.20 and 1.0 ?g/L (microgram per liter) ranged from 75 to 114 percent, with relative standard deviations of 15.8 percent or less for all compounds except flufenacet OXA, which had relative standard deviations ranging from 11.3 to 48.9 percent. Method detection levels (MDL's) using the updated HPLC/MS method varied from 0.009 to 0.045 ?g/L, with the flufenacet OXA MDL at 0.072 ?g/L. The updated HPLC/MS method is valuable for acquiring information about the fate and transport of the parent chloroacetanilide herbicides in water.

Short-stack modeling of degradation in solid oxide fuel cells. Part I. Contact degradation

NASA Astrophysics Data System (ADS)

Gazzarri, J. I.; Kesler, O.

As the first part of a two paper series, we present a two-dimensional impedance model of a working solid oxide fuel cell (SOFC) to study the effect of contact degradation on the impedance spectrum for the purpose of non-invasive diagnosis. The two dimensional modeled geometry includes the ribbed interconnect, and is adequate to represent co- and counter-flow configurations. Simulated degradation modes include: cathode delamination, interconnect oxidation, and interconnect-cathode detachment. The simulations show differences in the way each degradation mode impacts the impedance spectrum shape, suggesting that identification is possible. In Part II, we present a sensitivity analysis of the results to input parameter variability that reveals strengths and limitations of the method, as well as describing possible interactions between input parameters and concurrent degradation modes.

The use of gamma radiation for removal of pesticides from waste water

NASA Astrophysics Data System (ADS)

Dessouki, A. M.; Aly, H. F.; Sokker, H. H.

1999-01-01

In the present study, a try was made to explain the degradation kinetics due to irradiation of aqueous solutions of some active ingredient pesticides. These pesticides are as follows: one type of triazine herbicide Ametryn and one type of carbamate insecticide (Applaud) and two types of chlorinated organic pesticides, Aldrin and Chlorothalonil. Factors affecting the radiolysis of these pesticides such as the pesticide concentration, irradiation dose, dose rate and pH of the solutions were studied. Ametryn, Applaud, Chlorothalonil and Aldrin insecticides were degraded by γ-radiation and in the case of the chlorinated pesticides hydrochloric acid was detected. The pH effect has proved to vary according to the type of the pesticide and little degradation was observed in allkaline medium, while more degradation of the pesticides in the neutral medium was observed depending on the type of pesticide and on its chemical structure. However, the degradation in the acid medium was even higher. A drop in pH was observed and may be attributed to the degradation of the pesticide molecules to lower molecular weight compounds, such as organic acids. A combined treatment of gamma irradiation and conventional methods was applied and the effect of different additives such as nitrogen and oxygen showed that nitrogen did not enhance the degradation, while an enhancement in the degradation process was observed when oxygen was added. Experiments on the adsorption of these pesticides on certain polymeric materials and on Granular Activated Carbon (GAC), showed that GAC has the highest adsorption capacity. It may be concluded that the radiation degradation followed by adsorption of the toxic pesticide pollutants and their removal from wastewater down to concentrations not exceeding the maximum permissible concentration (MPC), according to international standards, proved to be better than the conventional methods of purification.

Two different spectrophotometric determinations of potential anticancer drug and its toxic metabolite

NASA Astrophysics Data System (ADS)

Farid, Nehal F.; Abdelwahab, Nada S.

2015-06-01

Flutamide is a hormone therapy used for men with advanced prostate cancer. Flutamide is highly susceptible to hydrolysis with the production of 3-(trifluoromethyl)aniline, which is reported to be one of its toxic metabolites, impurities and related substances according to BP and USP. Flutamide was found to be stable when exposed to oxidation by 30% hydrogen peroxide and direct sunlight for up to 4 h. Two accurate and sensitive spectrophotometric methods were used for determination of flutamide in bulk and in pharmaceutical formulations. Method (I) is the area under curve (AUC) spectrophotometric method that depends on measuring the AUC in the wavelength ranges of 275-305 nm and 350-380 nm and using Cramer's rule. The linearity range was found to be 1-35 μg/mL and 0.5-16 μg/mL for the drug and the degradate, respectively. In method (II), combination of the isoabsorptive and dual wavelength spectrophotometric methods was used for resolving the binary mixture. The absorbance at 249.2 nm (λiso) was used for determination of total mixture concentration, while the difference in absorbance between 232 nm and 341.2 nm was used for measuring the drug concentration. By subtraction, the degradate concentration was obtained. Beer's law was obeyed in the range of 2-35 μg/mL and 0.5-20 μg/mL for the drug and its degradate, respectively. The two methods were validated according to USP guidelines and were applied for determination of the drug in its pharmaceutical dosage form. Moreover AUC method was used for the kinetic study of the hydrolytic degradation of flutamide. The kinetic degradation of flutamide was found to follow pseudo-first order kinetics and is pH and temperature dependent. Activation energy, kinetic rate constants and t1/2 at different temperatures and pH values were calculated.

Application of normal fluorescence and stability-indicating derivative synchronous fluorescence spectroscopy for the determination of gliquidone in presence of its fluorescent alkaline degradation product

NASA Astrophysics Data System (ADS)

El-ghobashy, Mohamed R.; Yehia, Ali M.; Helmy, Aya H.; Youssef, Nadia F.

2018-01-01

Simple, smart and sensitive normal fluorescence and stability-indicating derivative synchronous spectrofluorimetric methods have been developed and validated for the determination of gliquidone in the drug substance and drug product. Normal spectrofluorimetric method of gliquidone was established in methanol at λ excitation 225 nm and λ emission 400 nm in concentration range 0.2-3 μg/ml with LOD equal 0.028. The fluorescence quantum yield of gliquidone was calculated using quinine sulfate as a reference and found to be 0.542. Stability-indicating first and third derivative synchronous fluorescence spectroscopy were successfully utilized to overcome the overlapped spectra in normal fluorescence of gliquidone and its alkaline degradation product. Derivative synchronous methods are based on using the synchronous fluorescence of gliquidone and its degradation product in methanol at Δ λ50 nm. Peak amplitude in the first derivative of synchronous fluorescence spectra was measured at 309 nm where degradation product showed zero-crossing without interference. The peak amplitudes in the third derivative of synchronous fluorescence spectra, peak to trough were measured at 316,329 nm where degradation product showed zero-crossing. The different experimental parameters affecting the normal and synchronous fluorescence intensity of gliquidone were studied and optimized. Moreover, the cited methods have been validated as per ICH guidelines. The peak amplitude-concentration plots of the derivative synchronous fluorescence were linear over the concentration range 0.05-2 μg/ml for gliquidone. Limits of detection were 0.020 and 0.022 in first and third derivative synchronous spectra, respectively. The adopted methods were successfully applied to commercial tablets and the results demonstrated that the derivative synchronous fluorescence spectroscopy is a powerful stability-indicating method, suitable for routine use with a short analysis time. Statistical comparison between the results obtained by normal fluorescence and derivative synchronous methods and the official one using student's t-test and F-ratio showed no significant difference regarding accuracy and precision.

Two different spectrophotometric determinations of potential anticancer drug and its toxic metabolite.

PubMed

Farid, Nehal F; Abdelwahab, Nada S

2015-06-15

Flutamide is a hormone therapy used for men with advanced prostate cancer. Flutamide is highly susceptible to hydrolysis with the production of 3-(trifluoromethyl)aniline, which is reported to be one of its toxic metabolites, impurities and related substances according to BP and USP. Flutamide was found to be stable when exposed to oxidation by 30% hydrogen peroxide and direct sunlight for up to 4h. Two accurate and sensitive spectrophotometric methods were used for determination of flutamide in bulk and in pharmaceutical formulations. Method (I) is the area under curve (AUC) spectrophotometric method that depends on measuring the AUC in the wavelength ranges of 275-305 nm and 350-380nm and using Cramer's rule. The linearity range was found to be 1-35 μg/mL and 0.5-16 μg/mL for the drug and the degradate, respectively. In method (II), combination of the isoabsorptive and dual wavelength spectrophotometric methods was used for resolving the binary mixture. The absorbance at 249.2 nm (λiso) was used for determination of total mixture concentration, while the difference in absorbance between 232 nm and 341.2 nm was used for measuring the drug concentration. By subtraction, the degradate concentration was obtained. Beer's law was obeyed in the range of 2-35 μg/mL and 0.5-20 μg/mL for the drug and its degradate, respectively. The two methods were validated according to USP guidelines and were applied for determination of the drug in its pharmaceutical dosage form. Moreover AUC method was used for the kinetic study of the hydrolytic degradation of flutamide. The kinetic degradation of flutamide was found to follow pseudo-first order kinetics and is pH and temperature dependent. Activation energy, kinetic rate constants and t1/2 at different temperatures and pH values were calculated. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of a validated stability-indicating densitometric thin-layer chromatographic method to stress degradation studies on moxifloxacin.

PubMed

Motwani, Sanjay K; Khar, Roop K; Ahmad, Farhan J; Chopra, Shruti; Kohli, K; Talegaonkar, S

2007-01-16

A simple, sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for densitometric determination of moxifloxacin both as a bulk drug and from pharmaceutical formulation was developed and validated as per the International Conference on Harmonization (ICH) guidelines. The method employed TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of n-propanol-ethanol-6M ammonia solution (4:1:2, v/v/v). Densitometric analysis of moxifloxacin was carried out in the absorbance mode at 298 nm. Compact spots for moxifloxacin were found at R(f) value of 0.58+/-0.02. The linear regression analysis data for the calibration plots showed good linear relationship with r=0.9925 in the working concentration range of 100-800 ng spot(-1). The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were 3.90 and 11.83 ng spot(-1), respectively. Drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment and photodegradation. All the peaks of degradation products were well resolved from the standard drug with significantly different R(f) values. Statistical analysis proves that the developed HPTLC method is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of the acidic and alkaline degradation processes at different temperatures. Arrhenius plot was constructed and apparent pseudo-first-order rate constant, half-life and activation energy were calculated. In addition the pH-rate profile for degradation of moxifloxacin in constant ionic strength buffer solutions within the pH range 1.2-10.8 was studied.

Prediction of troponin-T degradation using color image texture features in 10d aged beef longissimus steaks.

PubMed

Sun, X; Chen, K J; Berg, E P; Newman, D J; Schwartz, C A; Keller, W L; Maddock Carlin, K R

2014-02-01

The objective was to use digital color image texture features to predict troponin-T degradation in beef. Image texture features, including 88 gray level co-occurrence texture features, 81 two-dimension fast Fourier transformation texture features, and 48 Gabor wavelet filter texture features, were extracted from color images of beef strip steaks (longissimus dorsi, n = 102) aged for 10d obtained using a digital camera and additional lighting. Steaks were designated degraded or not-degraded based on troponin-T degradation determined on d 3 and d 10 postmortem by immunoblotting. Statistical analysis (STEPWISE regression model) and artificial neural network (support vector machine model, SVM) methods were designed to classify protein degradation. The d 3 and d 10 STEPWISE models were 94% and 86% accurate, respectively, while the d 3 and d 10 SVM models were 63% and 71%, respectively, in predicting protein degradation in aged meat. STEPWISE and SVM models based on image texture features show potential to predict troponin-T degradation in meat. © 2013.

FPGA implementation of predictive degradation model for engine oil lifetime

NASA Astrophysics Data System (ADS)

Idros, M. F. M.; Razak, A. H. A.; Junid, S. A. M. Al; Suliman, S. I.; Halim, A. K.

2018-03-01

This paper presents the implementation of linear regression model for degradation prediction on Register Transfer Logic (RTL) using QuartusII. A stationary model had been identified in the degradation trend for the engine oil in a vehicle in time series method. As for RTL implementation, the degradation model is written in Verilog HDL and the data input are taken at a certain time. Clock divider had been designed to support the timing sequence of input data. At every five data, a regression analysis is adapted for slope variation determination and prediction calculation. Here, only the negative value are taken as the consideration for the prediction purposes for less number of logic gate. Least Square Method is adapted to get the best linear model based on the mean values of time series data. The coded algorithm has been implemented on FPGA for validation purposes. The result shows the prediction time to change the engine oil.

Stress and Damage in Polymer Matrix Composite Materials Due to Material Degradation at High Temperatures

NASA Technical Reports Server (NTRS)

McManus, Hugh L.; Chamis, Christos C.

1996-01-01

This report describes analytical methods for calculating stresses and damage caused by degradation of the matrix constituent in polymer matrix composite materials. Laminate geometry, material properties, and matrix degradation states are specified as functions of position and time. Matrix shrinkage and property changes are modeled as functions of the degradation states. The model is incorporated into an existing composite mechanics computer code. Stresses, strains, and deformations at the laminate, ply, and micro levels are calculated, and from these calculations it is determined if there is failure of any kind. The rationale for the model (based on published experimental work) is presented, its integration into the laminate analysis code is outlined, and example results are given, with comparisons to existing material and structural data. The mechanisms behind the changes in properties and in surface cracking during long-term aging of polyimide matrix composites are clarified. High-temperature-material test methods are also evaluated.