Conformational analysis of a modified RGD adhesive sequence.

PubMed

Triguero, Jordi; Zanuy, David; Alemán, Carlos

2017-02-01

The conformational preferences of the Arg-GlE-Asp sequence, where GlE is an engineered amino acid bearing a 3,4-ethylenedioxythiophene (EDOT) ring as side group, have been determined combining density functional theory calculations with a well-established conformational search strategy. Although the Arg-GlE-Asp sequence was designed to prepare a conducting polymer-peptide conjugate with excellent electrochemical and bioadhesive properties, the behavior of such hybrid material as adhesive biointerface is improvable. Results obtained in this work prove that the bioactive characteristics of the parent Arg-Gly-Asp sequence become unstable in Arg-GlE-Asp because of both the steric hindrance caused by the EDOT side group and the repulsive interactions between the oxygen atoms belonging to the backbone amide groups and the EDOT side group. Detailed analyses of the conformational preferences identified in this work have been used to re-engineer the Arg-GlE-Asp sequence for the future development of a new electroactive conjugate with improved bioadhesive properties. The preparation of this new conjugate is in progress. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

PubMed Central

Dunstan, Rhys A.; Heinz, Eva; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Purcell, Anthony W.; Evans, Timothy J.; Praszkier, Judyta; Robins-Browne, Roy M.; Strugnell, Richard A.; Korotkov, Konstantin V.; Lithgow, Trevor

2013-01-01

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS. PMID:23326233

Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis.

PubMed

Park, Jong Nam; Park, Sang Kyun; Cho, Min Kyoung; Park, Mi-Kyung; Kang, Shin Ae; Kim, Dong-Hee; Yu, Hak Sun

2012-12-21

In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. Copyright © 2012 Elsevier B.V. All rights reserved.

Different HIV-1 env frames: gp120 and ASP (antisense protein) biosynthesis, and theirs co-variation tropic amino acid signatures in X4- and R5-viruses.

PubMed

Dimonte, Salvatore

2017-01-01

Antisense protein (ASP) is the new actor of viral life of Human Immunodeficiency Virus type 1 (HIV-1) although proposed above 20 years ago. The asp ORF is into complementary strand of the gp120/gp41 junction of env gene. The ASP biological role remains little known. Knowing the Env markers of viral tropism, a dataset of sequences (660 strains) was used to analyze the hypothetical ASP involvement in CCR5 (R5) and/or CXCR4 (X4) co-receptor interaction. Preliminarily, prevalence of ASP and gp120 V3 mutations was performed; following association among mutations were elaborate. The classical V3 tropic-signatures were confirmed, and 36 R5- and 22 X4-tropic ASP mutations were found. Moreover, by analyzing the ASP sequences, 36 out of 179 amino acid positions significantly associated with different co-receptor usage were found. Several statistically significant associations between gp120 V3 and ASP mutations were observed. The dendrogram showed the existence of a cluster associated with R5-usage and a large cluster associated with X4-usage. These results show that gp120 V3 and specific amino acid changes in ASP are associated together with CXCR4 and/or CCR5-usage. These findings implement previous observations on unclear ASP functions. J. Med. Virol. 89:112-122, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identification of angiotensin I in several vertebrate species: its structural and functional evolution.

PubMed

Takei, Yoshio; Joss, Jean M P; Kloas, Werner; Rankin, J Cliff

2004-02-01

In order to delineate further the molecular evolution of the renin-angiotensin system in vertebrates, angiotensin I (ANG I) has been isolated after incubation of plasma and kidney extracts of emu (Dromiceus novaehollandiae), axolotl (Ambystoma mexicanum), and sea lamprey (Petromyzon marinus). The identified sequences were [Asp1, Val5, Asn9] ANG I in emu, [Asp1, Val5, His9] ANG I in axolotl, and [Asn1, Val5, Thr9] ANG I in sea lamprey. These results confirmed the previous findings that tetrapods have Asp and fishes including cyclostomes have Asn at the N-terminus, and that the amino acid residue at position 9 of ANG I was highly variable but, those at other positions were well conserved among different species. Since Asp and Asn are convertible during incubation, angiotensinogen sequences were searched in the genome and/or EST database to determine the N-terminal amino acid residue from the gene. The screening detected 12 tetrapod (10 mammalian, one avian, and one amphibian) and seven teleostean angiotensinogen sequences. Among them, all tetrapods have [Asp1] ANG except for Xenopus, and all teleosts have [Asn1] ANG, thereby confirming the above rule. Comparison of the vasopressor activity in the eel revealed that [Asn1] ANG I and II were more potent than [Asp1] peptides, which was opposite to the previous results in mammals and birds, in which [Asp1] ANG I and II were more potent. Collectively, the present results support the general rule that tetrapods have [Asp1] ANG and fishes including cyclostomes have [Asn1] ANG. However, an aquatic anuran (Xenopus) has [Asn1] ANG in its gene despite another aquatic urodele (axolotl) has [Asp1] ANG. From the functional viewpoint, homologous [Asn1] ANG was more potent in fish as is homologous [Asp1] ANG in tetrapods, suggesting that ANG II molecule has undergone co-evolution with its receptor during vertebrate phylogeny.

Tetrapod-type [Asp1] angiotensin is present in a holostean fish, Amia calva.

PubMed

Takei, Y; Itahara, Y; Butler, D G; Watanabe, T X; Oudit, G Y

1998-05-01

The renin-angiotensin system has been identified in various vertebrates, from elasmobranchs to mammals. Tetrapod (amphibians to mammals) angiotensin (ANG) has Asp at the N-terminus, but Asp is replaced by Asn in elasmobranch and teleost fish. ANG I has been isolated from incubates of plasma and kidney extracts of the bowfin Amia calva, a holostean fish, using the eel vasopressor activity as an assay system; its sequence was found to be H-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Asn-Leu-OH after sequence analysis, mass spectrometry, and comparison with the synthetic peptide. This sequence is identical to bullfrog ANG I. [Asn1] ANG I was not detected. Thus the bowfin is the first fish species which contains only [Asp1] ANG I. The bowfin ANG I and II were no more vasopressor than eel peptides in the bowfin, indicating that bowfin ANG II receptors do not distinguish between [Asp1] and [Asn1] peptides. In the rat, bowfin ANG I and rat [Ile5, His9] ANG I have equipressor activities when examined in different animals, but the vasopressor activity of bowfin ANG I decreased following rat ANG I in the same animals, although the activity of rat ANG I was unaffected after bowfin ANG I. The present study directly demonstrates the presence of the renin-angiotensin system in a holostean fish and showed that its ANG II receptors have not yet fully coevolved with the homologous [Asp1] peptide.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies.

PubMed Central

Chow, L P; Liu, S L; Yu, C J; Liao, H K; Tsai, J J; Tang, T K

2000-01-01

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein. PMID:10677362

Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32.

PubMed Central

Slee, E A; Zhu, H; Chow, S C; MacFarlane, M; Nicholson, D W; Cohen, G M

1996-01-01

Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases. PMID:8670109

A ripple-spreading genetic algorithm for the aircraft sequencing problem.

PubMed

Hu, Xiao-Bing; Di Paolo, Ezequiel A

2011-01-01

When genetic algorithms (GAs) are applied to combinatorial problems, permutation representations are usually adopted. As a result, such GAs are often confronted with feasibility and memory-efficiency problems. With the aircraft sequencing problem (ASP) as a study case, this paper reports on a novel binary-representation-based GA scheme for combinatorial problems. Unlike existing GAs for the ASP, which typically use permutation representations based on aircraft landing order, the new GA introduces a novel ripple-spreading model which transforms the original landing-order-based ASP solutions into value-based ones. In the new scheme, arriving aircraft are projected as points into an artificial space. A deterministic method inspired by the natural phenomenon of ripple-spreading on liquid surfaces is developed, which uses a few parameters as input to connect points on this space to form a landing sequence. A traditional GA, free of feasibility and memory-efficiency problems, can then be used to evolve the ripple-spreading related parameters in order to find an optimal sequence. Since the ripple-spreading model is the centerpiece of the new algorithm, it is called the ripple-spreading GA (RSGA). The advantages of the proposed RSGA are illustrated by extensive comparative studies for the case of the ASP.

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.

Structural elucidation of the DFG-Asp in and DFG-Asp out states of TAM kinases and insight into the selectivity of their inhibitors.

PubMed

Messoussi, Abdellah; Peyronnet, Lucile; Feneyrolles, Clémence; Chevé, Gwénaël; Bougrin, Khalid; Yasri, Aziz

2014-10-10

Structural elucidation of the active (DFG-Asp in) and inactive (DFG-Asp out) states of the TAM family of receptor tyrosine kinases is required for future development of TAM inhibitors as drugs. Herein we report a computational study on each of the three TAM members Tyro-3, Axl and Mer. DFG-Asp in and DFG-Asp out homology models of each one were built based on the X-ray structure of c-Met kinase, an enzyme with a closely related sequence. Structural validation and in silico screening enabled identification of critical amino acids for ligand binding within the active site of each DFG-Asp in and DFG-Asp out model. The position and nature of amino acids that differ among Tyro-3, Axl and Mer, and the potential role of these residues in the design of selective TAM ligands, are discussed.

Structure characterization of lipocyclopeptide antibiotics, aspartocins A, B & C, by ESI-MSMS and ESI-nozzle-skimmer-MSMS.

PubMed

Siegel, Marshall M; Kong, Fangming; Feng, Xidong; Carter, Guy T

2009-12-01

Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions. Copyright 2009 John Wiley & Sons, Ltd.

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

PubMed Central

Zaprasis, Adrienne; Bleisteiner, Monika; Kerres, Anne; Hoffmann, Tamara

2014-01-01

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. PMID:25344233

Purification and characterisation of a novel antistaphylococcal peptide (ASP-1) from Bacillus sp. URID 12.1.

PubMed

Chalasani, Ajay Ghosh; Roy, Utpal; Nema, Sushma

2018-01-01

A strong antistaphylococcal peptide (ASP-1) from Bacillus subtilis URID 12.1 strain that is active against cefoxitin- and methicillin-resistant Staphylococcus aureus clinical isolates was purified to homogeneity by solvent extraction, silica gel-based adsorption chromatography and reversed-phase high-performance liquid chromatography. The peptide sequence of ASP-1 as determined by MALDI-TOF/MS and ESI-FTICR-MS was acetylated Phe-Thr-Ala-Val-Dhb-Phe-Ile/Leu. The peptide was further analysed by alkaline hydrolysis, ESI-Q-TOF-MS and an ion mobility assay, which detected the presence of a lactone ring in the intact peptide and a cyclic nature, subsequently revealing the linearised peptide sequence as acPhe-Leu-Phe-Thr-Val-Ala-Dhb. Based on the molecular mass (804.5 Da), peptide sequence and amino acid composition, ASP-1 was identified as a lactone ring-containing peptide similar to TL-119, a poorly studied cyclic depsipeptide. Circular dichroism spectroscopy revealed its predominantly random structure in aqueous solution and its β-sheet conformation in methanol. Minimum inhibitory concentrations (MICs) of the purified peptide against S. aureus and methicillin-resistant S. aureus (MRSA) ranged from 2 µg/mL to 64 µg/mL. At sub-MICs and 1× MIC, ASP-1 showed a strong antibiofilm characteristic. ASP-1 at a concentration of 128 µg/mL did not show haemolytic activity, and no cytotoxicity was observed against hepatic carcinoma and breast carcinoma cell lines at the same concentration. Peptide ASP-1 with anti-MRSA and antibiofilm abilities and non-haemolytic and non-cytotoxic properties has not been reported previously. These findings suggest that it may serve as a lead molecule for developing alternative topical antibacterial agents. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Screening of Pro-Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes.

PubMed

Lee, Hwa Kyoung; Lee, Yujean; Kim, Hyori; Lee, Hye-Eun; Chang, Hyejin; Nam, Ki Tae; Jeong, Dae Hong; Chung, Junho

2017-09-15

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X) 2 DP, AE(X) 3 DP, AE(X) 4 DP, AE(X) 5 DP, and AE(X) 6 DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X) 2 DP, AE(X) 3 DP, and AE(X) 4 DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

Development of allele-specific primer PCR for a swine TLR2 SNP and comparison of the frequency among several pig breeds of Japan and the Czech Republic.

PubMed

Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor

2012-05-01

In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.

FATE OF WATER SOLUBLE AZO DYES IN THE ACTIVATED SLUDGE PROCESS

EPA Science Inventory

The objective of this study was to determine the partitioning of water soluble azo dyes in the activated sludge process (ASP). Azo dyes are of concern because some of the dyes, dye precursors , and/or their degradation products such as aromatic amines (which are also dye precurso...

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee-royalisin-for specific detection of oxidized low-density lipoprotein.

PubMed

Sato, Akira; Unuma, Hiroto; Yamazaki, Yoji; Ebina, Keiichi

2018-06-01

The probes for detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox-LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox-LDL, we investigated the interaction (with ox-LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N-terminus to FITC in the presence of 6-amino-n-caproic acid (AC) linker, (FITC-AC)-royalisin P11, which contains both sequences, Phe-Lys-Asp and Asp-Lys-Tyr, similar to Tyr-Lys-Asp in (FITC)KP6. The (FITC-AC)-royalisin P11 bound with high specificity to ox-LDL in a dose-dependent manner, through the binding to major lipid components in ox-LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC-AC)-shuffled royalisin P11 peptide, in which sequences Phe-Lys-Asp and Asp-Lys-Tyr were modified to Lys-Phe-Asp and Asp-Tyr-Lys, respectively, hardly bound to LDL and ox-LDL. These findings strongly suggest that (FITC-AC)-royalisin P11 may be an effective fluorescent probe for specific detection of ox-LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox-LDL. Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.

An ecological vegetation-activated sludge process (V-ASP) for decentralized wastewater treatment: system development, treatment performance, and mathematical modeling.

PubMed

Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Li, Pu; Zhao, Ke

2016-05-01

An environment-friendly decentralized wastewater treatment process that is comprised of activated sludge process (ASP) and wetland vegetation, named as vegetation-activated sludge process (V-ASP), was developed for decentralized wastewater treatment. The long-term experimental results evidenced that the vegetation sequencing batch reactor (V-SBR) process had consistently stable higher removal efficiencies of organic substances and nutrients from domestic wastewater compared with traditional sequencing batch reactor (SBR). The vegetation allocated into V-SBR system could not only remove nutrients through its vegetation transpiration ratio but also provide great surface area for microorganism activity enhancement. This high vegetation transpiration ratio enhanced nutrients removal effectiveness from wastewater mainly by flux enhancement, oxygen and substrate transportation acceleration, and vegetation respiration stimulation. A mathematical model based on ASM2d was successfully established by involving the specific function of vegetation to simulate system performance. The simulation results on the influence of operational parameters on V-ASP treatment effectiveness demonstrated that V-SBR had a high resistance to seasonal temperature fluctuations and influent loading shocking.

Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

PubMed Central

Bennett, Hugh P. J.; Lowry, Philip J.; McMartin, Colin; Scott, Alexander P.

1974-01-01

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian α-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the β-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An α-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin. PMID:4375978

D-Aspartate drinking solution alleviates pain and cognitive impairment in neuropathic mice.

PubMed

Palazzo, Enza; Luongo, Livio; Guida, Francesca; Marabese, Ida; Romano, Rosaria; Iannotta, Monica; Rossi, Francesca; D'Aniello, Antimo; Stella, Luigi; Marmo, Federica; Usiello, Alessandro; de Bartolomeis, Andrea; Maione, Sabatino; de Novellis, Vito

2016-07-01

D-Aspartate (D-Asp) is a free D-amino acid detected in multiple brain regions and putative precursor of endogenous N-methyl-D-aspartate (NMDA) acting as agonist at NMDA receptors. In this study, we investigated whether D-Asp (20 mM) in drinking solution for 1 month affects pain responses and pain-related emotional, and cognitive behaviour in a model of neuropathic pain induced by the spared nerve injury (SNI) of the sciatic nerve in mice. SNI mice developed mechanical allodynia and motor coordination impairment 30 days after SNI surgery. SNI mice showed cognitive impairment, anxiety and depression-like behaviour, reduced sociability in the three chamber sociability paradigm, increased expression of NR2B subunit of NMDA receptor and Homer 1a in the medial prefrontal cortex (mPFC). The expression of (post synaptic density) PSD-95 and Shank 1was instead unaffected in the mPFC of the SNI mice. Treatment with D-Asp drinking solution, started right after the SNI (day 0), alleviated mechanical allodynia, improved cognition and motor coordination and increased social interaction. D-Asp also restored the levels of extracellular D-Asp, Homer 1a and NR2B subunit of the NMDA receptor to physiological levels and reduced Shank1 and PSD-95 protein levels in the mPFC. Amitriptyline, a tricyclic antidepressant used also to alleviate neuropathic pain in humans, reverted mechanical allodynia and cognitive impairment, and unlike D-Asp, was effective in reducing depression and anxiety-like behaviour in the SNI mice and increased PSD protein level. Altogether these findings demonstrate that D-Asp improves sensorial, motor and cognitive-like symptoms related to chronic pain possibly through glutamate neurotransmission normalization in neuropathic mice.

Bacterial communities and enzymatic activities in the vegetation-activated sludge process (V-ASP) and related advantages by comparison with conventional constructed wetland.

PubMed

Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke; Du, Changhang; Shao, Yunxian

2016-11-01

A new-developed vegetation-activated sludge process (V-ASP) was implemented for decentralized domestic wastewater treatment, and studied in lab-scale and full-scale. The main purpose of this work was the investigation of biomass activities and microbial communities in V-ASP by comparison with conventional constructed wetland (CW), to unveil the causations of its consistently higher pollutants removal efficiencies. Compared with CWs, V-ASP has greater vegetation nitrogen and phosphorus uptake rates, higher biomass and enzymatic activities, and more bacteria community diversity. The microbial community structure was comprehensively analyzed by using high-throughput sequencing. It was observed that Proteobacteria was dominated in both CWs and V-ASPs, while their subdivisions distribution was rather different. V-ASPs contained a higher nitrite-oxidizing bacteria (Nitrospira) abundances that resulted in a consistently better nitrogen removal efficiency. Hence, a long-term experiment of full-scale V-ASP displayed stably excellent capability in resistance of influent loading shocks and seasonal temperature effect. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

PubMed

Faro, C; Ramalho-Santos, M; Vieira, M; Mendes, A; Simões, I; Andrade, R; Veríssimo, P; Lin, X; Tang, J; Pires, E

1999-10-01

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.

[Peptide Ala-Glu-Asp-Gly and interferon gamma: their role in immune response during aging].

PubMed

Lin'kova, N S; Kuznik, B I; Khavinson, V Kh

2012-01-01

The decrease of lymphocyte interferon gamma expression during aging is one of the main mechanisms leading to the immunodeficiency state in the elderly. Cell penetrating geroprotective peptide Ala-Glu-Asp-Gly has the capability to activate the proliferation of lymphocytes in thymus during its aging. The nucleotide sequence which is complementary contacted with peptide Ala-Glu-Asp-Gly was found in promoter region of interferon gamma gene. Thus, the immune protection of this peptide can be explained by its activation of the interferon gamma production in T-cells.

NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics

NASA Astrophysics Data System (ADS)

Volpon, Laurent; Tsan, Pascale; Majer, Zsuzsa; Vass, Elemer; Hollósi, Miklós; Noguéra, Valérie; Lancelin, Jean-Marc; Besson, Françoise

2007-08-01

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven α-amino acids of configuration LDDLLDL and one β-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common β-amino fatty acid 8- L-Asn1- D-Tyr2- D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo ( L-Asp1- D-Tyr2- D-Asn3- L-Ser4- L-Gln5- D-Ser6- L-Thr7-β-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

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

Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

1985-11-01

The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitismmore » of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.« less

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

Xiong, J.-P.; Stehle, T.; Zhang, R.

The structural basis for the divalent cation-dependent binding of heterodimeric alpha beta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alpha Vbeta 3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alpha V and beta 3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. Themore » tertiary rearrangements take place in beta A, the ligand-binding domain of beta 3; in the complex, beta A acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alpha V relative to beta 3.« less

The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

PubMed

Lauer, Janelle L; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R; Fields, Gregg B

2014-01-24

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

Opposing effects of D-aspartic acid and nitric oxide on tuning of testosterone production in mallard testis during the reproductive cycle.

PubMed

Di Fiore, Maria M; Lamanna, Claudia; Assisi, Loredana; Botte, Virgilio

2008-07-04

D-Aspartic acid (D-Asp) and nitric oxide (NO) play an important role in tuning testosterone production in the gonads of male vertebrates. In particular, D-Asp promotes either the synthesis or the release of testosterone, whereas NO inhibits it. In this study, we have investigated for the first time in birds the putative effects of D-Asp and NO on testicular testosterone production in relation to two phases of the reproductive cycle of the adult captive wild-strain mallard (Anas platyrhynchos) drake. It is a typical seasonal breeder and its cycle consists of a short reproductive period (RP) in the spring (April-May) and a non reproductive period (NRP) in the summer (July), a time when the gonads are quiescent. The presence and the localization of D-Asp and NO in the testis and the trends of D-Asp, NO and testosterone levels were assessed during the main phases of the bird's reproductive cycle. Furthermore, in vitro experiments revealed the direct effect of exogenously administered D-Asp and NO on testosterone steroidogenesis. By using immunohistochemical (IHC) techniques, we studied the presence and the distributional pattern of D-Asp and NO in the testes of RP and NRP drakes. D-Asp levels were evaluated by an enzymatic method, whereas NO content, via nitrite, was assessed using biochemical measurements. Finally, immunoenzymatic techniques determined testicular testosterone levels. IHC analyses revealed the presence of D-Asp and NO in Leydig cells. The distributional pattern of both molecules was in some way correlated to the steroidogenic pathway, which is involved in autocrine testosterone production. Indeed, whereas NO was present only during the NRP, D-Asp was almost exclusively present during the RP. Consistently, the high testosterone testicular content occurring during RP was coupled to a high D-Asp level and a low NO content in the gonad. By contrast, in sexually inactive drakes (NRP), the low testosterone content in the gonad was coupled to a low D-Asp content and to a relatively high NO level. Consequently, to determine the exogenous effects of the two amino acids on testosterone synthesis, we carried out in vitro experiments using testis sections deriving from both the RP and NRP. When testis slices were incubated for 60 or 120 min with D-Asp, testosterone was enhanced, whereas in the presence of L-Arg, a precursor of NO, it was inhibited. Our results provide new insights into the involvement of D-Asp and NO in testicular testosterone production in the adult captive wild-strain mallard drake. The localization of these two molecules in the Leydig cells in different periods of the reproductive cycle demonstrates that they play a potential role in regulating local testosterone production.

Differentiating founder and chronic HIV envelope sequences

PubMed Central

Maher, Stephen; Mota, Talia; Suzuki, Kazuo; Kelleher, Anthony D.

2017-01-01

Significant progress has been made in characterizing broadly neutralizing antibodies against the HIV envelope glycoprotein Env, but an effective vaccine has proven elusive. Vaccine development would be facilitated if common features of early founder virus required for transmission could be identified. Here we employ a combination of bioinformatic and operations research methods to determine the most prevalent features that distinguish 78 subtype B and 55 subtype C founder Env sequences from an equal number of chronic sequences. There were a number of equivalent optimal networks (based on the fewest covarying amino acid (AA) pairs or a measure of maximal covariance) that separated founders from chronics: 13 pairs for subtype B and 75 for subtype C. Every subtype B optimal solution contained the founder pairs 178–346 Asn-Val, 232–236 Thr-Ser, 240–340 Lys-Lys, 279–315 Asp-Lys, 291–792 Ala-Ile, 322–347 Asp-Thr, 535–620 Leu-Asp, 742–837 Arg-Phe, and 750–836 Asp-Ile; the most common optimal pairs for subtype C were 644–781 Lys-Ala (74 of 75 networks), 133–287 Ala-Gln (73/75) and 307–337 Ile-Gln (73/75). No pair was present in all optimal subtype C solutions highlighting the difficulty in targeting transmission with a single vaccine strain. Relative to the size of its domain (0.35% of Env), the α4β7 binding site occurred most frequently among optimal pairs, especially for subtype C: 4.2% of optimal pairs (1.2% for subtype B). Early sequences from 5 subtype B pre-seroconverters each exhibited at least one clone containing an optimal feature 553–624 (Ser-Asn), 724–747 (Arg-Arg), or 46–293 (Arg-Glu). PMID:28187204

Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase

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

McGuire, M.C.; Nogueira, C.P.; Bartels, C.F.

1989-02-01

A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for allmore » 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of {sup 32}P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. The authors conclude that the Asp-70 {yields} Gly mutation accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.« less

High-Precision Timing of Several Millisecond Pulsars

NASA Astrophysics Data System (ADS)

Ferdman, R. D.; Stairs, I. H.; Backer, D. C.; Ramachandran, R.; Demorest, P.; Nice, D. J.; Lyne, A. G.; Kramer, M.; Lorimer, D.; McLaughlin, M.; Manchester, D.; Camilo, F.; D'Amico, N.; Possenti, A.; Burgay, M.; Joshi, B. C.; Freire, P. C.

2004-12-01

The highest precision pulsar timing is achieved by reproducing as accurately as possible the pulse profile as emitted by the pulsar, in high signal-to-noise observations. The best profile reconstruction can be accomplished with several-bit voltage sampling and coherent removal of the dispersion suffered by pulsar signals as they traverse the interstellar medium. The Arecibo Signal Processor (ASP) and its counterpart the Green Bank Astronomical Signal Processor (GASP) are flexible, state-of-the-art wide-bandwidth observing systems, built primarily for high-precision long-term timing of millisecond and binary pulsars. ASP and GASP are in use at the 300-m Arecibo telescope in Puerto Rico and the 100-m Green Bank Telescope in Green Bank, West Virginia, respectively, taking advantage of the enormous sensitivities of these telescopes. These instruments result in high-precision science through 4 and 8-bit sampling and perform coherent dedispersion on the incoming data stream in real or near-real time. This is done using a network of personal computers, over an observing bandwidth of 64 to 128 MHz, in each of two polarizations. We present preliminary results of timing and polarimetric observations with ASP/GASP for several pulsars, including the recently-discovered relativistic double-pulsar binary J0737-3039. These data are compared to simultaneous observations with other pulsar instruments, such as the new "spigot card" spectrometer on the GBT and the Princeton Mark IV instrument at Arecibo, the precursor timing system to ASP. We also briefly discuss several upcoming observations with ASP/GASP.

Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

PubMed Central

Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

2016-01-01

An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai. PMID:27399771

Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai.

PubMed

Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

2016-07-05

An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

The isolation and amino acid sequence of an adrenocorticotrophin from the pars distalis and a corticotrophin-like intermediate-lobe peptide from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

PubMed Central

Lowry, Philip J.; Bennett, Hugh P. J.; McMartin, Colin; Scott, Alexander P.

1974-01-01

An adrenocorticotrophic hormone (ACTH) was isolated from extracts of the pars distalis of the pituitary of the dogfish Squalus acanthias by gel filtration and ion-exchange chromatography. It had 15% of the potency of human ACTH in promoting cortico-steroidogenesis in isolated rat adrenal cells. Sequence analysis revealed it to be a nonatria-contapeptide with the following primary structure: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Met-Gly-Arg-Lys-Arg-Arg-Pro-Ile-Lys-Val-Tyr-Pro-Asn-Ser-Phe-Glu-Asp-Glu-Ser-Val-Glu-Asn-Met-Gly-Pro-Glu-Leu. The N-terminal tridecapeptide sequence was identical with the proposed structure of dogfish α-melanocyte-stimulating hormone (α-MSH). On comparison with human ACTH eleven amino acid differences were seen, nine of which are in the 20–39 region of the molecule which is not essential for the steroidogenic activity of ACTH. A peptide identical with the 18–39 portion of this new ACTH was similarly isolated from the neurointermediate lobe of the pituitary where considerable amounts of dogfish α-MSH were found. This supported our view that ACTH as well as having a distinct biological role of its own is also the precursor of α-MSH. PMID:4375977

Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii

PubMed Central

Chen, Yu; Bensing, Barbara A.; Seepersaud, Ravin; Mi, Wei; Liao, Maofu; Jeffrey, Philip D.; Shajahan, Asif; Sonon, Roberto N.; Azadi, Parastoo; Sullam, Paul M.; Rapoport, Tom A.

2018-01-01

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O-glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1–3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O-glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery. PMID:29462788

Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii.

PubMed

Chen, Yu; Bensing, Barbara A; Seepersaud, Ravin; Mi, Wei; Liao, Maofu; Jeffrey, Philip D; Shajahan, Asif; Sonon, Roberto N; Azadi, Parastoo; Sullam, Paul M; Rapoport, Tom A

2018-04-06

Many pathogenic bacteria, including Streptococcus gordonii , possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O -glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1-3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O -glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N -acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.

cDNA cloning and immunological characterization of a newly identified enolase allergen from Penicillium citrinum and Aspergillus fumigatus.

PubMed

Lai, Hsiu-Yu; Tam, Ming F; Tang, Ren-Bin; Chou, Hong; Chang, Ching-Yun; Tsai, Jaw-Ji; Shen, Horng-Der

2002-03-01

Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5'- and 3'-rapid amplification cDNA end reactions. Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens. Copyright 2002 S. Karger AG, Basel

Design of polyaspartic acid peptide-poly (ethylene glycol)-poly (ε-caprolactone) nanoparticles as a carrier of hydrophobic drugs targeting cancer metastasized to bone

PubMed Central

Liu, Jinsong; Zeng, Youyun; Shi, Shuai; Xu, Lihua; Zhang, Hualin; Pathak, Janak L; Pan, Yihuai

2017-01-01

Treatment of cancer metastasized to bone is still a challenge due to hydrophobicity, instability, and lack of target specificity of anticancer drugs. Poly (ethylene glycol)-poly (ε-caprolactone) polymer (PEG-PCL) is an effective, biodegradable, and biocompatible hydrophobic drug carrier, but lacks bone specificity. Polyaspartic acid with eight peptide sequences, that is, (Asp)8, has a strong affinity to bone surface. The aim of this study was to synthesize (Asp)8-PEG-PCL nanoparticles as a bone-specific carrier of hydrophobic drugs to treat cancer metastasized to bone. 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, and transmission electron microscopy data showed that (Asp)8-PEG-PCL nanoparticles (size 100 nm) were synthesized successfully. (Asp)8-PEG-PCL nanoparticles did not promote erythrocyte aggregation. Fluorescence microscopy showed clear uptake of Nile red-loaded (Asp)8-PEG-PCL nanoparticles by cancer cells. (Asp)8-PEG-PCL nanoparticles did not show cytotoxic effect on MG63 and human umbilical vein endothelial cells at the concentration of 10–800 μg/mL. (Asp)8-PEG-PCL nanoparticles bound with hydroxyapatite 2-fold more than PEG-PCL. Intravenously injected (Asp)8-PEG-PCL nanoparticles accumulated 2.7-fold more on mice tibial bone, in comparison to PEG-PCL. Curcumin is a hydrophobic anticancer drug with bone anabolic properties. Curcumin was loaded in the (Asp)8-PEG-PCL. (Asp)8-PEG-PCL showed 11.07% loading capacity and 95.91% encapsulation efficiency of curcumin. The curcumin-loaded (Asp)8-PEG-PCL nanoparticles gave sustained release of curcumin in high dose for >8 days. The curcumin-loaded (Asp)8-PEG-PCL nanoparticles showed strong antitumorigenic effect on MG63, MCF7, and HeLa cancer cells. In conclusion, (Asp)8-PEG-PCL nanoparticles were biocompatible, permeable in cells, a potent carrier, and an efficient releaser of hydrophobic anticancer drug and were bone specific. The curcumin-loaded (Asp)8-PEG-PCL nanoparticles showed strong antitumorigenic ability in vitro. Therefore, (Asp)8-PEG-PCL nanoparticles could be a potent carrier of hydrophobic anticancer drugs to treat the cancer metastasized to bone. PMID:28507436

Design of polyaspartic acid peptide-poly (ethylene glycol)-poly (ε-caprolactone) nanoparticles as a carrier of hydrophobic drugs targeting cancer metastasized to bone.

PubMed

Liu, Jinsong; Zeng, Youyun; Shi, Shuai; Xu, Lihua; Zhang, Hualin; Pathak, Janak L; Pan, Yihuai

2017-01-01

Treatment of cancer metastasized to bone is still a challenge due to hydrophobicity, instability, and lack of target specificity of anticancer drugs. Poly (ethylene glycol)-poly (ε-caprolactone) polymer (PEG-PCL) is an effective, biodegradable, and biocompatible hydrophobic drug carrier, but lacks bone specificity. Polyaspartic acid with eight peptide sequences, that is, (Asp) 8 , has a strong affinity to bone surface. The aim of this study was to synthesize (Asp) 8 -PEG-PCL nanoparticles as a bone-specific carrier of hydrophobic drugs to treat cancer metastasized to bone. 1 H nuclear magnetic resonance, Fourier transform infrared spectroscopy, and transmission electron microscopy data showed that (Asp) 8 -PEG-PCL nanoparticles (size 100 nm) were synthesized successfully. (Asp) 8 -PEG-PCL nanoparticles did not promote erythrocyte aggregation. Fluorescence microscopy showed clear uptake of Nile red-loaded (Asp) 8 -PEG-PCL nanoparticles by cancer cells. (Asp) 8 -PEG-PCL nanoparticles did not show cytotoxic effect on MG63 and human umbilical vein endothelial cells at the concentration of 10-800 μg/mL. (Asp) 8 -PEG-PCL nanoparticles bound with hydroxyapatite 2-fold more than PEG-PCL. Intravenously injected (Asp) 8 -PEG-PCL nanoparticles accumulated 2.7-fold more on mice tibial bone, in comparison to PEG-PCL. Curcumin is a hydrophobic anticancer drug with bone anabolic properties. Curcumin was loaded in the (Asp) 8 -PEG-PCL. (Asp) 8 -PEG-PCL showed 11.07% loading capacity and 95.91% encapsulation efficiency of curcumin. The curcumin-loaded (Asp) 8 -PEG-PCL nanoparticles gave sustained release of curcumin in high dose for >8 days. The curcumin-loaded (Asp) 8 -PEG-PCL nanoparticles showed strong antitumorigenic effect on MG63, MCF7, and HeLa cancer cells. In conclusion, (Asp) 8 -PEG-PCL nanoparticles were biocompatible, permeable in cells, a potent carrier, and an efficient releaser of hydrophobic anticancer drug and were bone specific. The curcumin-loaded (Asp) 8 -PEG-PCL nanoparticles showed strong antitumorigenic ability in vitro. Therefore, (Asp) 8 -PEG-PCL nanoparticles could be a potent carrier of hydrophobic anticancer drugs to treat the cancer metastasized to bone.

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

PubMed Central

Henderson, L E; Sowder, R; Copeland, T D; Smythers, G; Oroszlan, S

1984-01-01

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus. Images PMID:6333515

Comparative study of (Asp)7-CHOL-modified liposome prepared using pre-insertion and post-insertion methods for bone targeting in vivo.

PubMed

Zhang, Lijing; Cao, Hua; Zhang, Jiaxin; Yang, Chengli; Hu, Tingting; Li, Huili; Yang, Wu; He, Gu; Song, Xiangrong; Tong, Aiping; Guo, Gang; Li, Rui; Jiang, Yu; Liu, Jiyan; Cai, Lulu; Zheng, Yu

2017-02-01

Specific delivery of drugs to bone tissue is very challenging due to the architecture and structure of bone tissue. A seven-repeat sequence of aspartate, a representative bone-targeting oligopeptide, is preferentially used for targeted therapy for bone diseases. In this study, Asp7-cholesterol((Asp)7-CHOL) was synthesized and (Asp)7-CHOL-modified liposome loaded with doxorubicin (DOX) was successfully prepared using both pre-insertion (pre-L) and post-insertion (post-L) methods. The formulation was optimized according to particle size, zeta potential and the drug-loading efficiency of the liposome. In addition, the bone affinity of the (Asp)7-CHOL-modified liposome was evaluated using a hydroxyapatite (HA) absorption method. The results suggested that (Asp)7-CHOL-modified liposome show excellent HA absorption; pre-L showed slightly higher HA binding than post-L. However, post-L had a higher DOX entrapment efficiency than pre-L. In vivo imaging further demonstrated that pre-L showed a higher bone-targeting efficiency than post-L, which was consistent with in vitro results. In all, (Asp)7-CHOL-modified liposome showed excellent bone-targeting activity, suggesting their potential for use as a drug delivery system for bone disease-targeted therapies.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

PubMed

Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

2011-11-01

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.

PubMed Central

McGuire, M C; Nogueira, C P; Bartels, C F; Lightstone, H; Hajra, A; Van der Spek, A F; Lockridge, O; La Du, B N

1989-01-01

A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool. Images PMID:2915989

PRP5: a helicase-like protein required for mRNA splicing in yeast.

PubMed Central

Dalbadie-McFarland, G; Abelson, J

1990-01-01

A 96-kDa protein predicted by the DNA sequence of the Saccharomyces cerevisiae PRP5 gene contains a domain that bears a striking resemblance to a family of RNA helicases characterized by the conserved amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D). Previous work indicated that the product of the PRP5 gene is required for splicing and that spliceosome assembly does not occur in its absence. However, its precise role in splicing and the nature of its biochemical activity remained unknown. To examine the role of PRP5 in splicing, we cloned the gene by complementation of a temperature-sensitive mutation and determined its DNA sequence. We discuss here the possible roles for an RNA helicase in splicing and for the activity of the PRP5 protein. Images PMID:2349233

Characterization of Fine Particulate Matter (PM) and Secondary PM Precursor Gases in the Mexico City Metropolitan Area

DOE R&D Accomplishments Database

Molina, Luisa T.; Volkamer, Rainer; de Foy, Benjamin; Lei, Wenfang; Zavala, Miguel; Velasco, Erik; Molina; Mario J.

2008-10-31

This project was one of three collaborating grants funded by DOE/ASP to characterize the fine particulate matter (PM) and secondary PM precursors in the Mexico City Metropolitan Area (MCMA) during the MILAGRO Campaign. The overall effort of MCMA-2006, one of the four components, focused on i) examination of the primary emissions of fine particles and precursor gases leading to photochemical production of atmospheric oxidants and secondary aerosol particles; ii) measurement and analysis of secondary oxidants and secondary fine PM production, with particular emphasis on secondary organic aerosol (SOA), and iii) evaluation of the photochemical and meteorological processes characteristic of the Mexico City Basin. The collaborative teams pursued the goals through three main tasks: i) analyses of fine PM and secondary PM precursor gaseous species data taken during the MCMA-2002/2003 campaigns and preparation of publications; ii) planning of the MILAGRO Campaign and deployment of the instrument around the MCMA; and iii) analysis of MCMA-2006 data and publication preparation.

Molecular cloning, expression, and functional analysis of the copper amine oxidase gene in the endophytic fungus Shiraia sp. Slf14 from Huperzia serrata.

PubMed

Yang, Huilin; Peng, Silu; Zhang, Zhibin; Yan, Riming; Wang, Ya; Zhan, Jixun; Zhu, Du

2016-12-01

Huperzine A (HupA) is a drug used for the treatment of Alzheimer's disease. However, the biosynthesis of this medicinally important compound is not well understood. The HupA biosynthetic pathway is thought to be initiated by the decarboxylation of lysine to form cadaverine, which is then converted to 5-aminopentanal by copper amine oxidase (CAO). In this study, we cloned and expressed an SsCAO gene from a HupA-producing endophytic fungus, Shiraia sp. Slf14. Analysis of the deduced protein amino acid sequence showed that it contained the Asp catalytic base, conserved motif Asn-Tyr-Asp/Glu, and three copper-binding histidines. The cDNA of SsCAO was amplified and expressed in Escherichia coli BL21(DE3), from which a 76 kDa protein was obtained. The activity of this enzyme was tested, which provided more information about the SsCAO gene in the endophytic fungus. Gas Chromatograph-Mass Spectrometry (GC-MS) revealed that this SsCAO could accept cadaverine as a substrate to produce 5-aminopentanal, the precursor of HupA. Phylogenetic tree analysis indicated that the SsCAO from Shiraia sp. Slf14 was closely related to Stemphylium lycopersici CAO. This is the first report on the cloning and expression of a CAO gene from HupA-producing endophytic fungi. Functional characterization of this enzyme provides new insights into the biosynthesis of the HupA an anti-Alzheimer's drug. Copyright © 2016 Elsevier Inc. All rights reserved.

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

PubMed Central

Headey, Stephen J.; MacAskill, Ursula K.; Wright, Michele A.; Claridge, Jolyon K.; Edwards, Patrick J. B.; Farley, Peter C.; Christeller, John T.; Laing, William A.; Pascal, Steven M.

2010-01-01

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp32–Asp215 diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin. PMID:20538608

Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

PubMed

Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

2007-10-01

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling▿ †

PubMed Central

Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C.; Abe, Keietsu

2007-01-01

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity. PMID:17660287

Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor

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

Dame, J.B.; Williams, J.L.; McCutchan, T.F.

An antimalarial immunogenic stimulant is described comprising an immunogenic carrier and a peptide sequence of between 2 and 1000 consecutive repeats of a sequence Asn-X-Y-Pro, wherein X is Ala or Val and Y is Asn or Asp.

Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

PubMed

Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

2016-09-01

A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

Poly aspartic acid peptide-linked PLGA based nanoscale particles: potential for bone-targeting drug delivery applications.

PubMed

Jiang, Tao; Yu, Xiaohua; Carbone, Erica J; Nelson, Clarke; Kan, Ho Man; Lo, Kevin W-H

2014-11-20

Delivering drugs specifically to bone tissue is very challenging due to the architecture and structure of bone tissue. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) hold great promise for the delivery of therapeutics to bone tissue. The goal of the present research was to formulate a PLGA-based NP drug delivery system for bone tissue exclusively. Since poly-aspartic acids (poly-Asp) peptide sequence has been shown to bind to hydroxyapatite (HA), and has been suggested as a molecular tool for bone-targeting applications, we fabricated PLGA-based NPs linked with poly-Asp peptide sequence. Nanoparticles made of methoxy - poly(ethylene glycol) (PEG)-PLGA and maleimide-PEG-PLGA were prepared using a water-in-oil-in-water double emulsion and solvent evaporation method. Fluorescein isothiocyanate (FITC)-tagged poly-Asp peptide was conjugated to the surface of the nanoparticles via the alkylation reaction between the sulfhydryl groups at the N-terminal of the peptide and the CC double bond of maleimide at one end of the polymer chain to form thioether bonds. The conjugation of FITC-tagged poly-Asp peptide to PLGA NPs was confirmed by NMR analysis and fluorescent microscopy. The developed nanoparticle system is highly aqueous dispersible with an average particle size of ∼80 nm. In vitro binding analyses demonstrated that FITC-poly-Asp NPs were able to bind to HA gel as well as to mineralized matrices produced by human mesenchymal stem cells and mouse bone marrow stromal cells. Using a confocal microscopy technique, an ex vivo binding study of mouse major organ ground sections revealed that the FITC-poly-Asp NPs were able to bind specifically to the bone tissue. In addition, proliferation studies indicated that our FITC-poly-Asp NPs did not induce cytotoxicity to human osteoblast-like MG63 cell lines. Altogether, these promising results indicated that this nanoscale targeting system was able to bind to bone tissue specifically and might have a great potential for bone disease therapy in clinical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Project Report: Automatic Sequence Processor Software Analysis

NASA Technical Reports Server (NTRS)

Benjamin, Brandon

2011-01-01

The Mission Planning and Sequencing (MPS) element of Multi-Mission Ground System and Services (MGSS) provides space missions with multi-purpose software to plan spacecraft activities, sequence spacecraft commands, and then integrate these products and execute them on spacecraft. Jet Propulsion Laboratory (JPL) is currently is flying many missions. The processes for building, integrating, and testing the multi-mission uplink software need to be improved to meet the needs of the missions and the operations teams that command the spacecraft. The Multi-Mission Sequencing Team is responsible for collecting and processing the observations, experiments and engineering activities that are to be performed on a selected spacecraft. The collection of these activities is called a sequence and ultimately a sequence becomes a sequence of spacecraft commands. The operations teams check the sequence to make sure that no constraints are violated. The workflow process involves sending a program start command, which activates the Automatic Sequence Processor (ASP). The ASP is currently a file-based system that is comprised of scripts written in perl, c-shell and awk. Once this start process is complete, the system checks for errors and aborts if there are any; otherwise the system converts the commands to binary, and then sends the resultant information to be radiated to the spacecraft.

Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.

PubMed

Akin, C; Kirshenbaum, A S; Semere, T; Worobec, A S; Scott, L M; Metcalfe, D D

2000-02-01

The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.

Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic.

PubMed

Muneta, Yoshihiro; Minagawa, Yu; Kusumoto, Masahiro; Shinkai, Hiroki; Uenishi, Hirohide; Splichal, Igor

2012-06-01

In the present study, an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP-PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP-PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5. © 2012 The Societies and Blackwell Publishing Asia Pty Ltd.

Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

USDA-ARS?s Scientific Manuscript database

Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp...

Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance.

PubMed Central

Reyna, F; Huesca, M; González, V; Fuchs, L Y

1995-01-01

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined. PMID:7492118

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

Gallium metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides in complex mixtures

PubMed Central

Blacken, Grady R.; Sadílek, Martin; Tureček, František

2008-01-01

Metal affinity capture tandem mass spectrometry (MAC-MSMS) is evaluated in a comparative study of a lysine-derived nitrilotriacetic acid (Nα, Nα-bis-(carboxymethyl)lysine, LysNTA) and an aspartic-acid-related iminodiacetic acid (N-(4-aminobutyl)aspartic acid, AspIDA) as selective phosphopeptide detection reagents. Both LysNTA and AspIDA spontaneously form ternary complexes with GaIII and phosphorylated amino acids and phosphopeptides upon mixing in solution. Collision-induced dissociation of positive complex ions produced by electrospray produces common fragments (LysNTA + H)+ or (AspIDA + H)+ at m/z 263 and 205, respectively. MSMS precursor scans using these fragments as reporter ions allow one to selectively detect multiple charge states of phosphopeptides in mixtures. It follows from this comparative study that LysNTA is superior to AspIDA in detecting phosphopeptides, possibly because of the higher coordination number and greater stability constant for GaIII – phosphopeptide complexation of the former reagent. In a continuing development of MAC-MSMS for proteomics applications, we demonstrate its utility in a post-column reaction format. Using a simple post-column-reaction ‘T’ and syringe pump to deliver our chelating reagents, α-casein tryptic phosphopeptides can be selectively analyzed from a solution containing a twofold molar excess of bovine serum albumin. The MAC-MSMS method is shown to be superior to the commonly used neutral loss scan for the common loss of phosphoric acid. PMID:18265438

De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.

PubMed

Fassio, Anna; Esposito, Alessandro; Kato, Mitsuhiro; Saitsu, Hirotomo; Mei, Davide; Marini, Carla; Conti, Valerio; Nakashima, Mitsuko; Okamoto, Nobuhiko; Olmez Turker, Akgun; Albuz, Burcu; Semerci Gündüz, C Nur; Yanagihara, Keiko; Belmonte, Elisa; Maragliano, Luca; Ramsey, Keri; Balak, Chris; Siniard, Ashley; Narayanan, Vinodh; Ohba, Chihiro; Shiina, Masaaki; Ogata, Kazuhiro; Matsumoto, Naomichi; Benfenati, Fabio; Guerrini, Renzo

2018-06-01

V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients' lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients' lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

PubMed

Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

2014-03-10

Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts

PubMed Central

Rodriguez, Douglas E.; Thula-Mata, Taili; Toro, Edgardo J.; Yeh, Ya-Wen; Holt, Carl; Holliday, L. Shannon; Gower, Laurie B.

2013-01-01

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working toward this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite (HA) to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins (NCPs), to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid (RGD) ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates. PMID:24140612

Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation

PubMed Central

2017-01-01

Deamidation of asparagine is a spontaneous and irreversible post-translational modification associated with a growing list of human diseases. While pervasive, deamidation is often overlooked because it represents a relatively minor chemical change. Structural and functional characterization of this modification is complicated because deamidation of asparagine yields four isomeric forms of Asp. Herein, radical directed dissociation (RDD), in conjunction with mass spectrometry, is used to identify and quantify all four isomers in a series of model peptides that were subjected to various deamidation conditions. Although primary sequence significantly influences the rate of deamidation, it has little impact on the relative proportions of the product isomers. Furthermore, the addition of ammonia can be used to increase the rate of deamidation without significantly perturbing isomer populations. Conversely, external factors such as buffer conditions and temperature alter product distributions but exhibit less dramatic effects on the deamidation rate. Strikingly, the common laboratory and biologically significant bicarbonate buffer is found to strongly promote racemization, yielding increased amounts of d-Asp and d-isoAsp. These outcomes following deamidation have broad implications in human aging and should be considered during the development of protein-based therapeutics. PMID:28984444

Cloning and characterization of the novel D-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38.

PubMed

Nirasawa, Satoru; Nakahara, Kazuhiko; Takahashi, Saori

2018-02-27

Paenidase is the first microorganism-derived D-aspartyl endopeptidase that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds. Using peptide sequences obtained from the protein, we performed PCR with degenerate primers to amplify the paenidase I-encoding gene. Nucleotide sequencing revealed that mature paenidase I consists of 322 amino acid residues and that the protein is encoded as a pro-protein with a 197-amino-acid N-terminal extension compared to the mature protein. Paenidase I exhibits amino acid sequence similarity to several penicillin-binding proteins. In addition, paenidase I was classified into peptidase family S12 based on a MEROPS database search. Family S12 contains serine-type D-Ala-D-Ala carboxypeptidases that have three active site residues (Ser, Lys, and Tyr) in the conserved motifs Ser-Xaa-Thr-Lys and Tyr-Xaa-Asn. These motifs were conserved in the primary structure of paenidase I, and the role of these residues was confirmed by site-directed mutagenesis.

[Mutation analysis for a pedigree affected with keratitis-ichthyosis-deafness syndrome].

PubMed

Li, Lulu; Li, Yuan; Lin, Wei; Zhao, Xiuli

2017-10-10

To identify mutation of GJB2 gene and provide genetic counseling for a family affected with keratitis-ichthyosis-deafness (KID) syndrome. Genomic DNA was extracted from peripheral blood samples with a standard phenol-chloroform method. PCR and Sanger sequencing were used to analyze potential mutation in the proband. Suspected mutation was verified with a PCR-high-resolution melting (PCR-HRM) method. T-clone sequencing was applied to determine the parental origin of the mutation. A heterozygous mutation, c.148G>A (p.Asp50Asn), which is located in the exon 1 of the GJB2 gene, was found in the proband. The results was confirmed by HRM analysis. Cloning sequencing suggested that the mutation was derived from the father's germline. The hot-spot mutation c.148G>A (p.Asp50Asn) in the GJB2 gene probably underlies the KID syndrome in this Chinese family. A PCR-HRM method has been established to rapidly detect common mutations associated with this disease.

Impact of Iron-Enriched Aspergillus oryzae on Iron Bioavailability, Safety, and Gut Microbiota in Rats.

PubMed

Reddy, Manju B; Armah, Seth M

2018-06-20

Iron deficiency is a leading global nutritional problem. Ferrous sulfate (FeSO 4 ) is the most common iron source used for supplementation. Because of many side effects associated with its consumption, it is important to identify new forms of iron. The objectives of this study were to assess the bioavailability of iron-enriched Aspergillus oryzae, Aspiron (ASP), evaluate the toxicity of high-dose iron supplementation with ASP, and determine the ASP impact on gut microbiota in rats. In this study, we investigated iron bioavailability using the hemoglobin repletion test. Aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen levels were determined to evaluate the effect on liver and kidney functions. Protein carbonyls were measured to assess oxidative damage to proteins. Fecal samples at the end of the 14 day repletion period were used for 16S rRNA sequencing for gut microbiota analysis. The slope ratio method using a common intercept linear regression model was used to compare the bioavailability of ASP to FeSO 4 . Iron repletion increased hemoglobin concentrations with both ASP and FeSO 4 treatments compared to the control group, except in the lowest ASP group. The slope ratio indicated that relative iron bioavailability of ASP was 60% of that of FeSO 4 when hemoglobin change was compared to iron in the diet. Similar results were obtained when absolute iron intake was compared on the basis of food consumption. In comparison to the control, protein carbonyl concentrations were significantly ( p < 0.05) higher in the FeSO 4 group but not with the ASP group. Supplementation with both sources of iron reduced the Enterobacteriaceae population in the gut microbiota of the rats. A higher relative abundance of bacteria from the phylum Verrucomicrobia was also observed with the highest dose of ASP. Iron-enriched A. oryzae with 60% relative bioavailability of FeSO 4 did not show any signs of adverse effects after 14 days of iron supplementation. Future human studies are needed to understand the ASP detailed effect on gut microbiota.

Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 is an Inorganic Pyrophosphatase+

PubMed Central

Huang, Hua; Yury, Patskovsky; Toro, Rafael; Farelli, Jeremiah D.; Pandya, Chetanya; Almo, Steven C.; Allen, Karen N.; Dunaway-Mariano, Debra

2012-01-01

The explosion of protein sequence information requires that current strategies for function assignment must evolve to complement experimental approaches with computationally-based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown Haloalkanoate Dehalogenase superfamily member BT2127 (Uniprot accession # Q8A5V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics/structure/mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with kcat/Km value for pyrophosphate of ∼1 × 105 M−1 s−1), together with the gene context, supports the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of the wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure guided site directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. Based on this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172 and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue β-phosphoglucomutase control the leaving group size (phosphate vs. glucose-phosphate) and the position of the Asp acid/base in the open vs. closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases. PMID:21894910

Purification and structural characterization of vasoactive intestinal polypeptide from the trout and bowfin.

PubMed

Wang, Y; Conlon, J M

1995-04-01

Vasoactive intestinal polypeptide (VIP) was purified from extracts of the stomachs of the rainbow trout, Oncorhynchus mykiss, and the bowfin, Amia calva. The primary structure of VIP from both species was the same: His-Ser-Asp-Ala-Ile-Phe-Thr-Asp-Asn-Tyr10- Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Val-Lys20-Lys-Tyr-Leu-Asn-Ser-Val- Leu-Thr. This amino acid sequence shows only one amino acid substitution (Val5-->Ile) compared with the common sequence of VIP from the chicken, alligator, and European green frog. The structural identity of VIP from the trout and bowfin is consistent with the close phylogenetic relationship between the Salmoniformes and the Amiiformes and the data indicate that pressure to conserve the complete primary structure of VIP during vertebrate evolution has been very strong.

Dynamic characterization of HLA-B*44 Alleles: A comparative molecular dynamics simulation study.

PubMed

Ozbek, Pemra

2016-06-01

Human Leukocyte Antigens (HLA) are highly polymorphic proteins that play a key role in the immune system. HLA molecule is present on the cell membrane of antigen-presenting cells of the immune system and presents short peptides, originating from the proteins of invading pathogens or self-proteins, to the T-cell Receptor (TCR) molecule of the T-cells. In this study, peptide-binding characteristics of HLA-B*44:02, 44:03, 44:05 alleles bound to three nonameric peptides were studied using molecular dynamics simulations. Polymorphisms among these alleles (Asp116Tyr and Asp156Leu) result in major differences in the allele characteristics. While HLA-B*44:02 (Asp116, Asp156) and HLA-B*44:03 (Asp116, Leu156) depend on tapasin for efficient peptide loading, HLA-B*44:05 (Tyr116, Asp156) is tapasin independent. On the other hand, HLA-B*44:02 and HLA-B*44:03 mismatch is closely related to transplant rejection and acute-graft-versus-host disease. In order to understand the dynamic characteristics, the simulation trajectories were analyzed by applying Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) calculations and hydrogen bonding analysis. Binding dynamics of the three HLA-B*44 alleles and peptide sequences are comparatively discussed. In general, peptide binding stability is found to depend on the peptide rather than the allele type for HLA-B*44 alleles. Copyright © 2016 Elsevier Ltd. All rights reserved.

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia.

PubMed

Reis, Marta I R; do Vale, Ana; Pinto, Cristina; Nascimento, Diana S; Costa-Ramos, Carolina; Silva, Daniela S P; Silva, Manuel T; Dos Santos, Nuno M S

2007-03-01

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

Salt-bridging effects on short amphiphilic helical structure and introducing sequence-based short beta-turn motifs.

PubMed

Guarracino, Danielle A; Gentile, Kayla; Grossman, Alec; Li, Evan; Refai, Nader; Mohnot, Joy; King, Daniel

2018-02-01

Determining the minimal sequence necessary to induce protein folding is beneficial in understanding the role of protein-protein interactions in biological systems, as their three-dimensional structures often dictate their activity. Proteins are generally comprised of discrete secondary structures, from α-helices to β-turns and larger β-sheets, each of which is influenced by its primary structure. Manipulating the sequence of short, moderately helical peptides can help elucidate the influences on folding. We created two new scaffolds based on a modestly helical eight-residue peptide, PT3, we previously published. Using circular dichroism (CD) spectroscopy and changing the possible salt-bridging residues to new combinations of Lys, Arg, Glu, and Asp, we found that our most helical improvements came from the Arg-Glu combination, whereas the Lys-Asp was not significantly different from the Lys-Glu of the parent scaffold, PT3. The marked 3 10 -helical contributions in PT3 were lessened in the Arg-Glu-containing peptide with the beginning of cooperative unfolding seen through a thermal denaturation. However, a unique and unexpected signature was seen for the denaturation of the Lys-Asp peptide which could help elucidate the stages of folding between the 3 10 and α-helix. In addition, we developed a short six-residue peptide with β-turn/sheet CD signature, again to help study minimal sequences needed for folding. Overall, the results indicate that improvements made to short peptide scaffolds by fine-tuning the salt-bridging residues can enhance scaffold structure. Likewise, with the results from the new, short β-turn motif, these can help impact future peptidomimetic designs in creating biologically useful, short, structured β-sheet-forming peptides.

Canavan disease. Analysis of the nature of the metabolic lesions responsible for development of the observed clinical symptoms.

PubMed

Baslow, M H; Resnik, T R

1997-10-01

Canavan disease (CD), a rare recessive autosomal genetic disorder, is characterized by early onset and a progressive spongy degeneration of the brain involving loss of the axon's myelin sheath. After a relatively normal birth, homozygous individuals generally develop clinical symptoms within months, and usually die within several years of the onset of the disease. A biochemical defect associated with this disease results in reduced activity of the enzyme N-acetyl-L-aspartate amidohydrolase (aspartoacylase) and affected individuals have less ability to hydrolyze N-acetyl-L-asparate (NAA) in brain and other tissues. As a result of aspartoacylase deficiency, NAA builds up in extracellular fluids (ECF) and is excreted in urine. From an analysis of the NAA biochemical cycle in various tissues of many vertebrate species, evidence is presented that there may be two distinct NAA circulation patterns related to aspartoacylase activity. These include near-field circulations in the brain and the eye, and a far-field systemic circulation involving the liver and kidney, the purpose of which in each case is apparently to regenerate aspartate (Asp) in order for it to be recycled into NAA as part of the still unknown function of the NAA cycle. Based on the authors' analysis, they have also identified several metabolic outcomes of the genetic biochemical aspartoacylase lesion. First, there is a daily induced Asp deficit in the central nervous system (CNS) that is at least six times the static level of available free Asp. Second, there is up to a 50-fold drop in the intercompartmental NAA gradient, and third, the ability of the brain to perform its normal intercompartmental cycling of NAA to Asp is terminated, and as a result, the only remaining long-term source of Asp for NAA synthesis is via nutritional supplementation of Asp or its metabolic precursors. Finally, the authors identify a potential maternal-fetal interaction that may be responsible for observed normal fetal development in utero, and that provides a rationale for, and suggests how, CD might respond to far-field nutritional, transplantation, or genetic engineering techniques to alter the course of the disease.

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

PubMed

Bae, Yun Mi; Kim, Myung Hee; Yu, Gwang Sig; Um, Bong Ho; Park, Hee Kyung; Lee, Hyun-il; Lee, Kang Taek; Suh, Yung Doug; Choi, Joon Sig

2014-02-10

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification and characterization of the reptilian GnRH-II gene in the leopard gecko, Eublepharis macularius, and its evolutionary considerations.

PubMed

Ikemoto, Tadahiro; Park, Min Kyun

2003-10-16

To elucidate the molecular phylogeny and evolution of a particular peptide, one must analyze not the limited primary amino acid sequences of the low molecular weight mature polypeptide, but rather the sequences of the corresponding precursors from various species. Of all the structural variants of gonadotropin-releasing hormone (GnRH), GnRH-II (chicken GnRH-II, or cGnRH-II) is remarkably conserved without any sequence substitutions among vertebrates, but its precursor sequences vary considerably. We have identified and characterized the full-length complementary DNA (cDNA) encoding the GnRH-II precursor and determined its genomic structure, consisting of four exons and three introns, in a reptilian species, the leopard gecko Eublepharis macularius. This is the first report about the GnRH-II precursor cDNA/gene from reptiles. The deduced leopard gecko prepro-GnRH-II polypeptide had the highest identities with the corresponding polypeptides of amphibians. The GnRH-II precursor mRNA was detected in more than half of the tissues and organs examined. This widespread expression is consistent with the previous findings in several species, though the roles of GnRH outside the hypothalamus-pituitary-gonadal axis remain largely unknown. Molecular phylogenetic analysis combined with sequence comparison showed that the leopard gecko is more similar to fishes and amphibians than to eutherian mammals with respect to the GnRH-II precursor sequence. These results strongly suggest that the divergence of the GnRH-II precursor sequences seen in eutherian mammals may have occurred along with amniote evolution.

ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy

PubMed Central

Monga, Isha; Qureshi, Abid; Thakur, Nishant; Gupta, Amit Kumar; Kumar, Manoj

2017-01-01

Allele-specific siRNAs (ASP-siRNAs) have emerged as promising therapeutic molecules owing to their selectivity to inhibit the mutant allele or associated single-nucleotide polymorphisms (SNPs) sparing the expression of the wild-type counterpart. Thus, a dedicated bioinformatics platform encompassing updated ASP-siRNAs and an algorithm for the prediction of their inhibitory efficacy will be helpful in tackling currently intractable genetic disorders. In the present study, we have developed the ASPsiRNA resource (http://crdd.osdd.net/servers/aspsirna/) covering three components viz (i) ASPsiDb, (ii) ASPsiPred, and (iii) analysis tools like ASP-siOffTar. ASPsiDb is a manually curated database harboring 4543 (including 422 chemically modified) ASP-siRNAs targeting 78 unique genes involved in 51 different diseases. It furnishes comprehensive information from experimental studies on ASP-siRNAs along with multidimensional genetic and clinical information for numerous mutations. ASPsiPred is a two-layered algorithm to predict efficacy of ASP-siRNAs for fully complementary mutant (Effmut) and wild-type allele (Effwild) with one mismatch by ASPsiPredSVM and ASPsiPredmatrix, respectively. In ASPsiPredSVM, 922 unique ASP-siRNAs with experimentally validated quantitative Effmut were used. During 10-fold cross-validation (10nCV) employing various sequence features on the training/testing dataset (T737), the best predictive model achieved a maximum Pearson’s correlation coefficient (PCC) of 0.71. Further, the accuracy of the classifier to predict Effmut against novel genes was assessed by leave one target out cross-validation approach (LOTOCV). ASPsiPredmatrix was constructed from rule-based studies describing the effect of single siRNA:mRNA mismatches on the efficacy at 19 different locations of siRNA. Thus, ASPsiRNA encompasses the first database, prediction algorithm, and off-target analysis tool that is expected to accelerate research in the field of RNAi-based therapeutics for human genetic diseases. PMID:28696921

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

Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis.

PubMed

Mathupala, S P; Lowe, S E; Podkovyrov, S M; Zeikus, J G

1993-08-05

The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).

PubMed

Godkin, A; Friede, T; Davenport, M; Stevanovic, S; Willis, A; Jewell, D; Hill, A; Rammensee, H G

1997-06-01

HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and -DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.

Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide.

PubMed Central

Nicosia, R. F.; Bonanno, E.

1991-01-01

This study was designed to evaluate the effect of the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis. Images Figure 1 PMID:1707235

Determination of malathion and diazinon resistance by sequencing the Md alpha E7 gene from Guatemala, Colombia, Manhattan, and Thailand housefly (Musca domestica L.) strains.

PubMed

Taşkin, Vatan; Kence, Meral; Göçmen, Belgin

2004-04-01

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of alpha E7 gene, which is a member of alpha-esterase gene cluster, is probably playing a role in detoxification of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly137 to Asp substitution was found in the active center of the product of alpha E7 gene. In malathion (an OP) resistant M. domestica strains Trp251 to Ser substitution was identified in the active center of the Md alpha E7. In our research, to understand the allelic diversity of the Md alpha E7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA) and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp137, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp137 and Trp251 residues on their both alleles at these two different positions.

The Aspergillus Genome Database: multispecies curation and incorporation of RNA-Seq data to improve structural gene annotations.

PubMed

Cerqueira, Gustavo C; Arnaud, Martha B; Inglis, Diane O; Skrzypek, Marek S; Binkley, Gail; Simison, Matt; Miyasato, Stuart R; Binkley, Jonathan; Orvis, Joshua; Shah, Prachi; Wymore, Farrell; Sherlock, Gavin; Wortman, Jennifer R

2014-01-01

The Aspergillus Genome Database (AspGD; http://www.aspgd.org) is a freely available web-based resource that was designed for Aspergillus researchers and is also a valuable source of information for the entire fungal research community. In addition to being a repository and central point of access to genome, transcriptome and polymorphism data, AspGD hosts a comprehensive comparative genomics toolbox that facilitates the exploration of precomputed orthologs among the 20 currently available Aspergillus genomes. AspGD curators perform gene product annotation based on review of the literature for four key Aspergillus species: Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus and Aspergillus niger. We have iteratively improved the structural annotation of Aspergillus genomes through the analysis of publicly available transcription data, mostly expressed sequenced tags, as described in a previous NAR Database article (Arnaud et al. 2012). In this update, we report substantive structural annotation improvements for A. nidulans, A. oryzae and A. fumigatus genomes based on recently available RNA-Seq data. Over 26 000 loci were updated across these species; although those primarily comprise the addition and extension of untranslated regions (UTRs), the new analysis also enabled over 1000 modifications affecting the coding sequence of genes in each target genome.

Solid-phase synthesis of a nucleopeptide from the linking site of adenovirus-2 nucleoprotein, -Ser(p5'CATCAT)-Gly-Asp-. Convergent versus stepwise strategy.

PubMed Central

Robles, J; Pedroso, E; Grandas, A

1995-01-01

The synthesis of a nucleopeptide with the sequence -Ser(p5'CATCAT)-Gly-Asp- has been undertaken by either convergent or stepwise solid-phase strategies, both of which use base-labile permanent protecting groups. The coupling of phosphitylated protected peptides onto oligonucleotide-resins did not afford the desired nucleopeptide, which was nevertheless obtained after oligonucleotide elongation at the hydroxyl group of the resin-bound peptide and deprotection under mild basic conditions. A preliminary study on the stability of different nucleopeptides to bases is also reported. PMID:7479079

Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli.

PubMed

Ramesh, M V; Podkovyrov, S M; Lowe, S E; Zeikus, J G

1994-01-01

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.

Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli.

PubMed Central

Ramesh, M V; Podkovyrov, S M; Lowe, S E; Zeikus, J G

1994-01-01

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase. Images PMID:8117096

Two types of amorphous protein particles facilitate crystal nucleation.

PubMed

Yamazaki, Tomoya; Kimura, Yuki; Vekilov, Peter G; Furukawa, Erika; Shirai, Manabu; Matsumoto, Hiroaki; Van Driessche, Alexander E S; Tsukamoto, Katsuo

2017-02-28

Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

PubMed

Stayton, M M; Black, M; Bedbrook, J; Dunsmuir, P

1986-12-22

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

Meprin A and meprin {alpha} generate biologically functional IL-1{beta} from pro-IL-1{beta}

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

Herzog, Christian; University of Arkansas for Medical Sciences, Department of Medicine, Little Rock, AR 72205; Haun, Randy S.

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin {alpha} are capable of generating biologically active IL-1{beta} from its precursor pro-IL-1{beta}. Amino-acid sequencing analysis reveals that meprin A and meprin {alpha} cleave pro-IL-1{beta} at the His{sup 115}-Asp{sup 116} bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin {beta} site. The biological activity of the pro-IL-1{beta} cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1{beta} product produced by meprin {beta}more » or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1{beta}, meprin inhibitor actinonin significantly reduces levels of serum IL-1{beta}. Meprin A and meprin {alpha} may therefore play a critical role in the production of active IL-1{beta} during inflammation and tissue injury.« less

Fungal DNA in hotel rooms in Europe and Asia--associations with latitude, precipitation, building data, room characteristics and hotel ranking.

PubMed

Norbäck, Dan; Cai, Gui-Hong

2011-10-01

There is little information on the indoor environment in hotels. Analysis of fungal DNA by quantitative PCR (qPCR) is a new method which can detect general and specific sequences. Dust was collected through swab sampling of door frames in 69 hotel rooms in 20 countries in Europe and Asia (2007-2009). Five sequences were detected by qPCR: total fungal DNA, Aspergillus and Penicillium DNA (Asp/Pen DNA), Aspergillus versicolor (A. versicolor DNA), Stachybotrys chartarum (S. chartarum DNA) and Streptomyces spp. (Streptomyces DNA). Associations were analysed by multiple linear regression. Total fungal DNA (GM = 1.08 × 10(8) cell equivalents m(-2); GSD = 6.36) and Asp/Pen DNA (GM = 1.79 × 10(7) cell equivalents m(-2); GSD = 10.12) were detected in all rooms. A. versicolor DNA, S. chartarum DNA and Streptomyces DNA were detected in 84%, 28% and 47% of the samples. In total, 20% of the rooms had observed dampness/mould, and 30% had odour. Low latitude (range 1.5-64.2 degrees) was a predictor of Asp/Pen DNA. Seaside location, lack of mechanical ventilation, and dampness or mould were other predictors of total fungal DNA and Asp/Pen DNA. Hotel ranking (Trip Advisor) or self-rated quality of the interior of the hotel room was a predictor of total fungal DNA, A. versicolor DNA and Streptomyces DNA. Odour was a predictor of S. chartarum DNA. In conclusion, fungal DNA in swab samples from hotel rooms was related to latitude, seaside location, ventilation, visible dampness and indoor mould growth. Hotels in tropical areas may have 10-100 times higher levels of common moulds such as Aspergillus and Penicillium species, as compared to a temperate climate zone.

Molecular cloning and characterization of an α-amylase cDNA highly expressed in major feeding stages of the coffee berry borer, Hypothenemus hampei.

PubMed

Bezerra, C A; Macedo, L L P; Amorim, T M L; Santos, V O; Fragoso, R R; Lucena, W A; Meneguim, A M; Valencia-Jimenez, A; Engler, G; Silva, M C M; Albuquerque, E V S; Grossi-de-Sa, M F

2014-12-10

α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies. Copyright © 2014. Published by Elsevier B.V.

Identification of a precursor genomic segment that provided a sequence unique to glycophorin B and E genes

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

Onda, M.; Kudo, S.; Fukuda, M.

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE gene arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here the authors describe the identification ofmore » this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes. 37 refs., 5 figs.« less

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

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

PubMed Central

Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

1993-01-01

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 3 PMID:8471039

Toll like receptor-4 gene polymorphisms in patients with solitary cysticercus granuloma.

PubMed

Singh, Akhilesh; Garg, Ravindra Kumar; Jain, Amita; Malhotra, Hardeep Singh; Prakash, Shantanu; Verma, Rajesh; Sharma, Praveen Kumar

2015-08-15

Solitary cysticercus granuloma (SCG) of the brain is the most common type of neurocysticercosis in India. In this study, we evaluated TLR4 polymorphisms in patients with SCG. One-hundred-forty-three patients with SCG and 134 controls were enrolled. Assessment for TLR4 Asp299Gly and Thr399Ile polymorphism was done. TLR4 genotype was determined by PCR-sequencing chain termination method. The patients were followed for 6 months. Asp/Gly (P=0.024) and Thr/Ile (P=0.004) genotypes were significantly associated with the SCG. The Gly (Asp/Gly plus Gly/Gly) genotype (P=0.025) and Ile (Thr/Ile plus Ile/Ile) genotype (P=0.008) were significantly associated with the SCG. Gly/Gly and Ile/Ile genotypes were not significantly associated with SCG (P=0.767 for Gly/Gly, P=0.936 for Ile/Ile). At 6 months, TLR4 299Asp/Gly (P=0.02) and 399Ile/Thr (P=0.023) polymorphisms were significantly associated with the calcification or persistence of SCG. TLR4 polymorphisms are associated with the susceptibility to infection with SCG. Copyright © 2015 Elsevier B.V. All rights reserved.

Close evolutionary relatedness among functionally distantly related members of the (alpha/beta)8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region.

PubMed

Janecek, S

1995-12-11

A short conserved sequence equivalent to the fifth conserved sequence region of alpha-amylases (173_LPDLD, Aspergillus oryzae alpha-amylase) comprising the calcium-ligand aspartate, Asp-175, was identified in the amino acid sequences of several members of the family of (alpha/beta)8-barrel glycosyl hydrolases. Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26-28 amino acid residues in front of the well-known catalytic aspartate (Asp-206, A. oryzae alpha-amylase) located in the beta 4-strand of the barrel. The identification of this region revealed remarkable similarities between some alpha-amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo-1,6-glucosidase, amylomaltase and neopullulanase, respectively) on the other hand. The most interesting example was offered by B. subtilis alpha-amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively. These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the alpha-amylase-type (alpha/beta)8-barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

PubMed Central

Allison, J; Hall, L; MacIntyre, I; Craig, R K

1981-01-01

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein. Images Fig. 1. Fig. 2. Fig. 3. PMID:6896146

Novel mutations in CRB1 gene identified in a chinese pedigree with retinitis pigmentosa by targeted capture and next generation sequencing

PubMed Central

Lo, David; Weng, Jingning; Liu, xiaohong; Yang, Juhua; He, Fen; Wang, Yun; Liu, Xuyang

2016-01-01

PURPOSE To detect the disease-causing gene in a Chinese pedigree with autosomal-recessive retinitis pigmentosa (ARRP). METHODS All subjects in this family underwent a complete ophthalmic examination. Targeted-capture next generation sequencing (NGS) was performed on the proband to detect variants. All variants were verified in the remaining family members by PCR amplification and Sanger sequencing. RESULTS All the affected subjects in this pedigree were diagnosed with retinitis pigmentosa (RP). The compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations in the Crumbs homolog 1 (CRB1) gene were identified in all the affected patients but not in the unaffected individuals in this family. These mutations were inherited from their parents, respectively. CONCLUSION The novel compound heterozygous mutations in CRB1 were identified in a Chinese pedigree with ARRP using targeted-capture next generation sequencing. After evaluating the significant heredity and impaired protein function, the compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations are the causal genes of early onset ARRP in this pedigree. To the best of our knowledge, there is no previous report regarding the compound mutations. PMID:27806333

A comparative study of occurrence and fate of endocrine disruptors: diethyl phthalate and dibutyl phthalate in ASP- and SBR-based wastewater treatment plants.

PubMed

Saini, Gita; Pant, Shalini; Singh, Shri Om; Kazmi, A A; Alam, Tanveer

2016-11-01

Phthalates are endocrine-disrupting chemicals which affect endocrine system by bio-accumulation in aquatic organisms and produce adverse health effects in aquatic organisms as well as human beings, when come in contact. Present study focuses on occurrence and removal of two phthalates: diethylphthalate (DEP) and dibutylphthalate (DBP) in two full-scale wastewater treatment plants (WWTPs) i.e. sewage treatment plants (STPs) based on well-adopted technologies, activated sludge process (ASP) and sequencing batch reactor (SBR).Gas chromatography-mass spectrometry (GC-MS) analysis was performed for both wastewater and sludge sample for determination and identification of the concentration of these compounds in both STPs by monitoring the STPs for 9 months. It was observed that the concentration of DEP was less than DBP in the influent of ASP and SBR. Average concentrations of DEP and DBP in sludge sample of ASP were found to be 2.15 and 2.08 ng/g, whereas in SBR plant, these values were observed as 1.71 and 2.01 ng/g, respectively. Concerning the removal efficiency of DEP, SBR and ASP plants were found effective with removal efficiency of 91.51 and 91.03 %, respectively. However, in the case of DBP, SBR showed lower removal efficiency (85.42 %) as compared to ASP (92.67 %). Comparative study of both plants proposed that in ASP plant, DBP reduction was higher than the SBR. Fourier transformation infrared (FTIR) analysis also confirmed the same result of sludge analysis for both STPs. Sludge disposal studied with scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and thermo-gravimetric analysis (TGA) techniques confirmed that sludge of both STPs have high calorific value and can be used as fuel to make fuel-briquettes and bottom ash to make firebricks.

Modulation of calcium oxalate dihydrate growth by selective crystal-face binding of phosphorylated osteopontin and polyaspartate peptide showing occlusion by sectoral (compositional) zoning.

PubMed

Chien, Yung-Ching; Masica, David L; Gray, Jeffrey J; Nguyen, Sarah; Vali, Hojatollah; McKee, Marc D

2009-08-28

Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp(86-93)). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp(86-93) dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp(86-93) followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp(86-93) adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp(86-93) sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp(86-93) domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion).

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26

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

Gudmundsson, Sanna; Wilbe, Maria; Ekvall, Sara

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G >more » A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. Finally to our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome.« less

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26

DOE PAGES

Gudmundsson, Sanna; Wilbe, Maria; Ekvall, Sara; ...

2017-02-01

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G >more » A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. Finally to our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome.« less

Identification of an active acidic residue in the catalytic site of beta-hexosaminidase.

PubMed

Tse, R; Vavougios, G; Hou, Y; Mahuran, D J

1996-06-11

Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits. Both isozymes hydrolyze terminal beta-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo GM2 ganglioside is a substrate for only the heterodimeric A isozyme. Thus, mutations in either gene encoding its alpha or beta subunits can result in GM2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases ae believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alpha and beta subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA and HEXB genes. This region includes beta Arg211 (invariant in 15 sequences), which we have previously shown to be an active residue. This region also contains two invariant and one conserved acidic residues. A fourth acidic residue, Asp alpha 258, beta 290, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a beta cDNA, followed by cellular expression. Of these, only the beta Asp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the K(m). Mutagenesis of either beta Asp240 or beta Asp290 to Asn decreased kcat by 10- or 1.4-fold but also raised the K(m) of the enzyme 11- of 3-fold, respectively. The above results strongly suggest that beta Asp196 is a catalytic acid residue in beta-hexosaminidase.

ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy.

PubMed

Monga, Isha; Qureshi, Abid; Thakur, Nishant; Gupta, Amit Kumar; Kumar, Manoj

2017-09-07

Allele-specific siRNAs (ASP-siRNAs) have emerged as promising therapeutic molecules owing to their selectivity to inhibit the mutant allele or associated single-nucleotide polymorphisms (SNPs) sparing the expression of the wild-type counterpart. Thus, a dedicated bioinformatics platform encompassing updated ASP-siRNAs and an algorithm for the prediction of their inhibitory efficacy will be helpful in tackling currently intractable genetic disorders. In the present study, we have developed the ASPsiRNA resource (http://crdd.osdd.net/servers/aspsirna/) covering three components viz (i) ASPsiDb , (ii) ASPsiPred , and (iii) analysis tools like ASP-siOffTar ASPsiDb is a manually curated database harboring 4543 (including 422 chemically modified) ASP-siRNAs targeting 78 unique genes involved in 51 different diseases. It furnishes comprehensive information from experimental studies on ASP-siRNAs along with multidimensional genetic and clinical information for numerous mutations. ASPsiPred is a two-layered algorithm to predict efficacy of ASP-siRNAs for fully complementary mutant (Eff mut ) and wild-type allele (Eff wild ) with one mismatch by ASPsiPred SVM and ASPsiPred matrix , respectively. In ASPsiPred SVM , 922 unique ASP-siRNAs with experimentally validated quantitative Eff mut were used. During 10-fold cross-validation (10nCV) employing various sequence features on the training/testing dataset (T737), the best predictive model achieved a maximum Pearson's correlation coefficient (PCC) of 0.71. Further, the accuracy of the classifier to predict Eff mut against novel genes was assessed by leave one target out cross-validation approach (LOTOCV). ASPsiPred matrix was constructed from rule-based studies describing the effect of single siRNA:mRNA mismatches on the efficacy at 19 different locations of siRNA. Thus, ASPsiRNA encompasses the first database, prediction algorithm, and off-target analysis tool that is expected to accelerate research in the field of RNAi-based therapeutics for human genetic diseases. Copyright © 2017 Monga et al.

The potential role of toll-like receptor 4 Asp299Gly polymorphism and its association with recurrent cystic echinococcosis in postoperative patients.

PubMed

Noori, Jafar; Spotin, Adel; Ahmadpour, Ehsan; Mahami-Oskouei, Mahmoud; Sadeghi-Bazargani, Homayoun; Kazemi, Tohid; Sakhinia, Ebrahim; Aghebati-Maleki, Leili; Shahrivar, Firooz

2018-06-01

The study of pathogenesis mechanisms of larval stages in the Taeniidae has recently focused on host genetic factors, particularly toll-like receptor (TLR) variations. However, the potential role of TLR4 polymorphism in hydatidosis has not yet been sufficiently elucidated in postoperative patients. In this case-control investigation, 80 patients from Iran, including 40 with acute hydatidosis (AH) and 40 with recurrent hydatidosis (RH), and 80 ethnically matched controls were evaluated from February 2015 to February 2017. Hydatidosis patients were confirmed using radiological, immunological, and histopathological examinations. Genotyping of Asp299Gly and Thr399Ile of TLR4 single-nucleotide polymorphisms was determined by restriction fragment length polymorphism, sequencing, and phylogenetic strategies. The heterozygous mutant-type TLR4 Asp299Gly genotype indicated a tendency to be associated with the occurrence of RH (P = 0.060) and conferred a 3-fold risk for susceptibility. There was no difference in genotype frequency of Asp299Gly between patients with AH and healthy controls (P = 0.42; OR, 1.82; 95% CI, 0.11-30.1%). Interestingly, a frequency of the G allele (12%: Gly) was observed to be a risk factor for susceptibility to RH patients (P = 0.050; OR, 7.08; 95% CI, 0.97-51.5%). A relative genetic variability of TLR4 Asp299Gly was found in RH patients (haplotype diversity: 0.700) compared to AH patients and healthy controls (Hd: 0.000). The Asp299Gly genotype was dominantly identified in patients with hepatic hydatid cysts. The TLR4 Thr399Ile codon was not detected except in a patient with a pulmonary hydatid cyst. The current findings enhance our knowledge regarding the TLR4 Asp299Gly polymorphism potentially leading to the development of RH, by skewing the immune system towards a Th2 response. Identification of the Asp299Gly codon may be a diagnostic hallmark in RH patients who have undergone unsuccessful postoperative intervention. However, further studies with a higher case number are needed on ethnic population from various geographic regions, in order to confirm this hypothesis.

Comparison of non-volatile umami components in chicken soup and chicken enzymatic hydrolysate.

PubMed

Kong, Yan; Yang, Xiao; Ding, Qi; Zhang, Yu-Yu; Sun, Bao-Guo; Chen, Hai-Tao; Sun, Ying

2017-12-01

Umami taste is an important part to the taste of chicken. To isolate and identify non-volatile umami compounds, fractions from chicken soup and hydrolysate were prepared and analyzed. Amino acids were analyzed by amino acid analyzer. Organic acids and nucleotides were determined by ultra-performance liquid chromatography. Separation procedures utilizing ultrafiltration, Sephadex G-15 and reversed-phase high-performance liquid chromatography were used to isolate umami taste peptides. Combined with sensory evaluation and LC-Q-TOF-MS, the amino acid sequences of 12 oligopeptides were determined. The amount of taste compounds was higher in chicken enzymatic hydrolysate than that of chicken soup. Eight oligopeptides from chicken enzymatic hydrolysate were identified, including Ala-Asp, Ala-Met, His-Ser, Val-Glu, Ala-Glu, Asp-Ala-Gly, Glu-Asp and Ala-Glu-Ala. Four oligopeptides from chicken soup were identified, including Val-Thr, Ala-His, Ala-Phe and Thr-Glu. Copyright © 2017 Elsevier Ltd. All rights reserved.

HYDROPS FETALIS ASSOCIATED WITH HOMOZYGOSITY FOR Hb ADANA [α59(E8)Gly→Asp (α2)

PubMed Central

Nainggolan, Ita M.; Harahap, Alida; Setianingsih, Iswari

2010-01-01

We describe cases of hydrops fetalis associated with nondeletional α-thalassemia (α-thal), in three unrelated Indonesian families. The genotypes of the fetuses and their parents were generated by DNA sequencing and by a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP)-based method to rapidly identify mutations detected by sequencing. Two of the fetuses had hydrops fetalis and homozygous α59(E8)Gly→Asp (α2), also known as Hb Adana. The third fetus was also suspected to be homozygous for Hb Adana because both parents were carriers of this mutation. This study shows that homozygosity for Hb Adana is associated with hydrops fetalis in the Indonesian population. We discuss this mutation and its various phenotypes including compound heterozygosity with other α-thal mutations and describe a simple approach to genetic testing that will clarify the risk of hydrops fetalis in the offspring of couples carrying this nondeletional mutation. PMID:20642338

Phylogenetic Relationship of Necoclí Virus to Other South American Hantaviruses (Bunyaviridae: Hantavirus).

PubMed

Montoya-Ruiz, Carolina; Cajimat, Maria N B; Milazzo, Mary Louise; Diaz, Francisco J; Rodas, Juan David; Valbuena, Gustavo; Fulhorst, Charles F

2015-07-01

The results of a previous study suggested that Cherrie's cane rat (Zygodontomys cherriei) is the principal host of Necoclí virus (family Bunyaviridae, genus Hantavirus) in Colombia. Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences in this study confirmed that Necoclí virus is phylogenetically closely related to Maporal virus, which is principally associated with the delicate pygmy rice rat (Oligoryzomys delicatus) in western Venezuela. In pairwise comparisons, nonidentities between the complete amino acid sequence of the nucleocapsid protein of Necoclí virus and the complete amino acid sequences of the nucleocapsid proteins of other hantaviruses were ≥8.7%. Likewise, nonidentities between the complete amino acid sequence of the glycoprotein precursor of Necoclí virus and the complete amino acid sequences of the glycoprotein precursors of other hantaviruses were ≥11.7%. Collectively, the unique association of Necoclí virus with Z. cherriei in Colombia, results of the Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences, and results of the pairwise comparisons of amino acid sequences strongly support the notion that Necoclí virus represents a novel species in the genus Hantavirus. Further work is needed to determine whether Calabazo virus (a hantavirus associated with Z. brevicauda cherriei in Panama) and Necoclí virus are conspecific.

Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates.

PubMed Central

Carré-Eusèbe, D; Lederer, F; Lê, K H; Elsevier, S M

1991-01-01

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid. Images Fig. 1. Fig. 3. Fig. 4. PMID:1854346

[Characterization of microbial community in produced water from a petroleum reservoir subjected to alkali-surfactant-polymer ASP flooding].

PubMed

Hao, Qin Qin; Shi, Rong Jiu; Hao, Jin Sheng; Zhao, Jin Yi; Li, Guo Qiao; Zhao, Feng; Han, Si Qin; Zhang, Ying

2017-10-01

Injection of alkali, surfactant and polymer (ASP) into oil reservoir can substantially increase oil recovery compared with water-flooding strategy. However, the effects of these agents on the microbial diversity and community structure, which is important for water management and corrosion control in oil industry, are hitherto poorly understood. Here, we disclosed the microbial diversity and community structure in the produced water collected from four producing wells of an ASP-flooded oilfield at Daqing, China, using high-throughput sequencing technique. Results showed that the average pH in produced water was as high as 9.65. The microbial diversity varied from well to well, and the Shannon diversity index was between 2.00 to 3.56. The Proteobacteria (85.5%-98.3%), γ-proteobacteria (83.7%-97.8%), and alkaliphilic Nitrincola (51.8%-82.5%) were the most dominant phylogenetic taxa at the phylum, class, and genus levels, respectively. A total of 12 potentially sulfide-producing genera were detected, and the most abundant taxon was Sulfurospirillum (0.4%-7.4%). The microbial community of ASP-flooded petroleum reservoir was distinct, showing an alkaliphilic or alkalitolerant potential; a reduced diversity and more simple structure were observed compared with those of the water-flooded petroleum reservoirs that were previously reported.

Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

PubMed Central

Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban

1997-01-01

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature. PMID:9192645

Phylogenetic analysis reveals conservation and diversification of micro RNA166 genes among diverse plant species.

PubMed

Barik, Suvakanta; SarkarDas, Shabari; Singh, Archita; Gautam, Vibhav; Kumar, Pramod; Majee, Manoj; Sarkar, Ananda K

2014-01-01

Similar to the majority of the microRNAs, mature miR166s are derived from multiple members of MIR166 genes (precursors) and regulate various aspects of plant development by negatively regulating their target genes (Class III HD-ZIP). The evolutionary conservation or functional diversification of miRNA166 family members remains elusive. Here, we show the phylogenetic relationships among MIR166 precursor and mature sequences from three diverse model plant species. Despite strong conservation, some mature miR166 sequences, such as ppt-miR166m, have undergone sequence variation. Critical sequence variation in ppt-miR166m has led to functional diversification, as it targets non-HD-ZIPIII gene transcript (s). MIR166 precursor sequences have diverged in a lineage specific manner, and both precursors and mature osa-miR166i/j are highly conserved. Interestingly, polycistronic MIR166s were present in Physcomitrella and Oryza but not in Arabidopsis. The nature of cis-regulatory motifs on the upstream promoter sequences of MIR166 genes indicates their possible contribution to the functional variation observed among miR166 species. Copyright © 2013 Elsevier Inc. All rights reserved.

Direct assessment of the metabolic origin of carbon atoms in glutamate from illuminated leaves using 13 C-NMR.

PubMed

Abadie, Cyril; Lothier, Jérémy; Boex-Fontvieille, Edouard; Carroll, Adam; Tcherkez, Guillaume

2017-12-01

Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with 13 CO 2 and 13 C-NMR analyses to examine the labelling pattern and the appearance of multi-labelled species of Glu molecules to trace the origin of C-atoms found in Glu. We also compared this with 13 C-labelling patterns in Ala and Asp, which reflect citrate (and thus Glu) precursors, that is, pyruvate and oxaloacetate. Glu appeared to be less 13 C-labelled than Asp and Ala, showing that the Glu pool was mostly formed by 'old' carbon atoms. There were modest differences in intramolecular 13 C- 13 C couplings between Glu C-2 and Asp C-3, showing that oxaloacetate metabolism to Glu biosynthesis did not involve C-atom redistribution by the Krebs cycle. The apparent carbon allocation increased with carbon net photosynthesis. However, when expressed relative to CO 2 fixation, it was clearly higher at low CO 2 while it did not change in 2% O 2 , as compared to standard conditions. We conclude that Glu production from current photosynthetic carbon represents a small flux that is controlled by the gaseous environment, typically upregulated at low CO 2 . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

[Molecular cloning and characterization in silico of phospholipase A(2) transcript isolated from Lachesis muta peruvian snake venom].

PubMed

Jimenez, Karim L; Zavaleta, Amparo I; Izaguirre, Victor; Yarleque, Armando; Inga, Rosio R

2010-01-01

Isolate and characterize in silico gene phospholipase A(2) (PLA(2)) isolated from Lachesis muta venom of the Peruvian Amazon. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13,976 kDa and 5.66 respectively. The aminoacid sequence was called Lm-PLA(2)-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA(2) from Lachesis stenophrys (93%) and other PLA(2) snake venoms and over 80% of other sPLA(2) family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA(2)-Peru grouped with other acidic [Asp(49)] sPLA(2) previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA(2) group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. The nucleotide sequence corresponding to the first transcript of gene from PLA(2) cloned of Lachesis muta venom, snake from the Peruvian rainforest.

Design of Cyclic Peptide Based Glucose Receptors and Their Application in Glucose Sensing.

PubMed

Li, Chao; Chen, Xin; Zhang, Fuyuan; He, Xingxing; Fang, Guozhen; Liu, Jifeng; Wang, Shuo

2017-10-03

Glucose assay is of great scientific significance in clinical diagnostics and bioprocess monitoring, and to design a new glucose receptor is necessary for the development of more sensitive, selective, and robust glucose detection techniques. Herein, a series of cyclic peptide (CP) glucose receptors were designed to mimic the binding sites of glucose binding protein (GBP), and CPs' sequence contained amino acid sites Asp, Asn, His, Asp, and Arg, which constituted the first layer interactions of GBP. The properties of these CPs used as a glucose receptor or substitute for the GBP were studied by using a quartz crystal microbalance (QCM) technique. It was found that CPs can form a self-assembled monolayer at the Au quartz electrode surface, and the monolayer's properties were characterized by using cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. The CPs' binding affinity to saccharide (i.e., galactose, fructose, lactose, sucrose, and maltose) was investigated, and the CPs' sensitivity and selectivity toward glucose were found to be dependent upon the configuration,i.e., the amino acids sequence of the CPs. The cyclic unit with a cyclo[-CNDNHCRDNDC-] sequence gave the highest selectivity and sensitivity for glucose sensing. This work suggests that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of glucose receptors and would find huge application in biological, life science, and clinical diagnostics fields.

Somatic mosaicism of a CDKL5 mutation identified by next-generation sequencing.

PubMed

Kato, Takeshi; Morisada, Naoya; Nagase, Hiroaki; Nishiyama, Masahiro; Toyoshima, Daisaku; Nakagawa, Taku; Maruyama, Azusa; Fu, Xue Jun; Nozu, Kandai; Wada, Hiroko; Takada, Satoshi; Iijima, Kazumoto

2015-10-01

CDKL5-related encephalopathy is an X-linked dominantly inherited disorder that is characterized by early infantile epileptic encephalopathy or atypical Rett syndrome. We describe a 5-year-old Japanese boy with intractable epilepsy, severe developmental delay, and Rett syndrome-like features. Onset was at 2 months, when his electroencephalogram showed sporadic single poly spikes and diffuse irregular poly spikes. We conducted a genetic analysis using an Illumina® TruSight™ One sequencing panel on a next-generation sequencer. We identified two epilepsy-associated single nucleotide variants in our case: CDKL5 p.Ala40Val and KCNQ2 p.Glu515Asp. CDKL5 p.Ala40Val has been previously reported to be responsible for early infantile epileptic encephalopathy. In our case, the CDKL5 heterozygous mutation showed somatic mosaicism because the boy's karyotype was 46,XY. The KCNQ2 variant p.Glu515Asp is known to cause benign familial neonatal seizures-1, and this variant showed paternal inheritance. Although we believe that the somatic mosaic CDKL5 mutation is mainly responsible for the neurological phenotype in the patient, the KCNQ2 variant might have some neurological effect. Genetic analysis by next-generation sequencing is capable of identifying multiple variants in a patient. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions.

PubMed

Lee, Sun Ha; Moon, Sung Jin; Paeng, Jisun; Kang, Hye-Young; Nam, Bo Young; Kim, Seonghun; Kim, Chan Ho; Lee, Mi Jung; Oh, Hyung Jung; Park, Jung Tak; Han, Seung Hyeok; Yoo, Tae-Hyun; Kang, Shin-Wook

2015-08-01

Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocin-induced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis.

Engineering thermal stability of L-asparaginase by in vitro directed evolution.

PubMed

Kotzia, Georgia A; Labrou, Nikolaos E

2009-03-01

L-asparaginase (EC 3.5.1.1, L-ASNase) catalyses the hydrolysis of l-Asn, producing L-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the L-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental L-ASNases show 77% identity, but their half-inactivation temperature (T(m)) differs by 10 degrees C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T(m) of this variant was 55.8 degrees C, whereas the wild-type enzyme has a T(m) of 46.4 degrees C. At 50 degrees C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability.

Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

PubMed

Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

2000-02-29

Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.

Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates.

PubMed

Huang, Shih-Li; Jao, Chia-Ling; Ho, Kit-Pan; Hsu, Kuo-Chiang

2012-05-01

The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1,422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC(50) values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract. Copyright © 2012 Elsevier Inc. All rights reserved.

Location of alkali metal binding sites in endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu), from multistep collisionally activated decompositions.

PubMed

Ngoka, L C; Gross, M L

2000-02-01

We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide. Copyright 2000 John Wiley & Sons, Ltd.

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach*

PubMed Central

Kim, Young-Min; Kiso, Yoshiaki; Muraki, Tomoe; Kang, Min-Sun; Nakai, Hiroyuki; Saburi, Wataru; Lang, Weeranuch; Kang, Hee-Kwon; Okuyama, Masayuki; Mori, Haruhide; Suzuki, Ryuichiro; Funane, Kazumi; Suzuki, Nobuhiro; Momma, Mitsuru; Fujimoto, Zui; Oguma, Tetsuya; Kobayashi, Mikihiko; Kim, Doman; Kimura, Atsuo

2012-01-01

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr451–Val1082), a portion of which shares identity (35% at Ala39–Ser1304 of PsDex) with Pro32–Ala755 of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837–Met932 for PsDex and Tyr404–Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39–Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide mutants decreased activity (11,500 to 140,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp340 and Glu412 as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3. PMID:22461618

Long QT syndrome type 5-Lite: Defining the clinical phenotype associated with the potentially proarrhythmic p.Asp85Asn-KCNE1 common genetic variant.

PubMed

Lane, Conor; Giudicessi, John R; Ye, Dan; Tester, David J; Rohatgi, Ram K; Bos, J Martijn; Ackerman, Michael J

2018-04-03

Long QT syndrome (LQTS) genetic test reports commonly exclude potentially proarrhythmic common variants such as p.Asp85Asn-KCNE1. The purpose of this study was to determine whether a discernible phenotype is associated with p.Asp85Asn-KCNE1 and whether relatively common KCNE1 variants underlie transient QT prolongation pedigrees with negative commercial LQTS genetic tests. Retrospective review was used to compare demographics, symptomatology, and QT parameters of individuals with p.Asp85Asn-KCNE1 in the absence of other rare/ultra-rare variants in LQTS-susceptibility genes and those who underwent comprehensive LQTS genetic testing. Compared to the Genome Aggregation Database, p.Asp85Asn-KCNE1 was more prevalent in individuals undergoing LQTS genetic testing (33/1248 [2.6%] vs 1552/126,652 [1.2%]; P = .0001). In 19 of 33 patients (58%), only p.Asp85Asn-KCNE1 was observed. These patients were predominantly female (90% vs 62%; P = .02) and were less likely to experience syncope (0% vs 34%; P = .0007), receive β-blockers (53% vs 85%; P = .001), or require an implantable cardioverter-defibrillator (5.3% vs 33%; P = .01). However, they exhibited a similar degree of QT prolongation (QTc 460 ms vs 467 ms; P = NS). Whole exome sequencing of 2 commercially genotype-negative pedigrees revealed that p.Asp85Asn-KCNE1 and p.Arg36His-KCNE1 traced with a transient QT prolongation phenotype. Functional characterization of p.Arg36His-KCNE1 demonstrated loss of function, with a 47% reduction in peak I Ks current density in the heterozygous state. We provide further evidence that relatively common variants in KCNE1 may result in a mild QT phenotype designated as "LQT5-Lite" to distinguish such potentially proarrhythmic common variants (ie, functional risk alleles) from rare pathogenic variants that truly confer monogenic disease susceptibility, albeit with incomplete penetrance. Copyright © 2018. Published by Elsevier Inc.

Characterization of gonadotrophin-releasing hormone precursor cDNA in the Old World mole-rat Cryptomys hottentotus pretoriae: high degree of identity with the New World guinea pig sequence.

PubMed

Kalamatianos, T; du Toit, L; Hrabovszky, E; Kalló, I; Marsh, P J; Bennett, N C; Coen, C W

2005-05-01

Regulation of pituitary gonadotrophins by the decapeptide gonadotrophin-releasing hormone 1 (GnRH1) is crucial for the development and maintenance of reproductive functions. A common amino acid sequence for this decapeptide, designated as 'mammalian' GnRH, has been identified in all mammals thus far investigated with the exception of the guinea pig, in which there are two amino acid substitutions. Among hystricognath rodents, the members of the family Bathyergidae regulate reproduction in response to diverse cues. Thus, highveld mole-rats (Cryptomys hottentotus pretoriae) are social bathyergids in which breeding is restricted to a particular season in the dominant female, but continuously suppressed in subordinate colony members. Elucidation of reproductive control in these animals will be facilitated by characterization of their GnRH1 gene. A partial sequence of GnRH1 precursor cDNA was isolated and characterized. Comparative analysis revealed the highest degree of identity (86%) to guinea pig GnRH1 precursor mRNA. Nevertheless, the deduced amino acid sequence of the mole-rat decapeptide is identical to the 'mammalian' sequence rather than that of guinea pigs. Successful detection of GnRH1-synthesizing neurones using either a guinea pig GnRH1 riboprobe or an antibody against the 'mammalian' decapeptide is consistent with the guinea pig-like sequence for the precursor and the classic 'mammalian' form for the decapeptide. The high degree of identity in the GnRH1 precursor sequence between this Old World mole-rat and the New World guinea pig is consistent with the theory that caviomorphs and phiomorphs originated from a common ancestral line in the Palaeocene to mid Eocene, some 63-45 million years ago.

NOS3 Glu298Asp genotype and blood pressure response to endurance training: the HERITAGE family study.

PubMed

Rankinen, T; Rice, T; Pérusse, L; Chagnon, Y C; Gagnon, J; Leon, A S; Skinner, J S; Wilmore, J H; Rao, D C; Bouchard, C

2000-11-01

Endothelium-dependent vasodilation is a mechanism that may affect blood pressure response to endurance training. Because NO plays a central role in this process, the endothelial NO synthase gene is a good candidate for the regulation of exercise blood pressure. We investigated the associations between an endothelial NO synthase gene polymorphism (Glu298Asp) and endurance training-induced changes in resting and submaximal exercise blood pressure in 471 white subjects of the HERITAGE Family Study. Two submaximal exercise tests at 50 W were conducted both before and after a 20-week endurance training program. Steady-state exercise blood pressure was measured twice in each test with an automated unit. The Glu298Asp polymorphism was typed with a PCR-based method and digestion with BAN:II. Both systolic and diastolic blood pressure at 50 W decreased in response to the training program, whereas resting blood pressure remained unchanged. The decrease in diastolic blood pressure at 50 W was greater (P=0.0005, adjusted for age, gender, baseline body mass index, and baseline diastolic blood pressure at 50 W) in the Glu/Glu homozygotes (4.4 [SEM 0.4] mm Hg, n=187) than in the heterozygotes (3.1 [0.4] mm Hg, n=213) and the Asp/Asp homozygotes (1.3 [0.7] mm Hg, n=71). The genotype accounted for 2.3% of the variance in diastolic blood pressure at 50 W training response. Both the Glu298 homozygotes and the heterozygotes had a greater (P=0.013) training-induced reduction in rate-pressure product at 50 W than the Asp298 homozygotes. These data suggest that DNA sequence variation in the endothelial NO synthase gene locus is associated with the endurance training-induced decreases in submaximal exercise diastolic blood pressure and rate-pressure product in sedentary normotensive white subjects.

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis.

PubMed

Bulau, Patrick; Okuno, Atsuro; Thome, Elke; Schmitz, Tina; Peter-Katalinic, Jasna; Keller, Rainer

2005-11-01

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.

Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells.

PubMed

Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

2016-01-01

One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg-Gly-Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration.

Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.

PubMed

Goto, Yoshikuni; Hattori, Akira; Mizutani, Shigehiko; Tsujimoto, Masafumi

2007-12-21

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

Identification and characterization of an autolysin gene, atlA, from Streptococcus criceti.

PubMed

Tamura, Haruki; Yamada, Arisa; Kato, Hirohisa

2012-10-01

AtlA of Streptococcus mutans is a major autolysin and belongs to glycoside hydrolase family 25 with cellosyl of Streptomyces coelicolor. The autolysin gene (atlA) encoding AtlA was identified from S. criceti. AtlA of S. criceti comprises the signal sequence in the N-terminus, the putative cell-wall-binding domain in the middle, and the catalytic domain in the C-terminus. Homology modeling analysis of the catalytic domain of AtlA showed the resemblance of the spatial arrangement of five amino acids around the predicted catalytic cavity to that of cellosyl. Recombinant AtlA and its four point mutants, D655A, D747A, W831A, and D849A, were evaluated on zymogram of S. criceti cells. Lytic activity was destroyed in the mutants D655A and D747A and diminished in the mutants W831A and D849A. These results suggest that Asp655 and Asp747 residues are critical for lytic activity and Trp831 and Asp849 residues are also associated with enzymatic activity.

Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus.

PubMed Central

Ko, E P; Akatsuka, H; Moriyama, H; Shinmyo, A; Hata, Y; Katsube, Y; Urabe, I; Okada, H

1992-01-01

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase. Images Fig. 1. Fig. 4 Fig. 5 PMID:1359880

Novel adipokinetic hormones in the kissing bugs Rhodnius prolixus, Triatoma infestans, Dipetalogaster maxima and Panstrongylus megistus.

PubMed

Marco, Heather G; Simek, Petr; Clark, Kevin D; Gäde, Gerd

2013-03-01

Peptides of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family were isolated and sequenced from the retrocerebral corpora cardiaca of four kissing bugs which are all vectors of the protozoan Trypanosoma cruzi responsible for Chagas' disease. The sequence of three novel AKHs were deduced from the multiple MS(N) electrospray mass data: the octapeptide pGlu-Leu-Thr-Phe-Ser-Thr-Asp-Trp amide (denoted Rhopr-AKH) in Rhodnius prolixus and Panstrongylus megistus, the nonapeptide pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly amide (denoted Triin-AKH) in Triatoma infestans and the decapeptide pGlu-Leu-Thr-Phe-Ser-Asp-Gly-Trp-Gly-Asn amide (denoted Dipma-AKH) in Dipetalogaster maxima. The sequences were confirmed by identical behavior of natural and synthetic forms in reversed-phase HPLC and by CID-MS mass spectra. Conspecific injections of a dose of 10 pmol of the respective synthetic peptides resulted in a small but significant increase of the lipid concentration in the hemolymph. These experiments suggest that AKHs in kissing bugs act to regulate lipid metabolism, possibly during dispersal flights which is one of the mechanisms whereby the insects reach new outbreak areas. Copyright © 2012 Elsevier Inc. All rights reserved.

Simultaneous and Sequential MS/MS Scan Combinations and Permutations in a Linear Quadrupole Ion Trap.

PubMed

Snyder, Dalton T; Szalwinski, Lucas J; Cooks, R Graham

2017-10-17

Methods of performing precursor ion scans as well as neutral loss scans in a single linear quadrupole ion trap have recently been described. In this paper we report methodology for performing permutations of MS/MS scan modes, that is, ordered combinations of precursor, product, and neutral loss scans following a single ion injection event. Only particular permutations are allowed; the sequences demonstrated here are (1) multiple precursor ion scans, (2) precursor ion scans followed by a single neutral loss scan, (3) precursor ion scans followed by product ion scans, and (4) segmented neutral loss scans. (5) The common product ion scan can be performed earlier in these sequences, under certain conditions. Simultaneous scans can also be performed. These include multiple precursor ion scans, precursor ion scans with an accompanying neutral loss scan, and multiple neutral loss scans. We argue that the new capability to perform complex simultaneous and sequential MS n operations on single ion populations represents a significant step in increasing the selectivity of mass spectrometry.

Characterization of the Role of a Highly Conserved Sequence in ATP Binding Cassette Transporter G (ABCG) Family in ABCG1 Stability, Oligomerization, and Trafficking

PubMed Central

2013-01-01

ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol and oxysterol efflux onto lipidated lipoproteins and plays an important role in macrophage reverse cholesterol transport. Here, we identified a highly conserved sequence present in the five ABCG transporter family members. The conserved sequence is located between the nucleotide binding domain and the transmembrane domain and contains five amino acid residues from Asn at position 316 to Phe at position 320 in ABCG1 (NPADF). We found that cells expressing mutant ABCG1, in which Asn316, Pro317, Asp319, and Phe320 in the conserved sequence were replaced with Ala simultaneously, showed impaired cholesterol efflux activity compared with wild type ABCG1-expressing cells. A more detailed mutagenesis study revealed that mutation of Asn316 or Phe 320 to Ala significantly reduced cellular cholesterol and 7-ketocholesterol efflux conferred by ABCG1, whereas replacement of Pro317 or Asp319 with Ala had no detectable effect. To confirm the important role of Asn316 and Phe320, we mutated Asn316 to Asp (N316D) and Gln (N316Q), and Phe320 to Ile (F320I) and Tyr (F320Y). The mutant F320Y showed the same phenotype as wild type ABCG1. However, the efflux of cholesterol and 7-ketocholesterol was reduced in cells expressing ABCG1 mutant N316D, N316Q, or F320I compared with wild type ABCG1. Further, mutations N316Q and F320I impaired ABCG1 trafficking while having no marked effect on the stability and oligomerization of ABCG1. The mutant N316Q and F320I could not be transported to the cell surface efficiently. Instead, the mutant proteins were mainly localized intracellularly. Thus, these findings indicate that the two highly conserved amino acid residues, Asn and Phe, play an important role in ABCG1-dependent export of cellular cholesterol, mainly through the regulation of ABCG1 trafficking. PMID:24320932

Examination of AVPR1a as an autism susceptibility gene.

PubMed

Wassink, T H; Piven, J; Vieland, V J; Pietila, J; Goedken, R J; Folstein, S E; Sheffield, V C

2004-10-01

Impaired reciprocal social interaction is one of the core features of autism. While its determinants are complex, one biomolecular pathway that clearly influences social behavior is the arginine-vasopressin (AVP) system. The behavioral effects of AVP are mediated through the AVP receptor 1a (AVPR1a), making the AVPR1a gene a reasonable candidate for autism susceptibility. We tested the gene's contribution to autism by screening its exons in 125 independent autistic probands and genotyping two promoter polymorphisms in 65 autism affected sibling pair (ASP) families. While we found no nonconservative coding sequence changes, we did identify evidence of linkage and of linkage disequilibrium. These results were most pronounced in a subset of the ASP families with relatively less severe impairment of language. Thus, though we did not demonstrate a disease-causing variant in the coding sequence, numerous nontraditional disease-causing genetic abnormalities are known to exist that would escape detection by traditional gene screening methods. Given the emerging biological, animal model, and now genetic data, AVPR1a and genes in the AVP system remain strong candidates for involvement in autism susceptibility and deserve continued scrutiny.

Substrate binding ability of chemically inactivated pectinase for the substrate pectic acid.

PubMed

Chiba, Y; Kobayashi, M

1995-07-01

Pectinase (polygalacturonase) was purified from a commercial pectinase preparation from a mold. Substrate binding of pectinase was measured by centrifugal affinity chromatography using an immobilized substrate, pectic acid. Desorption of pectinase from the affinity matrix with the substrate pectin and pectic acid gave Kd values of 5.3 and 8.5 mg/ml, respectively. Chemical modification of pectinase by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and diethyl pyrocarbonate (DEP) caused a loss of most of the enzyme activity, but the substrate binding ability was not impaired. Thus, the pectinase preparation was digested with lysyl endopeptidase and the resulting peptides were treated with pectic acid-affinity gel. Three peptide fragments, which were recovered from the affinity column and sequenced, were identical to sequences in the second pectinase gene from Aspergillus niger. The first peptide contained 17 amino acids, Asp101-Ser117, and the second and third peptides corresponded to 18 amino acids of Asn152-Asp169. These results indicate that the inactivated pectinase retained substrate binding ability and would function as an acidic polysaccharide recognizing protein.

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme III/sup mtl/ of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme II/sup mtl/ of Escherichia coli

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

Reiche, B.; Frank, R.; Deutscher, J.

1988-08-23

Enzyme III/sup mtl/ is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, the authors report the isolation of III/sup mtl/ from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of III/sup mtl/ with (/sup 32/P)PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase GLu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp-Asp. The correspondingmore » peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which they assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the III/sup mtl/ proteins was found to be 15,000. They have also determined the N-terminal sequence of both proteins. Comparison of the III/sup mtl/ peptide sequences and the C-terminal part of the enzyme II/sup mtl/ of Escherichia coli reveals considerable sequence homology, which supports the suggestion that II/sup mtl/ of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II.« less

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.

Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus

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

Juvvadi, Praveen Rao; Belina, Detti; Soderblom, Erik J.

2013-02-15

Highlights: ► In vivo interactions of the novel septin AspE were identified by GFP-Trap® affinity purification. ► Septins AspA, AspB, AspC and AspD interacted with AspE in vivo. ► Actin and tubulin interacted with AspE in vivo. ► AspE is phosphorylated at six serine residues in vivo. -- Abstract: We previously analyzed the differential localization patterns of five septins (AspA–E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE–EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated inmore » vivo and interacts with the other septins, AspA, AspB, AspC and AspD. The other major proteins interacting with AspE included the cytoskeletal proteins, actin and tubulin, which may be involved in the organization and transport of the septins. This is the first report analyzing the phosphorylation of AspE and localizing the sites of phosphorylation, and opens opportunities for further analysis on the role of post-translational modifications in the assembly and organization of A. fumigatus septins. This study also describes the previously unknown interaction of AspE with the actin-microtubule network. Furthermore, the novel GFP-Trap® affinity purification method used here complements widely-used GFP localization studies in fungal systems.« less

Aqueous Phase Synthesis of CuIn Alloy Nanoparticles and Their Application for a CIS (CuInSe2)-Based Printable Solar Battery

PubMed Central

Takahashi, Hideyuki; Fujiki, Hironari; Yokoyama, Shun; Kai, Takayuki; Tohji, Kazuyuki

2018-01-01

To apply CuInSe2 (CIS)-based printable solar batteries; an aqueous phase synthesis method of Cu-In (CI) alloy nanoparticles is studied. Metal complexes in the original solution are restricted to homogenized species by utilizing calculations. For example; [(Cu2+)(ASP2−)2] [ASP: the “body (C4H5O4N)” of aspartic acid (C4H7O4N)] is predominant in the pH 6–13 region (CASP/CCu > 6); while In complexes can be restricted to [(In3+)(OH−)(EDTA4−)] (pH 10–12; CEDTA/CIn = 2) and/or [(In3+)(ASP2−)2] (pH 7–9; CASP/CIn = 5). These results indicate that the added amount of complex reagents should be determined by calculations and not the stoichiometric ratio. The reduction potential of homogenized metal complex is measured by cyclic voltammetry (CV) measurements and evaluated by Nernst’s equation using the overall stability constants. CuIn alloy nanoparticles with a small amount of byproduct (In nanoparticles) are successfully synthesized. The CI precursor films are spin-coated onto the substrate using a 2-propanol dispersion. Then the films are converted into CIS solar cells; which show a maximum conversion efficiency of 2.30%. The relationship between the open circuit potential; short circuit current density; and fill factor indicate that smoothing of the CIS films and improving the crystallinity and thickness increase the solar cell conversion efficiency. PMID:29642413

Molecular characterization of an ependymin precursor from goldfish brain.

PubMed

Königstorfer, A; Sterrer, S; Eckerskorn, C; Lottspeich, F; Schmidt, R; Hoffmann, W

1989-01-01

Ependymins are thought to be implicated in fundamental processes involved in plasticity of the goldfish CNS. Gas-phase sequencing of purified ependymins beta and gamma revealed that they share the same N-terminal sequence. Each sequence displays microheterogeneities at several positions. Based on the protein sequences obtained, we constructed synthetic oligonucleotides and used them as hybridization probes for screening cDNA libraries of goldfish brain. In this article we describe the full-length sequence of a mRNA encoding a precursor of ependymins. A cleavable signal sequence characteristic of secretory proteins is located at the N-terminal end, followed directly by the ependymin sequence. Also, two potential N-glycosylation sites were detected. A computer search revealed that ependymins form a novel family of unique proteins.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms

PubMed Central

Abylkassimova, Nikara; Hugall, Andrew F.; O'Hara, Timothy D.; Elphick, Maurice R.

2017-01-01

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales. PMID:28878039

Mutation analysis of pre-mRNA splicing genes in Chinese families with retinitis pigmentosa

PubMed Central

Pan, Xinyuan; Chen, Xue; Liu, Xiaoxing; Gao, Xiang; Kang, Xiaoli; Xu, Qihua; Chen, Xuejuan; Zhao, Kanxing; Zhang, Xiumei; Chu, Qiaomei; Wang, Xiuying

2014-01-01

Purpose Seven genes involved in precursor mRNA (pre-mRNA) splicing have been implicated in autosomal dominant retinitis pigmentosa (adRP). We sought to detect mutations in all seven genes in Chinese families with RP, to characterize the relevant phenotypes, and to evaluate the prevalence of mutations in splicing genes in patients with adRP. Methods Six unrelated families from our adRP cohort (42 families) and two additional families with RP with uncertain inheritance mode were clinically characterized in the present study. Targeted sequence capture with next-generation massively parallel sequencing (NGS) was performed to screen mutations in 189 genes including all seven pre-mRNA splicing genes associated with adRP. Variants detected with NGS were filtered with bioinformatics analyses, validated with Sanger sequencing, and prioritized with pathogenicity analysis. Results Mutations in pre-mRNA splicing genes were identified in three individual families including one novel frameshift mutation in PRPF31 (p.Leu366fs*1) and two known mutations in SNRNP200 (p.Arg681His and p.Ser1087Leu). The patients carrying SNRNP200 p.R681H showed rapid disease progression, and the family carrying p.S1087L presented earlier onset ages and more severe phenotypes compared to another previously reported family with p.S1087L. In five other families, we identified mutations in other RP-related genes, including RP1 p. Ser781* (novel), RP2 p.Gln65* (novel) and p.Ile137del (novel), IMPDH1 p.Asp311Asn (recurrent), and RHO p.Pro347Leu (recurrent). Conclusions Mutations in splicing genes identified in the present and our previous study account for 9.5% in our adRP cohort, indicating the important role of pre-mRNA splicing deficiency in the etiology of adRP. Mutations in the same splicing gene, or even the same mutation, could correlate with different phenotypic severities, complicating the genotype–phenotype correlation and clinical prognosis. PMID:24940031

Low-Temperature Molecular Layer Deposition Using Monofunctional Aromatic Precursors and Ozone-Based Ring-Opening Reactions.

PubMed

Svärd, Laura; Putkonen, Matti; Kenttä, Eija; Sajavaara, Timo; Krahl, Fabian; Karppinen, Maarit; Van de Kerckhove, Kevin; Detavernier, Christophe; Simell, Pekka

2017-09-26

Molecular layer deposition (MLD) is an increasingly used deposition technique for producing thin coatings consisting of purely organic or hybrid inorganic-organic materials. When organic materials are prepared, low deposition temperatures are often required to avoid decomposition, thus causing problems with low vapor pressure precursors. Monofunctional compounds have higher vapor pressures than traditional bi- or trifunctional MLD precursors, but do not offer the required functional groups for continuing the MLD growth in subsequent deposition cycles. In this study, we have used high vapor pressure monofunctional aromatic precursors in combination with ozone-triggered ring-opening reactions to achieve sustained sequential growth. MLD depositions were carried out by using three different aromatic precursors in an ABC sequence, namely with TMA + phenol + O 3 , TMA + 3-(trifluoromethyl)phenol + O 3 , and TMA + 2-fluoro-4-(trifluoromethyl)benzaldehyde + O 3 . Furthermore, the effect of hydrogen peroxide as a fourth step was evaluated for all studied processes resulting in a four-precursor ABCD sequence. According to the characterization results by ellipsometry, infrared spectroscopy, and X-ray reflectivity, self-limiting MLD processes could be obtained between 75 and 150 °C with each of the three aromatic precursors. In all cases, the GPC (growth per cycle) decreased with increasing temperature. In situ infrared spectroscopy indicated that ring-opening reactions occurred in each ABC sequence. Compositional analysis using time-of-flight elastic recoil detection indicated that fluorine could be incorporated into the film when 3-(trifluoromethyl)phenol and 2-fluoro-4-(trifluoromethyl)benzaldehyde were used as precursors.

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

PubMed Central

Magazin, M; Minth, C D; Funckes, C L; Deschenes, R; Tavianini, M A; Dixon, J E

1982-01-01

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels. Images PMID:6127673

A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

PubMed

Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

1995-04-01

The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin.

PubMed

Roy, M; Lee, R W; Kaarsholm, N C; Thøgersen, H; Brange, J; Dunn, M F

1990-06-12

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.

Identification of the Regulon of AphB and Its Essential Roles in LuxR and Exotoxin Asp Expression in the Pathogen Vibrio alginolyticus.

PubMed

Gao, Xiating; Liu, Yang; Liu, Huan; Yang, Zhen; Liu, Qin; Zhang, Yuanxing; Wang, Qiyao

2017-10-15

In Vibrio species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in Vibrio alginolyticus , a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the V. alginolyticus genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of asp and the master quorum-sensing (QS) regulator luxR DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the luxR promoter region overlaps that of LuxR-binding site I, which mediates the positive control of luxR promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in V. alginolyticus IMPORTANCE In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in V. alginolyticus , a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the aphB gene itself was identified to be positively autoregulated, and AphB also positively controlled asp and luxR expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression. Copyright © 2017 American Society for Microbiology.

In-vitro and in-vivo phenotype of type Asia 1 foot-and-mouth disease viruses utilizing two non-RGD receptor recognition sites

PubMed Central

2011-01-01

Background Foot-and-mouth disease virus (FMDV) uses a highly conserved Arg-Gly-Asp (RGD) triplet for attachment to host cells and this motif is believed to be essential for virus viability. Previous sequence analyses of the 1D-encoding region of an FMDV field isolate (Asia1/JS/CHA/05) and its two derivatives indicated that two viruses, which contained an Arg-Asp-Asp (RDD) or an Arg-Ser-Asp (RSD) triplet instead of the RGD integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. To examine the influence of single amino acid substitutions in the receptor binding site of the RDD-containing FMD viral genome on virus viability and the ability of non-RGD FMDVs to cause disease in susceptible animals, we constructed an RDD-containing FMDV full-length cDNA clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals. Results Amino acid sequence analysis of the 1D-coding region of different derivatives derived from the Asia1/JS/CHA/05 field isolate revealed that the RDD mutants became dominant or achieved population equilibrium with coexistence of the RGD and RSD subpopulations at an early phase of type Asia1 FMDV quasispecies evolution. Furthermore, the RDD and RSD sequences remained genetically stable for at least 20 passages. Using reverse genetics, the RDD-, RSD-, and RGD-containing FMD viruses were rescued from full-length cDNA clones, and single amino acid substitution in RDD-containing FMD viral genome did not affect virus viability. The genetically engineered viruses replicated stably in BHK-21 cells and had similar growth properties to the parental virus. The RDD parental virus and two non-RGD recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia. Conclusions FMDV quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site. The RDD-containing FMD viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. The viruses expressing non-RGD receptor binding sites can replicate stably in vitro and produce typical FMD clinical disease in susceptible animals. PMID:21711567

STUDIES ON THE IMMUNE RESPONSE TO A CHARACTERIZED ANTIGENIC DETERMINANT OF THE TOBACCO MOSAIC VIRUS PROTEIN

PubMed Central

Spitler, Lynn; Benjamini, E.; Young, Janis D.; Kaplan, Harvey; Fudenberg, H. H.

1970-01-01

The following peptides have previously been shown to bind specifically with antibodies to TMVP: (a) An eicosapeptide representing residues 93–112 of TMVP and having the sequence Ileu-Ileu-Glu-Val-Glu-AspNH2-GluNH2-Ala-AspNH2-Pro-Thr-Thr-Ala-Glu-Thr-Leu-Asp-Ala-Thr-Arg. (b) Its C-terminal decapeptide. (c) Its C-terminal pentapeptide. (d) N-octanoyl-C-terminal-tripeptide. (e) (Lys)4-C-terminal-pentapeptide. (f) (Lys)7 C-terminal-pentapeptide. The present communication deals with the investigation of several parameters of the immunological activity of the peptides. The results show that none of the peptides tested were immunogenic in guinea pigs, nor did they stimulate the incorporation of 14C-thymidine by spleen cells derived from TMVP-primed animals. Results also showed that all of the peptides tested could elicit specific delayed and immediate skin reactions in TMVP-sensitized guinea pigs, and furthermore, that the peptides could specifically inhibit the migration of peritoneal exudate cells derived from these animals. The elicitation of delayed skin reactions and the ability to inhibit migration of peritoneal exudate cells were independent of carrier specificity. PMID:5409944

Identification of Novel Placentally Expressed Aspartic Proteinase in Humans

PubMed Central

Majewska, Marta; Lipka, Aleksandra; Panasiewicz, Grzegorz; Gowkielewicz, Marek; Jozwik, Marcin; Majewski, Mariusz Krzysztof; Szafranska, Bozena

2017-01-01

This study presents pioneering data concerning the human pregnancy-associated glycoprotein-Like family, identified in the genome, of the term placental transcriptome and proteome. RNA-seq allowed the identification of 1364 bp hPAG-L/pep cDNA with at least 56.5% homology with other aspartic proteinases (APs). In silico analyses revealed 388 amino acids (aa) of full-length hPAG-L polypeptide precursor, with 15 aa-signal peptide, 47 aa-blocking peptide and 326 aa-mature protein, and two Asp residues (D), specific for a catalytic cleft of the APs (VVFDTGSSNLWV91-102 and AIVDTGTSLLTG274-285). Capillary sequencing identified 9330 bp of the hPAG-L gene (Gen Bank Acc. No. KX533473), composed of nine exons and eight introns. Heterologous Western blotting revealed the presence of one dominant 60 kDa isoform of the hPAG-L amongst cellular placental proteins. Detection with anti-pPAG-P and anti-Rec pPAG2 polyclonals allowed identification of the hPAG-L proteins located within regions of chorionic villi, especially within the syncytiotrophoblast of term singleton placentas. Our novel data extend the present knowledge about the human genome, as well as placental transcriptome and proteome during term pregnancy. Presumably, this may contribute to establishing a new diagnostic tool for examination of some disturbances during human pregnancy, as well as growing interest from both scientific and clinical perspectives. PMID:28594357

Identification of Novel Placentally Expressed Aspartic Proteinase in Humans.

PubMed

Majewska, Marta; Lipka, Aleksandra; Panasiewicz, Grzegorz; Gowkielewicz, Marek; Jozwik, Marcin; Majewski, Mariusz Krzysztof; Szafranska, Bozena

2017-06-08

This study presents pioneering data concerning the human pregnancy-associated glycoprotein-Like family, identified in the genome, of the term placental transcriptome and proteome. RNA-seq allowed the identification of 1364 bp hPAG-L/pep cDNA with at least 56.5% homology with other aspartic proteinases (APs). In silico analyses revealed 388 amino acids (aa) of full-length hPAG-L polypeptide precursor, with 15 aa-signal peptide, 47 aa-blocking peptide and 326 aa-mature protein, and two Asp residues (D), specific for a catalytic cleft of the APs (VVFDTGSSNLWV91-102 and AIVDTGTSLLTG274-285). Capillary sequencing identified 9330 bp of the hPAG-L gene (Gen Bank Acc. No. KX533473), composed of nine exons and eight introns. Heterologous Western blotting revealed the presence of one dominant 60 kDa isoform of the hPAG-L amongst cellular placental proteins. Detection with anti-pPAG-P and anti-Rec pPAG2 polyclonals allowed identification of the hPAG-L proteins located within regions of chorionic villi, especially within the syncytiotrophoblast of term singleton placentas. Our novel data extend the present knowledge about the human genome, as well as placental transcriptome and proteome during term pregnancy. Presumably, this may contribute to establishing a new diagnostic tool for examination of some disturbances during human pregnancy, as well as growing interest from both scientific and clinical perspectives.

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40T*

PubMed Central

Wang, Weijun; Yan, Ruoyu; Nocek, Boguslaw P.; Vuong, Thu V.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Gatenholm, Paul; Toriz, Guillermo; Tenkanen, Maija; Savchenko, Alexei; Master, Emma R.

2016-01-01

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C+ domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C+ in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes. PMID:27129264

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40 T

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

Wang, Weijun; Yan, Ruoyu; Nocek, Boguslaw P.

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as -glucuronidases that release the - (132)-linked (Me)GlcAp side groups. Herein, a family GH115 enzyme from the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domainmore » architecture, with an additional insertion C domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.« less

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics*

PubMed Central

Guo, Xiaofeng; Trudgian, David C.; Lemoff, Andrew; Yadavalli, Sivaramakrishna; Mirzaei, Hamid

2014-01-01

Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases. PMID:24696503

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

PubMed

Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

1994-07-08

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Investigation of phase evolution of CaCu{sub 3}Ti{sub 4}O{sub 12} (CCTO) by in situ synchrotron high-temperature powder diffraction

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

Ouyang, Xin; Huang, Saifang; School of Materials Science and Technology, China University of Geosciences

2014-03-15

In situ synchrotron X-ray powder diffraction was used to study the high-temperature phase evolution of CaCu{sub 3}Ti{sub 4}O{sub 12} (CCTO) precursors prepared via solid-state and sol–gel methods. After the precursors are heated to 1225 °C, the CCTO phase is the main phase observed in the calcined powder, with the presence of some minor impurities. Comparing the two precursors, we found that the onset temperature for the CCTO phase formation is 800 °C in the sol–gel precursor, lower than that in the solid-state precursor (875 °C). Intermediate phases were only observed in the sol–gel precursor. Both precursors are able to bemore » calcined to sub-micrometric sized powders. Based on the synchrotron data along with differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), the phase formation sequence and mechanism during calcination are proposed in this study. -- Graphical abstract: The in situ synchrotron HT-XRD patterns of CCTO sol–gel and solid-state precursor. Highlights: • Phase formation sequence/mechanism in two CCTO precursors has been established. • Formation temperature of CCTO via sol–gel method is lower than solid-state method. • Intermediate phases are only observed in the sol–gel precursor. • Both precursors are able to be calcined into sub-micrometric sized powders.« less

Characterization of Urtica dioica agglutinin isolectins and the encoding gene family.

PubMed

Does, M P; Ng, D K; Dekker, H L; Peumans, W J; Houterman, P M; Van Damme, E J; Cornelissen, B J

1999-01-01

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA-isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA-isolectins. The deduced amino acid sequences share 78.9-98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.

Maintenance of coat protein N-terminal net charge and not primary sequence is essential for zucchini yellow mosaic virus systemic infectivity.

PubMed

Kimalov, Boaz; Gal-On, Amit; Stav, Ran; Belausov, Eduard; Arazi, Tzahi

2004-11-01

Zucchini yellow mosaic virus (ZYMV) surface exposed coat protein (CP) N-terminal domain (Nt) is 43 aa long and contains an equal number of positively and negatively charged amino acid residues (CP-Nt net charge = 0). A ZYMV-AGII truncation mutant lacking the first 20 aa of its CP-Nt (AGII-CP Delta 20; CP-Nt net charge = +2) was found to be systemically non-infectious even though AGII mutants harbouring larger CP-Nt deletions were previously demonstrated to be fully infectious. Nevertheless, AGII-CP Delta 20 infectivity was restored by fusion to its CP-Nt two Asp residues or a negatively charged Myc peptide, both predicted to neutralize CP-Nt net positive charge. To evaluate further the significance of CP-Nt net charge for AGII infectivity, a series of CP-Nt net charge mutants was generated and analysed for systemic infectivity of squash plants. AGII-CP(KKK) harbouring a CP-Nt amino fusion of three Lys residues (CP-Nt net charge = +3) was not systemically infectious. Addition of up to four Asp residues to CP-Nt did not abolish virus infectivity, although certain mutants were genetically unstable and had delayed infectivity. Addition of five negatively charged residues abolished infectivity (AGII-CP(DDDDD); CP-Nt net charge = -5) even though a recombinant CP(DDDDD) could assemble into potyviral-like particle in bacteria. Neutralization of CP-Nt net charge by fusing Asp or Lys residues recovered infectivity of AGII-CP(KKK) and AGII-CP(DDDDD). GFP-tagging of these mutants has demonstrated that both viruses have defective cell-to-cell movement. Together, these findings suggest that maintenance of CP-Nt net charge and not primary sequence is essential for ZYMV infectivity.

EEC- and ADULT-associated TP63 mutations exhibit functional heterogeneity toward P63 responsive sequences.

PubMed

Monti, Paola; Russo, Debora; Bocciardi, Renata; Foggetti, Giorgia; Menichini, Paola; Divizia, Maria T; Lerone, Margherita; Graziano, Claudio; Wischmeijer, Anita; Viadiu, Hector; Ravazzolo, Roberto; Inga, Alberto; Fronza, Gilberto

2013-06-01

TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability. © 2013 Wiley Periodicals, Inc.

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

PubMed

Yang, Xiaoxue; Cong, Hua; Song, Jinzhu; Zhang, Junzheng

2013-11-01

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

PubMed Central

Shen, Jana K.

2010-01-01

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pKas allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pKa for Asp8 in the denatured state of wild-type, which is due to a nonnative interaction between Asp8 and Lys12. Interestingly, the simulation also shows a nonnative interaction between Asp8 and Glu48 in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape. PMID:20682271

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.

PubMed

Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D; Proudfoot, Michael; Makarova, Kira S; Kudritska, Marina; Kochinyan, Samvel; Wang, Shuren; Chruszcz, Maksymilian; Minor, Wladek; Koonin, Eugene V; Edwards, Aled M; Savchenko, Alexei; Yakunin, Alexander F

2008-07-18

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

A Novel High-Molecular-Mass Bacteriocin Produced by Enterococcus faecium: Biochemical Features and Mode of Action.

PubMed

Vasilchenko, A S; Vasilchenko, A V; Valyshev, A V; Rogozhin, E A

2018-02-08

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.

Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis of hospital origin: pbp4 gene polymorphism and genetic diversity.

PubMed

Conceição, Natália; da Silva, Lucas Emanuel Pinheiro; Darini, Ana Lúcia da Costa; Pitondo-Silva, André; de Oliveira, Adriana Gonçalves

2014-12-01

Despite the spread of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates in diverse countries, the mechanisms leading to this unusual resistance phenotype have not yet been investigated. The aim of this study was to evaluate whether polymorphism in the pbp4 gene is associated with penicillin resistance in PRASEF isolates and to determine their genetic diversity. E. faecalis isolates were recovered from different clinical specimens of hospitalized patients from February 2006 to June 2010. The β-lactam minimal inhibitory concentrations (MICs) were determined by E-test®. The PCR-amplified pbp4 gene was sequenced with an automated sequencer. The genetic diversities of the isolates were established by PFGE (pulsed-field gel electrophoresis) and MLST (multilocus sequencing typing). Seventeen non-producing β-lactamase PRASEF and 10 penicillin-susceptible, ampicillin-susceptible E. faecalis (PSASEF) strains were analyzed. A single-amino-acid substitution (Asp-573→Glu) in the penicillin-binding domain was significantly found in all PRASEF isolates by sequencing of the pbp4 gene but not in the penicillin-susceptible isolates. In contrast to the PSASEF isolates, a majority of the PRASEFs had similar PFGE profiles. Six representative PRASEF isolates were resolved by MLST into ST9 and ST524 and belong to the globally dispersed clonal complex 9 (CC9). In conclusion, it appears quite likely that the amino acid alteration (Asp-573→Glu) found in the PBP4 of the Brazilian PRASEF isolates may account for their reduced susceptibility to penicillin, although other resistance mechanisms remain to be investigated. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of septins across kingdoms reveals orthology and new motifs.

PubMed

Pan, Fangfang; Malmberg, Russell L; Momany, Michelle

2007-07-01

Septins are cytoskeletal GTPase proteins first discovered in the fungus Saccharomyces cerevisiae where they organize the septum and link nuclear division with cell division. More recently septins have been found in animals where they are important in processes ranging from actin and microtubule organization to embryonic patterning and where defects in septins have been implicated in human disease. Previous studies suggested that many animal septins fell into independent evolutionary groups, confounding cross-kingdom comparison. In the current work, we identified 162 septins from fungi, microsporidia and animals and analyzed their phylogenetic relationships. There was support for five groups of septins with orthology between kingdoms. Group 1 (which includes S. cerevisiae Cdc10p and human Sept9) and Group 2 (which includes S. cerevisiae Cdc3p and human Sept7) contain sequences from fungi and animals. Group 3 (which includes S. cerevisiae Cdc11p) and Group 4 (which includes S. cerevisiae Cdc12p) contain sequences from fungi and microsporidia. Group 5 (which includes Aspergillus nidulans AspE) contains sequences from filamentous fungi. We suggest a modified nomenclature based on these phylogenetic relationships. Comparative sequence alignments revealed septin derivatives of already known G1, G3 and G4 GTPase motifs, four new motifs from two to twelve amino acids long and six conserved single amino acid positions. One of these new motifs is septin-specific and several are group specific. Our studies provide an evolutionary history for this important family of proteins and a framework and consistent nomenclature for comparison of septin orthologs across kingdoms.

Purification and characterization of angiotensin I converting enzyme inhibition peptides from sandworm Sipunculus nudus

NASA Astrophysics Data System (ADS)

Sun, Xueping; Wang, Man; Liu, Buming; Sun, Zhenliang

2017-10-01

Three angiotensin I converting enzyme (ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC50 values of the purified peptides for ACE inhibition activity were 34.72 μmol L-1, 20.55 μmol L-1 and 22.77 μmol L-1, respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.

An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

PubMed

Freimuth, P; Anderson, C W

1993-03-01

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Automatic Command Sequence Generation

NASA Technical Reports Server (NTRS)

Fisher, Forest; Gladded, Roy; Khanampompan, Teerapat

2007-01-01

Automatic Sequence Generator (Autogen) Version 3.0 software automatically generates command sequences for the Mars Reconnaissance Orbiter (MRO) and several other JPL spacecraft operated by the multi-mission support team. Autogen uses standard JPL sequencing tools like APGEN, ASP, SEQGEN, and the DOM database to automate the generation of uplink command products, Spacecraft Command Message Format (SCMF) files, and the corresponding ground command products, DSN Keywords Files (DKF). Autogen supports all the major multi-mission mission phases including the cruise, aerobraking, mapping/science, and relay mission phases. Autogen is a Perl script, which functions within the mission operations UNIX environment. It consists of two parts: a set of model files and the autogen Perl script. Autogen encodes the behaviors of the system into a model and encodes algorithms for context sensitive customizations of the modeled behaviors. The model includes knowledge of different mission phases and how the resultant command products must differ for these phases. The executable software portion of Autogen, automates the setup and use of APGEN for constructing a spacecraft activity sequence file (SASF). The setup includes file retrieval through the DOM (Distributed Object Manager), an object database used to store project files. This step retrieves all the needed input files for generating the command products. Depending on the mission phase, Autogen also uses the ASP (Automated Sequence Processor) and SEQGEN to generate the command product sent to the spacecraft. Autogen also provides the means for customizing sequences through the use of configuration files. By automating the majority of the sequencing generation process, Autogen eliminates many sequence generation errors commonly introduced by manually constructing spacecraft command sequences. Through the layering of commands into the sequence by a series of scheduling algorithms, users are able to rapidly and reliably construct the desired uplink command products. With the aid of Autogen, sequences may be produced in a matter of hours instead of weeks, with a significant reduction in the number of people on the sequence team. As a result, the uplink product generation process is significantly streamlined and mission risk is significantly reduced. Autogen is used for operations of MRO, Mars Global Surveyor (MGS), Mars Exploration Rover (MER), Mars Odyssey, and will be used for operations of Phoenix. Autogen Version 3.0 is the operational version of Autogen including the MRO adaptation for the cruise mission phase, and was also used for development of the aerobraking and mapping mission phases for MRO.

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

PubMed

Amiche, M; Ducancel, F; Lajeunesse, E; Boulain, J C; Ménez, A; Nicolas, P

1993-03-31

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

PubMed

Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

2017-01-26

Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

PubMed Central

Huili, Wang; Xiaokai, Zhao; Meili, Lin; Dahlgren, Randy A.; Wei, Chen; Jaiopeng, Zhou; Chengyang, Xu; Chunlei, Jin; Yi, Xu; Xuedong, Wang; Li, Ding; Qiyu, Bao

2013-01-01

Arthrospira (Spirulina) platensis (ASP) is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C) using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328). The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones), energy metabolism (photosynthesis, respiratory electron transport), translation (ribosomal structure and biogenesis) and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and translation levels. The remaining 12 genes showed inconsistent protein expression with transcription level and accounted for 31.6% of the total target genes. PMID:24349519

Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

PubMed

Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

2015-12-10

Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

NASA Astrophysics Data System (ADS)

Qi, Fei; Guo, Huarong; Wang, Jian

2008-02-01

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

The Highly Conserved Asp23 Family Protein YqhY Plays a Role in Lipid Biosynthesis in Bacillus subtilis

PubMed Central

Tödter, Dominik; Gunka, Katrin; Stülke, Jörg

2017-01-01

In most bacteria, fatty acid biosynthesis is an essential process that must be controlled by the availability of precursors and by the needs of cell division. So far, no mechanisms controlling synthesis of malonyl-coenzyme A (CoA), the committed step in fatty acid synthesis, have been identified in the Gram-positive model bacterium Bacillus subtilis. We have studied the localization and function of two highly expressed proteins of unknown function, YqhY and YloU. Both proteins are members of the conserved and widespread Asp23 family. While the deletion of yloU had no effect, loss of the yqhY gene induced the rapid acquisition of suppressor mutations. The vast majority of these mutations affect subunits of the acetyl-CoA carboxylase (ACCase) complex, the enzyme that catalyzes the formation of malonyl-CoA. Moreover, lack of yqhY is accompanied by the formation of lipophilic clusters in the polar regions of the cells indicating an increased activity of ACCase. Our results suggest that YqhY controls the activity of ACCase and that this control results in inhibition of ACCase activity. Hyperactivity of the enzyme complex in the absence of YqhY does then provoke mutations that cause reduced ACCase activity. PMID:28579978

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

PubMed

Pane, Katia; Verrillo, Mariavittoria; Avitabile, Angela; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Di Maro, Antimo; Rega, Camilla; Amoresano, Angela; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

2018-04-18

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB L , an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion.

PubMed

Chen, Tianbao; Gagliardo, Ron; Walker, Brian; Zhou, Mei; Shaw, Chris

2005-12-01

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.

Evaluation of Ga-DOTA-(D-Asp)n as bone imaging agents: D-aspartic acid peptides as carriers to bone.

PubMed

Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Makino, Akira; Kozaka, Takashi; Kiyono, Yasushi; Shiba, Kazuhiro; Odani, Akira

2017-10-25

67 Ga-DOTA-(L-Asp) 11 and 67 Ga-DOTA-(L-Asp) 14 , which have been developed as bone imaging agents, showed a high accumulation in bone and a rapid blood clearance in mice. However, peptides composed of D-amino acids are more stable in vivo than those composed of their L-equivalents. In this study, 67 Ga-DOTA-(D-Asp) n (n = 2, 5, 8, 11, or 14) were synthesized using the Fmoc-based solid-phase methodology and evaluated. In hydroxyapatite binding assay, binding of 67 Ga-DOTA-(D-Asp) n tended to increase with increasing length of the amino acid chain. 67 Ga-DOTA-(D-Asp) 11 and 67 Ga-DOTA-(D-Asp) 14 caused a high accumulation of radioactivity in the bones of the mice. However, the results for 67 Ga-DOTA-(D-Asp) n and 67 Ga-DOTA-(L-Asp) n were comparable. In urine analyses, the proportion of intact complex after injection of 67 Ga-DOTA-(D-Asp) 14 was significantly higher than that of 67 Ga-DOTA-(L-Asp) 14 . Although 67 Ga-DOTA-(D-Asp) 14 was more stable than 67 Ga-DOTA-(L-Asp) 14 , the properties of 67 Ga-DOTA-(D-Asp) n and 67 Ga-DOTA-(L-Asp) n as bone imaging agents may be comparable.

Quantitative determination of free D-Asp, L-Asp and N-methyl-D-aspartate in mouse brain tissues by chiral separation and Multiple Reaction Monitoring tandem mass spectrometry.

PubMed

Fontanarosa, Carolina; Pane, Francesca; Sepe, Nunzio; Pinto, Gabriella; Trifuoggi, Marco; Squillace, Marta; Errico, Francesco; Usiello, Alessandro; Pucci, Piero; Amoresano, Angela

2017-01-01

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

Heterozygous Hb Hope [beta136(H14)Gly --> Asp] in association with heterozygous beta0-thalassemia with apparent homozygous expression, in a Spanish patient.

PubMed

Beneitez, David; Carrera, Alícia; Duran-Suárez, Joan Ramón; Paz, Victoria; León, Antonio; García Talavera, Juan

2006-01-01

Hb Hope [beta136(H14)Gly --> Asp (GGT --> GAT)] has been found alone or in combination with other globin gene mutations in several African-American families, as well as in Japanese, Thai, Laotian, Cuban and Mauritanian families. We report the hematological and molecular characteristics of a heterozygous association of Hb Hope with beta0-thalassemia (thal) in a Spanish patient, in whom the level of expression of abnormal hemoglobin (Hb) by cation exchange high performance liquid chromatography (HPLC) and electrophoresis suggested initially a homozygous expression of the abnormal Hb, although sequencing of the polymerase chain reaction (PCR)-amplified beta-globin gene demonstrated a heterozygous genotype for Hb Hope. To the best of our knowledge, this is the first description of a case of Hb Hope in a Spanish family.

Crystal structure of human nicotinamide riboside kinase.

PubMed

Khan, Javed A; Xiang, Song; Tong, Liang

2007-08-01

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

Associations between XPD Asp312Asn Polymorphism and Risk of Head and Neck Cancer: A Meta-Analysis Based on 7,122 Subjects

PubMed Central

Hu, Yuan Yuan; Yuan, Hua; Jiang, Guang Bing; Chen, Ning; Wen, Li; Leng, Wei Dong; Zeng, Xian Tao; Niu, Yu Ming

2012-01-01

Background To investigate the association between XPD Asp312Asn polymorphism and head and neck cancer risk through this meta-analysis. Methods We performed a meta-analysis of 9 published case-control studies including 2,670 patients with head and neck cancer and 4,452 controls. An odds ratio (OR) with a 95% confidence interval (CI) was applied to assess the association between XPD Asp312Asn polymorphism and head and neck cancer risk. Results Overall, no significant association between XPD Asp312Asn polymorphism and head and neck cancer risk was found in this meta-analysis (Asn/Asn vs. Asp/Asp: OR = 0.95, 95%CI = 0.80–1.13, P = 0.550, P heterogeneity = 0.126; Asp/Asn vs. Asp/Asp: OR = 1.11, 95%CI = 0.99–1.24, P = 0.065, P heterogeneity = 0.663; Asn/Asn+Asp/Asn vs. Asp/Asp: OR = 1.07, 95%CI = 0.97–1.19, P = 0.189, P heterogeneity = 0.627; Asn/Asn vs. Asp/Asp+Asp/Asn: OR = 0.87, 95%CI = 0.68–1.10, P = 0.243, P heterogeneity = 0.089). In the subgroup analysis by HWE, ethnicity, and study design, there was still no significant association detected in all genetic models. Conclusions This meta-analysis demonstrates that XPD Asp312Asn polymorphism may not be a risk factor for developing head and neck cancer. PMID:22536360

Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

PubMed Central

Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

2016-01-01

Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

Thermodynamics, morphology, and kinetics of early-stage self-assembly of π-conjugated oligopeptides

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

None, None

Synthetic oligopeptides containing π-conjugated cores self-assemble novel materials with attractive electronic and photophysical properties. All-atom, explicit solvent molecular dynamics simulations of Asp-Phe-Ala-Gly-OPV3-Gly-Ala-Phe-Asp peptides were used to parameterize an implicit solvent model to simulate early-stage self-assembly. Under low-pH conditions, peptides assemble into β-sheet-like stacks with strongly favorable monomer association free energies of ΔF ≈ -25kBT. Aggregation at high-pH produces disordered aggregates destabilized by Coulombic repulsion between negatively charged Asp termini (ΔF ≈ -5kBT). In simulations of hundreds of monomers over 70 ns we observe the spontaneous formation of up to undecameric aggregates under low-pH conditions. Modeling assembly as a continuous-time Markovmore » process, we infer transition rates between different aggregate sizes and microsecond relaxation times for early-stage assembly. Our data suggests a hierarchical model of assembly in which peptides coalesce into small clusters over tens of nanoseconds followed by structural ripening and diffusion limited aggregation on longer time scales. This work provides new molecular-level understanding of early-stage assembly, and a means to study the impact of peptide sequence and aromatic core chemistry upon the thermodynamics, assembly kinetics, and morphology of the supramolecular aggregates.« less

Cyclopeptide Dmt-[D-Lys-p-CF3-Phe-Phe-Asp]NH2, a novel G protein-biased agonist of the mu opioid receptor.

PubMed

Piekielna-Ciesielska, Justyna; Ferrari, Federica; Calo', Girolamo; Janecka, Anna

2018-03-01

Opioid peptides and alkaloid drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor which belongs to the G protein-coupled receptor (GPCR) family. A new important pharmacological concept in the field of GPCRs is biased agonism. Two mu receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH 2 (C-36) and Dmt-c[D-Lys-Phe-p-CF 3 -Phe-Asp]NH 2 (F-81), were evaluated in terms of their ability to promote or block mu receptor/G protein and mu receptor/β-arrestin interactions. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that C-36 activated both, G protein and β-arrestin pathways. Incorporation of trifluoromethyl group into the aromatic ring of phenylalanine in the sequence of F-81 led to activation of G-protein pathway rather than β-arrestin recruitment. Opioid cyclopeptide F-81 turned out to be a biased G protein mu receptor agonist. Such biased ligands are able to separate the biological actions of an activated receptor and have the potential to become more effective drug candidates with fewer side effects. Copyright © 2017 Elsevier Inc. All rights reserved.

Simultaneous measurement of Aspartate, NAA, and NAAG using HERMES spectral editing at 3 Tesla.

PubMed

Chan, Kimberly L; Saleh, Muhammad G; Oeltzschner, Georg; Barker, Peter B; Edden, Richard A E

2017-07-15

It has previously been shown that the HERMES method ('Hadamard Encoding and Reconstruction of MEGA-Edited Spectroscopy') can be used to simultaneously edit pairs of metabolites (such as N-acetyl-aspartate (NAA) and N-acetyl aspartyl glutamate (NAAG), or glutathione and GABA). In this study, HERMES is extended for the simultaneous editing of three overlapping signals, and illustrated for the example of NAA, NAAG and Aspartate (Asp). Density-matrix simulations were performed in order to optimize the HERMES sequence. The method was tested in NAA and Asp phantoms, and applied to the centrum semiovale of the nine healthy control subjects that were scanned at 3T. Both simulations and phantom experiments showed similar metabolite multiplet patterns with good segregation of all three metabolites. In vivo measurements show consistent relative signal intensities and multiplet patterns with concentrations in agreement with literature values. Simulations indicate co-editing of glutathione, glutamine, and glutamate, but their signals do not significantly overlap with the detected aspartyl resonances. This study demonstrates that a four-step Hadamard-encoded editing scheme can be used to simultaneously edit three otherwise overlapping metabolites, and can measure NAA, NAAG, and Asp in vivo in the brain at 3T with minimal crosstalk. Copyright © 2017 Elsevier Inc. All rights reserved.

Thermodynamics, morphology, and kinetics of early-stage self-assembly of π-conjugated oligopeptides

DOE PAGES

None, None

2016-03-22

Synthetic oligopeptides containing π-conjugated cores self-assemble novel materials with attractive electronic and photophysical properties. All-atom, explicit solvent molecular dynamics simulations of Asp-Phe-Ala-Gly-OPV3-Gly-Ala-Phe-Asp peptides were used to parameterize an implicit solvent model to simulate early-stage self-assembly. Under low-pH conditions, peptides assemble into β-sheet-like stacks with strongly favorable monomer association free energies of ΔF ≈ -25kBT. Aggregation at high-pH produces disordered aggregates destabilized by Coulombic repulsion between negatively charged Asp termini (ΔF ≈ -5kBT). In simulations of hundreds of monomers over 70 ns we observe the spontaneous formation of up to undecameric aggregates under low-pH conditions. Modeling assembly as a continuous-time Markovmore » process, we infer transition rates between different aggregate sizes and microsecond relaxation times for early-stage assembly. Our data suggests a hierarchical model of assembly in which peptides coalesce into small clusters over tens of nanoseconds followed by structural ripening and diffusion limited aggregation on longer time scales. This work provides new molecular-level understanding of early-stage assembly, and a means to study the impact of peptide sequence and aromatic core chemistry upon the thermodynamics, assembly kinetics, and morphology of the supramolecular aggregates.« less

Lauroyl-L-aspartate decreased food intake and body temperature in neonatal chicks.

PubMed

Erwan, E; Chowdhury, V S; Ito, K; Furuse, M

2013-11-15

We hypothesized that the effects of L- and D-amino acids might be influenced when conjugated with fatty acid. Thus, the effects of oral administration of lauroyl-L-aspartate (Lau-L-Asp) as well as lauroyl-D-aspartate (Lau-D-Asp) were examined. In Experiment 1, oral administration of both Lau-L-Asp and Lau-D-Asp decreased food intake while L- or D-Asp did not influence food intake. Interestingly, only Lau-L-Asp decreased body temperature. Experiment 2 was conducted to determine whether non-conjugated mixture of L-Asp plus lauric acid has same effects under ad libitum feeding conditions. Lau-L-Asp decreased food intake and body temperature, but L-Asp plus lauric acid did not show any effect studied. In Experiment 3, we found that Lau-L-Asp declined food intake as well as time-dependently suppressed the body temperature in fasted chicks. However, L-Asp plus lauric acid did not show any effect. These results suggest that Lau-L-Asp may exert anorexigenic and hypothermic actions in chicks. © 2013.

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

PubMed Central

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

1994-01-01

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

The effects of acylation stimulating protein supplementation VS antibody neutralization on energy expenditure in wildtype mice.

PubMed

Paglialunga, Sabina; Fisette, Alexandre; Munkonda, Mercedes; Gao, Ying; Richard, Denis; Cianflone, Katherine

2010-04-23

Acylation stimulating protein (ASP) is an adipogenic hormone that stimulates triglyceride (TG) synthesis and glucose transport in adipocytes. Previous studies have shown that ASP-deficient C3 knockout mice are hyperphagic yet lean, as they display increased oxygen consumption and fatty acid oxidation compared to wildtype mice. In the present study, antibodies against ASP (Anti-ASP) and human recombinant ASP (rASP) were tested in vitro and in vivo. Continuous administration for 4 weeks via osmotic mini-pump of Anti-ASP or rASP was evaluated in wildtype mice on a high-fat diet (HFD) to examine their effects on body weight, food intake and energy expenditure. In mature murine adipocytes, rASP significantly stimulated fatty acid uptake (+243% vs PBS, P < 0.05) while Anti-ASP neutralized the rASP response. Mice treated with Anti-ASP showed elevated energy expenditure (P < 0.0001), increased skeletal muscle glucose oxidation (+141%, P < 0.001), reduced liver glycogen (-34%, P < 0.05) and glucose-6-phosphate content (-64%, P = 0.08) compared to control mice. There was no change in body weight, food intake, fasting insulin, adiponectin, CRP or TG levels compared to controls. Interestingly, HFD mice treated with rASP showed the opposite phenotype with reduced energy expenditure (P < 0.0001) and increased body weight (P < 0.05), cumulative food intake (P < 0.0001) and liver glycogen content (+59%, P < 0.05). Again, there was no change in circulating insulin, adiponectin, CRP or TG levels, however, plasma free fatty acids were reduced (-48%, P < 0.05). In vitro, Anti-ASP effectively neutralized ASP stimulated fatty acid uptake. In vivo, Anti-ASP treatment increased whole body energy utilization while rASP increased energy storage. Therefore, ASP is a potent anabolic hormone that may also be a mediator of energy expenditure.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

PubMed

Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

2014-07-01

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

CID-miRNA: A web server for prediction of novel miRNA precursors in human genome

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

Tyagi, Sonika; Vaz, Candida; Gupta, Vipin

2008-08-08

microRNAs (miRNA) are a class of non-protein coding functional RNAs that are thought to regulate expression of target genes by direct interaction with mRNAs. miRNAs have been identified through both experimental and computational methods in a variety of eukaryotic organisms. Though these approaches have been partially successful, there is a need to develop more tools for detection of these RNAs as they are also thought to be present in abundance in many genomes. In this report we describe a tool and a web server, named CID-miRNA, for identification of miRNA precursors in a given DNA sequence, utilising secondary structure-based filteringmore » systems and an algorithm based on stochastic context free grammar trained on human miRNAs. CID-miRNA analyses a given sequence using a web interface, for presence of putative miRNA precursors and the generated output lists all the potential regions that can form miRNA-like structures. It can also scan large genomic sequences for the presence of potential miRNA precursors in its stand-alone form. The web server can be accessed at (http://mirna.jnu.ac.in/cidmirna/)« less

Dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn inhibits the proliferation rate of HL-60 cells.

PubMed

Marsili, V; Nardicchi, V; Lupidi, G; Brozzetti, A; Gianfranceschi, G L

1996-12-01

Small acidic phosphorylated chromatin peptides show regulatory activity on gene expression. The peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn, synthesized on the basis of structural and biochemical studies, shows functional properties in vitro (phosphorylation by casein kinase II, control of DNA transcription by RNA polymerase II, inhibition of proliferation and promotion of differentiation in some cell lines) very similar to those of native chromatin peptides. In this report we show that the dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn remarkably inhibits cell growth of the HL-60 cell line. The biological effect of the peptide seems to be considerably higher than that shown by the nondansylated peptide, and it cannot be attributed to a toxic effect of the Dns group. The measurement of uptake of 3H-labelled Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn demonstrates that it is unable to pass through the HL-60 cell membrane. It is our considered opinion that the addition of hydrophobic groups to the peptide N-terminus should increase the biological activity by improving its transport through the cellular membrane.

Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.

PubMed

van de Water, B; Tijdens, I B; Verbrugge, A; Huigsloot, M; Dihal, A A; Stevens, J L; Jaken, S; Mulder, G J

2000-08-18

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide.

PubMed

Laughlin, Taylor D; Miles, Jeremy R; Wright-Johnson, Elane C; Rempel, Lea A; Lents, Clay A; Pannier, Angela K

2017-11-01

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17β-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.

A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

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

Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D.

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure ofmore » SSO1404 was solved at 1.6{angstrom} resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.« less

Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.

PubMed

Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y

2016-09-01

This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR). © 2016 The Fisheries Society of the British Isles.

Using Markov chains of nucleotide sequences as a possible precursor to predict functional roles of human genome: a case study on inactive chromatin regions.

PubMed

Lee, K-E; Lee, E-J; Park, H-S

2016-08-30

Recent advances in computational epigenetics have provided new opportunities to evaluate n-gram probabilistic language models. In this paper, we describe a systematic genome-wide approach for predicting functional roles in inactive chromatin regions by using a sequence-based Markovian chromatin map of the human genome. We demonstrate that Markov chains of sequences can be used as a precursor to predict functional roles in heterochromatin regions and provide an example comparing two publicly available chromatin annotations of large-scale epigenomics projects: ENCODE project consortium and Roadmap Epigenomics consortium.

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1.

PubMed

Castagna, M; Soragna, A; Mari, S A; Santacroce, M; Betté, S; Mandela, P G; Rudnick, G; Peres, A; Sacchi, V F

2007-10-01

KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K(+) and Li(+) in addition to Na(+). We have previously demonstrated that Asp338 is essential for KAAT1 activation by K(+) and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na(+) and K(+). However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na(+)-dependent transport and the block in K(+)-dependent transport that characterize the D338E mutant. K(+)-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)(3), we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na(+) binding site in the recently solved crystal structure of the NSS transporter LeuT(Aa) (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.

On an early gene for membrane-integral inorganic pyrophosphatase in the genome of an apparently pre-luca extremophile, the archaeon Candidatus Korarchaeum cryptofilum.

PubMed

Baltscheffsky, Herrick; Persson, Bengt

2014-02-01

A gene for membrane-integral inorganic pyrophosphatase (miPPase) was found in the composite genome of the extremophile archaeon Candidatus Korarchaeum cryptofilum (CKc). This korarchaeal genome shows unusual partial similarity to both major archaeal phyla Crenarchaeota and Euryarchaeota. Thus this Korarchaeote might have retained features that represent an ancestral archaeal form, existing before the occurrence of the evolutionary bifurcation into Crenarchaeota and Euryarchaeota. In addition, CKc lacks five genes that are common to early genomes at the LUCA border. These two properties independently suggest a pre-LUCA evolutionary position of this extremophile. Our finding of the miPPase gene in the CKc genome points to a role for the enzyme in the energy conversion of this very early archaeon. The structural features of its miPPase indicate that it can pump protons through membranes. An miPPase from the extremophile bacterium Caldicellulosiruptor saccharolyticus also has a sequence indicating a proton pump. Recent analysis of the three-dimensional structure of the miPPase from Vigna radiata has resulted in the recognition of a strongly acidic substrate (orthophosphate: Pi, pyrophosphate: PPi) binding pocket, containing 11 Asp and one Glu residues. Asp (aspartic acid) is an evolutionarily very early proteinaceous amino acid as compared to the later appearing Glu (glutamic acid). All the Asp residues are conserved in the miPPase of CKc, V. radiata and other miPPases. The high proportion of Asp, as compared to Glu, seems to strengthen our argument that biological energy conversion with binding and activities of orthophosphate (Pi) and energy-rich pyrophosphate (PPi) in connection with the origin and early evolution of life may have started with similar or even more primitive acidic peptide funnels and/or pockets.

Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

PubMed

Marie-Claire, C; Ruffet, E; Antonczak, S; Beaumont, A; O'Donohue, M; Roques, B P; Fournié-Zaluski, M C

1997-11-11

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

PubMed

Fidler, Gabor; Kocsube, Sandor; Leiter, Eva; Biro, Sandor; Paholcsek, Melinda

2017-08-01

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Prevalence of plasmid-mediated qnr determinants and gyrase alteration in Klebsiella pneumoniae isolated from a university teaching hospital in Malaysia.

PubMed

Saiful Anuar, A S; Mohd Yusof, M Y; Tay, S T

2013-07-01

The ciprofloxacin resistance of Klebsiella (K.) pneumoniae is mediated primarily through alterations in type II topoisomerase (gyrA) gene and plasmid-mediated quinolone resistance-conferring genes (qnr). This study aimed to define the prevalence of plasmid-mediated quinolone resistance-conferring genes (qnr) and type II topoisomerase (gyrA) alterations of a population of ciprofloxacin-resistant (n = 21), intermediate (n = 8), and sensitive (n = 18) K. pneumoniae isolates obtained from a teaching hospital at Kuala Lumpur, Malaysia. A multiplex PCR assay was performed for simultaneous detection of qnrA, qnrB and qnrS. Sequence analysis of the amplified gyrA and gyrB regions of the isolates were performed. The findings in this study revealed the emergence of a high prevalence (48.9%) of qnr determinants in our isolates. Four variants of plasmid-mediated qnr determinants (qnrB1, qnrB6, qnrB10 and qnrS1) were detected from 11 (52.4%) ciprofloxacin-resistant, 5 (62.5%) intermediate and 7 (38.9%) sensitive isolates. gyrA alterations were detected from 18 (85.7%) ciprofloxacin-resistant isolates. Single gyrA alterations, Ser83→Tyr, Ser83→Ile, and Asp87→Gly, and double alterations, Ser83→Phe plus Asp87→Ala and Ser83→Tyr plus Asp87→Asn were detected. While ciprofloxacin resistance was significantly associated with gyrA alteration (Ser83, p = 0.003; Asp87, p = 0.005; double alteration, p = 0.016), no significant association of ciprofloxacin resistance was noted with the presence of qnr determinants (p = 0.283). The findings in this study demonstrate the emergence of qnr determinants and gyrA alterations contributed to the development and spread of fluoroquinolone resistance in the Malaysian isolates.

Glu298Asp eNOS gene polymorphism causes attenuation in nonexercising muscle vasodilatation.

PubMed

Dias, Rodrigo G; Alves, Maria-Janieire N N; Pereira, Alexandre C; Rondon, Maria Urbana P B; Dos Santos, Marcelo R; Krieger, José E; Krieger, Marta H; Negrão, Carlos E

2009-04-10

The influence of Glu298Asp endothelial nitric oxide synthase (eNOS) polymorphism in exercise-induced reflex muscle vasodilatation is unknown. We hypothesized that nonexercising forearm blood flow (FBF) responses during handgrip isometric exercise would be attenuated in individuals carrying the Asp298 allele. In addition, these responses would be mediated by reduced eNOS function and NO-mediated vasodilatation or sympathetic vasoconstriction. From 287 volunteers previously genotyped, we selected 33 healthy individuals to represent three genotypes: Glu/Glu [n = 15, age 43 +/- 3 yr, body mass index (BMI) 22.9 +/- 0.3 kg/m(2)], Glu/Asp (n = 9, age 41 +/- 3 yr, BMI 23.7 +/- 1.0 kg/m(2)), and Asp/Asp (n = 9, age 40 +/- 4 yr, BMI 23.5 +/- 0.9 kg/m(2)). Heart rate (HR), mean blood pressure (MBP), and FBF (plethysmography) were recorded for 3 min at baseline and 3 min during isometric handgrip exercise. Baseline HR, MBP, FBF, and forearm vascular conductance (FVC) were similar among genotypes. FVC responses to exercise were significantly lower in Asp/Asp when compared with Glu/Asp and Glu/Glu (Delta = 0.07 +/- 0.14 vs. 0.64 +/- 0.20 and 0.57 +/- 0.09 units, respectively; P = 0.002). Further studies showed that intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA) did not change FVC responses to exercise in Asp/Asp, but significantly reduced FVC in Glu/Glu (Delta = 0.79 +/- 0.14 vs. 0.14 +/- 0.09 units). Thus the differences between Glu/Glu and Asp/Asp were no longer observed (P = 0.62). l-NMMA + phentolamine increased similarly FVC responses to exercise in Glu/Glu and Asp/Asp (P = 0.43). MBP and muscle sympathetic nerve activity increased significant and similarly throughout experimental protocols in Glu/Glu and Asp/Asp. Individuals who are homozygous for the Asp298 allele of the eNOS enzyme have attenuated nonexercising muscle vasodilatation in response to exercise. This genotype difference is due to reduced eNOS function and NO-mediated vasodilatation, but not sympathetic vasoconstriction.

TREATMENT INTENSIFICATION WITH INSULIN DEGLUDEC/INSULIN ASPART TWICE DAILY: RANDOMIZED STUDY TO COMPARE SIMPLE AND STEP-WISE TITRATION ALGORITHMS.

PubMed

Gerety, Gregg; Bebakar, Wan Mohamad Wan; Chaykin, Louis; Ozkaya, Mesut; Macura, Stanislava; Hersløv, Malene Lundgren; Behnke, Thomas

2016-05-01

This 26-week, multicenter, randomized, open-label, parallel-group, treat-to-target trial in adults with type 2 diabetes compared the efficacy and safety of treatment intensification algorithms with twice-daily (BID) insulin degludec/insulin aspart (IDegAsp). Patients randomized 1:1 to IDegAsp BID used either a 'Simple' algorithm (twice-weekly dose adjustments based on a single prebreakfast and pre-evening meal self-monitored plasma glucose [SMPG] measurement; IDegAsp[BIDSimple], n = 136) or a 'Stepwise' algorithm (once-weekly dose adjustments based on the lowest of 3 pre-breakfast and 3 pre-evening meal SMPG values; IDegAsp[BIDStep-wise], n = 136). After 26 weeks, mean change from baseline in glycated hemoglobin (HbA1c) with IDegAsp[BIDSimple] was noninferior to IDegAsp[BIDStep-wise] (-15 mmol/mol versus -14 mmol/mol; 95% confidence interval [CI] upper limit, <4 mmol/mol) (baseline HbA1c: 66.3 mmol/mol IDegAsp[BIDSimple] and 66.6 mmol/mol IDegAsp[BIDStep-wise]). The proportion of patients who achieved HbA1c <7.0% (<53 mmol/mol) at the end of the trial was 66.9% with IDegAsp[BIDSimple] and 62.5% with IDegAsp[BIDStep-wise]. Fasting plasma glucose levels were reduced with each titration algorithm (-1.51 mmol/L IDegAsp[BIDSimple] versus -1.95 mmol/L IDegAsp[BIDStep-wise]). Weight gain was 3.8 kg IDegAsp[BIDSimple] versus 2.6 kg IDegAsp[BIDStep-wise], and rates of overall confirmed hypoglycemia (5.16 episodes per patient-year of exposure [PYE] versus 8.93 PYE) and nocturnal confirmed hypoglycemia (0.78 PYE versus 1.33 PYE) were significantly lower with IDegAsp[BIDStep-wise] versus IDegAsp[BIDSimple]. There were no significant differences in insulin dose increments between groups. Treatment intensification with IDegAsp[BIDSimple] was noninferior to IDegAsp[BIDStep-wise]. Both titration algorithms were well tolerated; however, the more conservative step-wise algorithm led to less weight gain and fewer hypoglycemic episodes. Clinicaltrials.gov: NCT01680341.

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain... peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the...terminal signal peptide (SP), which directs the precursor to the endoplasmic retic- ulum (ER) for further processing [11]. The SP, which has been

Atherogenic dyslipidemia in children: evaluation of clinical, biochemical and genetic aspects.

PubMed

Montali, Anna; Truglio, Gessica; Martino, Francesco; Ceci, Fabrizio; Ferraguti, Giampiero; Ciociola, Ester; Maranghi, Marianna; Gianfagna, Francesco; Iacoviello, Licia; Strom, Roberto; Lucarelli, Marco; Arca, Marcello

2015-01-01

The precursors of atherogenic dyslipidemia (AD) are not well defined. Therefore, we investigated 62 non-obese, non-diabetic AD and 221 normolipemic children. Anthropometric parameters, blood pressure and biochemical measures were obtained in index children, their parents and all available siblings. The heritability (h(2)) of anthropometric and biochemical traits was estimated by SOLAR. Rare and common variants in APOA1 and LPL genes were screened by re-sequencing. Compared to normolipemic, AD children showed increased body mass index, waist circumference, plasma glucose, insulin, ApoB, HOMA-IR, hs-CRP and lower adiponectin (p<0.001 for all). Metabolic syndrome was present in 40% of AD while absent in controls. All traits (except adiponectin and hs-CRP) showed a strong familial aggregation, with plasma glucose having the highest heritability (89%). Overall, 4 LPL loss-of-function mutations were detected (p.Asp9Asn, p.Ser45Asn, p.Asn291Ser, p.Leu365Val) and their cumulative prevalence was higher in AD than in control children (0.073 vs. 0.026; P=0.038). The LPL p.S447* gain-of-function mutation, resulted to be less frequent in AD than in control children (0.064 vs. 0.126; P=0.082). No variant in the APOA1 gene was found. Our data indicate that AD is a rather common dyslipidemia in childhood; it associates with metabolic abnormalities typical of insulin resistant state and shows a strong familial aggregation. LPL variants may contribute to the development of AD phenotype.

Common variants of the vitamin D binding protein gene and adverse health outcomes.

PubMed

Malik, Suneil; Fu, Lei; Juras, David James; Karmali, Mohamed; Wong, Betty Y L; Gozdzik, Agnes; Cole, David E C

2013-01-01

The vitamin D binding protein (DBP) is the major plasma carrier for vitamin D and its metabolites, but it is also an actin scavenger, and is the precursor to the immunomodulatory protein, Gc-MAF. Two missense variants of the DBP gene - rs7041 encoding Asp432Glu and rs4588 encoding Thr436Lys - change the amino acid sequence and alter the protein function. They are common enough to generate population-wide constitutive differences in vitamin D status, based on assay of the serum metabolite, 25-hydroxyvitamin D (25OHD). Whether these variants also influence the role of vitamin D in an immunologic milieu is not known. However, the issue is relevant, given the immunomodulatory effects of DBP and the role of protracted innate immune-related inflammation in response to tissue injury or repeated infection. Indeed, DBP and vitamin D may jointly or independently contribute to a variety of adverse health outcomes unrelated to classical notions of their function in bone and mineral metabolism. This review summarizes the reports to date of associations between DBP variants, and various chronic and infectious diseases. The available information leads us to conclude that DBP variants are a significant and common genetic factor in some common disorders, and therefore, are worthy of closer attention. In view of the heightened interest in vitamin D as a public health target, well-designed studies that look simultaneously at vitamin D and its carrier in relation to genotypes and adverse health outcome should be encouraged.

The Aspergillus fumigatus septins play pleiotropic roles in septation, conidiation, and cell wall stress, but are dispensable for virulence.

PubMed

Vargas-Muñiz, José M; Renshaw, Hilary; Richards, Amber D; Lamoth, Frédéric; Soderblom, Erik J; Moseley, M Arthur; Juvvadi, Praveen R; Steinbach, William J

2015-08-01

Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA-E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Colantonio, Patrizia; Leboffe, Loris; Bolli, Alessandro

Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H are reported. Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with K{sub i}{sup 0} = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 andmore » 25 deg. C). Moreover, Z-tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z-tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P{sub 2} position.« less

In vitro activity of almond skin polyphenols for scavenging free radicals and inducing quinone reductase.

PubMed

Chen, C-Y Oliver; Blumberg, Jeffrey B

2008-06-25

Observational studies and clinical trials suggest nut intake, including almonds, is associated with an enhancement in antioxidant defense and a reduction in the risk of cancer and cardiovascular disease. Almond skins are rich in polyphenols (ASP) that may contribute to these putative benefits. To assess their potential mechanisms of action, we tested the in vitro effect of ASP extracted with methanol (M) or a gastrointestinal juice mimic (GI) alone or in combination with vitamins C (VC) or E (VE) (1-10 micromol/L) on scavenging free radicals and inducing quinone reductase (QR). Flavonoid profiles from ASP-M and -GI extracts were different from one another. ASP-GI was more potent in scavenging HOCl and ONOO (-) radicals than ASP-M. In contrast, ASP-M increased and ASP-GI decreased QR activity in Hepa1c1c7 cells. Adding VC or VE to ASP produced a combination- and dose-dependent action on radical scavenging and QR induction. In comparison to their independent actions, ASP-M plus VC were less potent in scavenging DPPH, HOCl, ONOO (-), and O 2 (-) (*). However, the interaction between ASP-GI plus VC promoted their radical scavenging activity. Combining ASP-M plus VC resulted in a synergistic interaction, inducing QR activity, but ASP-GI plus VC had an antagonistic effect. On the basis of their total phenolic content, the measures of total antioxidant activity of ASP-M and -GI were comparable. Thus, in vitro, ASP act as antioxidants and induce QR activity, but these actions are dependent upon their dose, method of extraction, and interaction with antioxidant vitamins.

Sequence-based design of bioactive small molecules that target precursor microRNAs.

PubMed

Velagapudi, Sai Pradeep; Gallo, Steven M; Disney, Matthew D

2014-04-01

Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

Sequence-based design of bioactive small molecules that target precursor microRNAs

PubMed Central

Velagapudi, Sai Pradeep; Gallo, Steven M.; Disney, Matthew D.

2014-01-01

Oligonucleotides are designed to target RNA using base pairing rules, however, they are hampered by poor cellular delivery and non-specific stimulation of the immune system. Small molecules are preferred as lead drugs or probes, but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA precursors and identified bioactive small molecules that inhibit biogenesis by binding to nuclease processing sites (41% hit rate). Amongst 29 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Importantly, microRNA profiling shows that 1 only significantly effects microRNA-96 biogenesis and is more selective than an oligonucleotide. PMID:24509821

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

PubMed

Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

2016-08-01

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids.

PubMed

Chung, Si-Yin; Reed, Shawndrika

2015-01-01

The objective of this study was to determine if D-amino acids (D-aas) bind and inhibit immunoglobulin E (IgE) binding to peanut allergens. D-aas such as D-Asp (aspartic acid), D-Glu (glutamic acid), combined D-[Asp/Glu] and others were each prepared in a cocktail of 9 other D-aas, along with L-amino acids (L-aas) and controls. Each sample was mixed with a pooled plasma from peanut-allergic donors, and tested by ELISA (enzyme-linked immunosorbent assay) and Western blots for IgE binding to peanut allergens. Results showed that D-[Asp/Glu] (4 mg/ml) inhibited IgE binding (75%) while D-Glu, D-Asp and other D-aas had no inhibitory effect. A higher inhibition was seen with D-[Asp/Glu] than with L-[Asp/Glu]. We concluded that IgE was specific for D-[Asp/Glu], not D-Asp or D-Glu, and that D-[Asp/Glu] was more reactive than was L-[Asp/Glu] in IgE inhibition. The finding indicates that D-[Asp/Glu] may have the potential for removing IgE or reducing IgE binding to peanut allergens in vitro. Published by Elsevier Ltd.

Terbium-Aspartic Acid Nanocrystals with Chirality-Dependent Tunable Fluorescent Properties.

PubMed

Ma, Baojin; Wu, Yu; Zhang, Shan; Wang, Shicai; Qiu, Jichuan; Zhao, Lili; Guo, Daidong; Duan, Jiazhi; Sang, Yuanhua; Li, Linlin; Jiang, Huaidong; Liu, Hong

2017-02-28

Terbium-aspartic acid (Tb-Asp) nanocrystals with chirality-dependent tunable fluorescent properties can be synthesized through a facile synthesis method through the coordination between Tb and Asp. Asp with different chirality (dextrorotation/d and levogyration/l) changes the stability of the coordination center following fluorescent absorption/emission ability differences. Compared with l-Asp, d-Asp can coordinate Tb to form a more stable center, following the higher quantum yield and longer fluorescence life. Fluorescence intensity of Tb-Asp linearly increases with increase ratio of d-Asp in the mixed chirality Tb-Asp system, and the fluorescent properties of Tb-Asp nanocrystals can be tuned by adjusting the chirality ratio. Tb-Asp nanocrystals possess many advantage, such as high biocompatibility, without any color in visible light irradiation, monodispersion with very small size, and long fluorescent life. Those characteristics will give them great potential in many application fields, such as low-cost antifake markers and advertisements using inkjet printers or for molds when dispersed in polydimethylsiloxane. In addition, europium can also be used to synthesize Eu-Asp nanoparticles. Importantly, the facile, low-cost, high-yield, mass-productive "green" process provides enormous advantages for synthesis and application of fluorescent nanocrystals, which will have great impact in nanomaterial technology.

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

PubMed

Heilmann, L J; Trewitt, P M; Kumaran, A K

1993-01-01

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

PubMed

Otto, H; Marti, T; Holz, M; Mogi, T; Stern, L J; Engel, F; Khorana, H G; Heyn, M P

1990-02-01

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.

Whole-Genome Sequencing for Optimized Patient Management

PubMed Central

Bainbridge, Matthew N.; Wiszniewski, Wojciech; Murdock, David R.; Friedman, Jennifer; Gonzaga-Jauregui, Claudia; Newsham, Irene; Reid, Jeffrey G.; Fink, John K.; Morgan, Margaret B.; Gingras, Marie-Claude; Muzny, Donna M.; Hoang, Linh D.; Yousaf, Shahed; Lupski, James R.; Gibbs, Richard A.

2012-01-01

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins. PMID:21677200

MicroRNA-like RNAs from the same miRNA precursors play a role in cassava chilling responses.

PubMed

Zeng, Changying; Xia, Jing; Chen, Xin; Zhou, Yufei; Peng, Ming; Zhang, Weixiong

2017-12-07

MicroRNAs (miRNAs) are known to play important roles in various cellular processes and stress responses. MiRNAs can be identified by analyzing reads from high-throughput deep sequencing. The reads realigned to miRNA precursors besides canonical miRNAs were initially considered as sequencing noise and ignored from further analysis. Here we reported a small-RNA species of phased and half-phased miRNA-like RNAs different from canonical miRNAs from cassava miRNA precursors detected under four distinct chilling conditions. They can form abundant multiple small RNAs arranged along precursors in a tandem and phased or half-phased fashion. Some of these miRNA-like RNAs were experimentally confirmed by re-amplification and re-sequencing, and have a similar qRT-PCR detection ratio as their cognate canonical miRNAs. The target genes of those phased and half-phased miRNA-like RNAs function in process of cell growth metabolism and play roles in protein kinase. Half-phased miR171d.3 was confirmed to have cleavage activities on its target gene P-glycoprotein 11, a broad substrate efflux pump across cellular membranes, which is thought to provide protection for tropical cassava during sharp temperature decease. Our results showed that the RNAs from miRNA precursors are miRNA-like small RNAs that are viable negative gene regulators and may have potential functions in cassava chilling responses.

Does mental illness stigma contribute to adolescent standardized patients' discomfort with simulations of mental illness and adverse psychosocial experiences?

PubMed

Hanson, Mark D; Johnson, Samantha; Niec, Anne; Pietrantonio, Anna Marie; High, Bradley; MacMillan, Harriet; Eva, Kevin W

2008-01-01

Adolescent mental illness stigma-related factors may contribute to adolescent standardized patients' (ASP) discomfort with simulations of psychiatric conditions/adverse psychosocial experiences. Paradoxically, however, ASP involvement may provide a stigma-reduction strategy. This article reports an investigation of this hypothetical association between simulation discomfort and mental illness stigma. ASPs were randomly assigned to one of two simulation conditions: one was associated with mental illness stigma and one was not. ASP training methods included carefully written case simulations, educational materials, and active teaching methods. After training, ASPs completed the adapted Project Role Questionnaire to rate anticipated role discomfort with hypothetical adolescent psychiatric conditions/adverse psychosocial experiences and to respond to open-ended questions regarding this discomfort. A mixed design ANOVA was used to compare comfort levels across simulation conditions. Narrative responses to an open-ended question were reviewed for relevant themes. Twenty-four ASPs participated. A significant effect of simulation was observed, indicating that ASPs participating in the simulation associated with mental illness stigma anticipated greater comfort with portraying subsequent stigma-associated roles than did ASPs in the simulation not associated with stigma. ASPs' narrative responses regarding their reasons for anticipating discomfort focused upon the role of knowledge-related factors. ASPs' work with a psychiatric case simulation was associated with greater anticipated comfort with hypothetical simulations of psychiatric/adverse psychosocial conditions in comparison to ASPs lacking a similar work experience. The ASPs provided explanations for this anticipated discomfort that were suggestive of stigma-related knowledge factors. This preliminary research suggests an association between ASP anticipated role discomfort and mental illness stigma, and that ASP work may contribute to stigma reduction.

Prevalence, Characteristics, and Perception of Nursery Antibiotic Stewardship Coverage in the United States.

PubMed

Cantey, Joseph B; Vora, Niraj; Sunkara, Mridula

2017-09-01

Prolonged or unnecessary antibiotic use is associated with adverse outcomes in infants. Antibiotic stewardship programs (ASPs) aim to prevent these adverse outcomes and optimize antibiotic prescribing. However, data evaluating ASP coverage of nurseries are limited. The objectives of this study were to describe the characteristics of nurseries with and without ASP coverage and to determine perceptions of and barriers to nursery ASP coverage. The 2014 American Hospital Association annual survey was used to randomly select a level III neonatal intensive care unit from all 50 states. A level I and level II nursery from the same city as the level III nursery were then randomly selected. Hospital, nursery, and ASP characteristics were collected. Nursery and ASP providers (pharmacists or infectious disease providers) were interviewed using a semistructured template. Transcribed interviews were analyzed for themes. One hundred forty-six centers responded; 104 (71%) provided nursery ASP coverage. In multivariate analysis, level of nursery, university affiliation, and number of full-time equivalent ASP staff were the main predictors of nursery ASP coverage. Several themes were identified from interviews: unwanted coverage, unnecessary coverage, jurisdiction issues, need for communication, and a focus on outcomes. Most providers had a favorable view of nursery ASP coverage. Larger, higher-acuity nurseries in university-affiliated hospitals are more likely to have ASP coverage. Low ASP staffing and a perceived lack of importance were frequently cited as barriers to nursery coverage. Most nursery ASP coverage is viewed favorably by providers, but nursery providers regard it as less important than ASP providers. © The Author 2016. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Human mass balance, metabolite profile and identification of metabolic enzymes of [¹⁴C]ASP015K, a novel oral janus kinase inhibitor.

PubMed

Oda, Kazuo; Cao, Ying J; Sawamoto, Taiji; Nakada, Naoyuki; Fisniku, Ogert; Nagasaka, Yasuhisa; Sohda, Kin-Ya

2015-01-01

1. The human mass balance of (14)C-labelled ASP015K ([(14)C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [(14)C]ASP015K (100 mg, 3.7 MBq) in solution. [(14)C]ASP015K was rapidly absorbed with tmax of 1.6 and 1.8 h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5'-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components. 2. In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT). 3. The in vitro intrinsic clearance (CLint_in vitro) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CLint_in vitro of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.

Aversive Startle Potentiation and Fear Pathology: Mediating Role of Threat Sensitivity and Moderating Impact of Depression

PubMed Central

Yancey, James R.; Vaidyanathan, Uma; Patrick, Christopher J.

2015-01-01

Enhanced startle during exposure to unpleasant cues (aversive startle potentiation; ASP) appears in the RDoC matrix as a physiological index of acute threat response. Increased ASP has been linked to focal fear disorders and to scale measures of dispositional fearfulness (i.e., threat sensitivity; THT+). However, some studies have reported reduced ASP for fear pathology accompanied by major depressive disorder (MDD) or pervasive distress. The current study evaluated whether (a) THT+ as indexed by reported dispositional fearfulness mediates the relationship between fear disorders (when unaccompanied by depression) and ASP, and (b) depression moderates relations of THT+ and fear disorders with ASP. Fear disorder participants without MDD showed enhanced ASP whereas those with MDD (or other distress conditions) showed evidence of reduced ASP. Continuous THT+ scores also predicted ASP, and this association: (a) was likewise moderated by depression/distress, and (b) accounted for the relationship between ASP and fear pathology without MDD. These findings point to a role for the RDoC construct of acute threat, operationalized dispositionally, in enhanced ASP shown by individuals with fear pathology unaccompanied by distress pathology. PMID:25448265

Molecular characterization, transcriptional profiling, and antibacterial potential of G-type lysozyme from seahorse (Hippocampus abdominalis).

PubMed

Ko, Jiyeon; Wan, Qiang; Bathige, S D N K; Lee, Jehee

2016-11-01

Lysozymes are a family of enzymes that catalyze the hydrolysis of bacterial cell wall, acting as antimicrobial effectors of the innate immune system. In the present study, an ortholog of goose-type lysozyme (ShLysG) from the big-belly seahorse (Hippocampus abdominalis) was identified and characterized structurally and functionally. The full-length cDNA sequence (1213 bp) of ShLysG is comprised of an open reading frame made up of 552 bp, encoding a polypeptide of 184 amino acid (aa) with a predicted molecular mass of 20 kDa. In silico analysis of ShLysG revealed the absence of signal peptide and the presence of a characteristic bacterial soluble lytic transglycosylase (SLT) domain bearing three catalytic residues (Glu 71 , Asp 84 , and Asp 95 ) and seven N-acetyl-d-glucosamine binding sites (Glu 71 , Asp 95 , Tyr 98 , His 99 , Ile 117 , Tyr 145 , and Asn 146 ). Homology analysis demonstrated that the aa sequence of ShLysG shared 60.7-67.4% identity and 72.6-79.3% similarity with the orthologs of other teleosts. Phylogenetic analysis of ShLysG indicated a closest relationship with the ortholog from Gadus morhua. In healthy seahorse, ShLysG mRNA showed a constitutive expression in all the tissues examined, with the highest expression in kidney and the least expression in liver. The ShLysG mRNA levels were also shown significant elevation upon the bacterial and pathogen-associated molecular pattern (PAMPs) challenges. Furthermore, lytic activities of ShLysG recombinant protein were detected against several Gram-negative and Gram-positive bacterial species. Taken together, these results suggest that ShLysG might possess a potential immune defensive role against invading microbial pathogens in seahorse. Copyright © 2016 Elsevier Ltd. All rights reserved.

Exome sequencing identifies titin mutations causing hereditary myopathy with early respiratory failure (HMERF) in families of diverse ethnic origins.

PubMed

Toro, Camilo; Olivé, Montse; Dalakas, Marinos C; Sivakumar, Kumaraswami; Bilbao, Juan M; Tyndel, Felix; Vidal, Noemí; Farrero, Eva; Sambuughin, Nyamkhishig; Goldfarb, Lev G

2013-03-20

Hereditary myopathy with early respiratory failure (HMERF) was described in several North European families and recently linked to a titin gene (TTN) mutation. We independently studied HMERF-like diseases with the purpose to identify the cause, refine diagnostic criteria, and estimate the frequency of this disease among myopathy patients of various ethnic origins. Whole exome sequencing analysis was carried out in a large U.S. family that included seven members suffering from skeletal muscle weakness and respiratory failure. Subsequent mutation screening was performed in further 45 unrelated probands with similar phenotypes. Studies included muscle strength evaluation, nerve conduction studies and concentric needle EMG, respiratory function test, cardiologic examination, and muscle biopsy. A novel TTN p.Gly30150Asp mutation was identified in the highly conserved A-band of titin that co-segregated with the disease in the U.S. family. Screening of 45 probands initially diagnosed as myofibrillar myopathy (MFM) but excluded based on molecular screening for the known MFM genes led to the identification of a previously reported TTN p.Cys30071Arg mutation in one patient. This same mutation was also identified in a patient with suspected HMERF. The p.Gly30150Asp and p.Cys30071Arg mutations are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin. Missense mutations in TTN are the cause of HMERF in families of diverse origins. A comparison of phenotypic features of HMERF caused by the three known TTN mutations in various populations allowed to emphasize distinct clinical/pathological features that can serve as the basis for diagnosis. The newly identified p.Gly30150Asp and the p.Cys30071Arg mutation are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus.

PubMed

Higuchi, Shigesada; Murayama, Nobuhiro; Saguchi, Ken-ichi; Ohi, Hiroaki; Fujita, Yoshiaki; da Silva, Nelson Jorge; de Siqueira, Rodrigo José Bezerra; Lahlou, Saad; Aird, Steven D

2006-10-01

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.

Specific patterns of gyrA mutations determine the resistance difference to ciprofloxacin and levofloxacin in Klebsiella pneumoniae and Escherichia coli

PubMed Central

2013-01-01

Background Wide use of ciprofloxacin and levofloxacin has often led to increased resistance. The resistance rate to these two agents varies in different clinical isolates of Enterobacteriaceae. Mutations of GyrA within the quinolone resistance-determining regions have been found to be the main mechanism for quinolone resistance in Enterobacteriaceae. It has been shown that only some of the mutations in the gyrA gene identified from clinical sources were involved in fluoroquinolone resistance. Whether different patterns of gyrA mutation are related to antimicrobial resistance against ciprofloxacin and levofloxacin is unclear. Methods The minimum inhibitory concentration (MIC) of ciprofloxacin and levofloxacin were determined by the agar dilution method followed by PCR amplification and sequencing of the quinolone resistance determining region of gyrA to identify all the mutation types. The correlation between fluoroquinolone resistance and the individual mutation type was analyzed. Results Resistance differences between ciprofloxacin and levofloxacin were found in 327 isolates of K. pneumoniae and E. coli in Harbin, China and in the isolates reported in PubMed publications. GyrA mutations were found in both susceptible and resistant isolates. For the isolates with QRDR mutations, the resistance rates to ciprofloxacin and levofloxacin were also statistically different. Among the 14 patterns of alterations, two single mutations (Ser83Tyr and Ser83Ile), and three double mutations (Ser83Leu+Asp87Asn, Ser83Leu+Asp87Tyr and Ser83Phe+Asp87Asn) were associated with both ciprofloxacin and levofloxacin resistance. Two single mutations (Ser83Phe and Ser83Leu) were related with ciprofloxacin resistance but not to levofloxacin. Resistance difference between ciprofloxacin and levofloxacin in isolates harboring mutation Ser83Leu+Asp87Asn were of statistical significance among all Enterobacteriaceae (P<0.001). Conclusions Resistance rate to ciprofloxacin and levofloxacin were statistically different among clinical isolates of Enterobacteriaceae harboring GyrA mutations. Ser83Leu+Asp87Asn may account for the antimicrobial resistance difference between ciprofloxacin and levofloxacin. PMID:23295059

Accelerated Evolution of the Pituitary Adenylate Cyclase-Activating Polypeptide Precursor Gene During Human Origin

PubMed Central

Wang, Yin-qiu; Qian, Ya-ping; Yang, Su; Shi, Hong; Liao, Cheng-hong; Zheng, Hong-Kun; Wang, Jun; Lin, Alice A.; Cavalli-Sforza, L. Luca; Underhill, Peter A.; Chakraborty, Ranajit; Jin, Li; Su, Bing

2005-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. PMID:15834139

Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities.

PubMed

Imamura, Takahisa; Murakami, Yoji; Nitta, Hidetoshi

2017-09-26

Aeromonas sobria serine protease (ASP) is secreted from Aeromonas sobria, a pathogen causing gastroenteritis and sepsis. ASP resembles Saccharomyces cerevisiae Kex2, a member of the subtilisin family, and preferentially cleaves peptide bonds at the C-terminal side of paired basic amino acid residues; also accepting unpaired arginine at the P1 site. Unlike Kex2, however, ASP lacks an intramolecular chaperone N-terminal propeptide, instead utilizes the external chaperone ORF2 for proper folding, therefore, ASP and its homologues constitute a new subfamily in the subtilisin family. Through activation of the kallikrein/kinin system, ASP induces vascular leakage, and presumably causes edema and septic shock. ASP accelerates plasma clotting by α-thrombin generation from prothrombin, whereas it impairs plasma clottability by fibrinogen degradation, together bringing about blood coagulation disorder that occurs in disseminated intravascular coagulation, a major complication of sepsis. From complement C5 ASP liberates C5a that induces neutrophil recruitment and superoxide release, and mast cell degranulation, which are associated with pus formation, tissue injury and diarrhea, respectively. Nicked two-chain ASP also secreted from A. sobria is more resistant to inactivation by α2-macroglobulin than single-chain ASP, thereby raising virulence activities. Thus, ASP is a potent virulence factor and may participate in the pathogenesis of A. sobria infection.

Polyamine is a critical determinant of Pseudomonas chlororaphis O6 for GacS-dependent bacterial cell growth and biocontrol capacity.

PubMed

Park, Ju Yeon; Kang, Beom Ryong; Ryu, Choong-Min; Anderson, Anne J; Kim, Young Cheol

2018-05-01

The Gac/Rsm network regulates, at the transcriptional level, many beneficial traits in biocontrol-active pseudomonads. In this study, we used Phenotype MicroArrays, followed by specific growth studies and mutational analysis, to understand how catabolism is regulated by this sensor kinase system in the biocontrol isolate Pseudomonas chlororaphis O6. The growth of a gacS mutant was decreased significantly relative to that of the wild-type on ornithine and arginine, and on the precursor of these amino acids, N-acetyl-l-glutamic acid. The gacS mutant also showed reduced production of polyamines. Expression of the genes encoding arginine decarboxylase (speA) and ornithine decarboxylases (speC) was controlled at the transcriptional level by the GacS sensor of P. chlororaphis O6. Polyamine production was reduced in the speC mutant, and was eliminated in the speAspeC mutant. The addition of exogenous polyamines to the speAspeC mutant restored the in vitro growth inhibition of two fungal pathogens, as well as the secretion of three biological control-related factors: pyrrolnitrin, protease and siderophore. These results extend our knowledge of the regulation by the Gac/Rsm network in a biocontrol pseudomonad to include polyamine synthesis. Collectively, our studies demonstrate that bacterial polyamines act as important regulators of bacterial cell growth and biocontrol potential. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Evidence for association of a common variant of the endothelial nitric oxide synthase gene (Glu298→Asp polymorphism) to the presence, extent, and severity of coronary artery disease

PubMed Central

Colombo, M G; Andreassi, M G; Paradossi, U; Botto, N; Manfredi, S; Masetti, S; Rossi, G; Clerico, A; Biagini, A

2002-01-01

Background: Genetic variants of endothelial nitric oxide synthase (eNOS) could influence individual susceptibility to coronary artery disease. Objective: To assess whether Glu298→Asp polymorphism of the eNOS gene is associated with the occurrence and severity of angiographically defined coronary artery disease in the Italian population. Methods: Polymerase chain reaction/restriction fragment length polymorphism analysis was done to detect the Glu298→Asp variant of the eNOS gene in 201 patients with coronary artery disease and 114 controls. The severity of coronary artery disease was expressed by the number of affected vessels and by the Duke scoring system. Results: The frequencies of the eNOS Glu/Glu, Glu/Asp, and Asp/Asp genotypes in the coronary artery disease group were significantly different from those of controls (45.3%, 38.8%, and 15.9% v 42.1%, 51.8%, and 6.1%, respectively; χ2 = 8.589, p = 0.0136). In comparison with subjects who had a Glu298 allele in the eNOS gene, the risk of coronary artery disease was increased among Asp/Asp carriers (odds ratio 2.9, 95% confidence interval 1.2 to 6.8, p = 0.01) and was independent of the other common risk factors (p = 0.04). There was a significant association between the eNOS Glu298→Asp variant and both the number of stenosed vessels (mean (SEM), 2.3 (0.1) for Asp/Asp v 1.9 (0.1) and 1.8 (0.1) for Glu/Glu and Glu/Asp, respectively; p = 0.01) and the Duke score (56.1 (3.1) for Asp/Asp v 46.7 (2.0) and 46.1 (1.9) for Glu/Glu and Glu/Asp, respectively; p = 0.02). Conclusions: Glu298→Asp polymorphism of the eNOS gene appears to be associated with the presence, extent, and severity of angiographically assessed coronary artery disease. PMID:12010932

Change in plasma acylation stimulating protein during euglycaemic-hyperinsulinaemic clamp in overweight and obese postmenopausal women: a MONET study.

PubMed

St-Pierre, David H; Cianflone, Katherine; Smith, Jessica; Coderre, Lise; Karelis, Antony D; Imbeault, Pascal; Lavoie, Jean-Marc; Rabasa-Lhoret, Rémi

2009-04-01

Acylation-stimulating protein (ASP) has been shown to positively stimulate fatty acid esterification and glucose uptake in adipocytes. In vitro studies demonstrate that insulin stimulates ASP secretion from adipocytes. Individuals with obesity and/or metabolic disturbances (insulin resistance and type 2 diabetes) have increased plasma ASP. The present study was designed to evaluate whether ASP levels are influenced by the metabolic profiles of overweight and obese postmenopausal women during a euglycaemic/hyperinsulinaemic clamp (EHC). Patients The study population consisted of 76 overweight and obese sedentary postmenopausal women. We evaluated insulin sensitivity, plasma ASP levels, body composition including visceral adipose tissue area, blood lipid profiles, liver enzymes, peak aerobic capacity, resting metabolic rate (RMR) and total energy expenditure (TEE). We observed wide interindividual variations of ASP levels during the EHC. Therefore, subjects were divided into three groups based on ASP changes. Negative ASP Responders (NAR; n = 24) showed a -20% or greater decrease in ASP levels while Positive ASP Responders (PAR; n = 42) displayed ASP fluctuations superior to +20%. Ten subjects had little or no ASP change and were considered as Zero ASP responders (ZAR). PAR women displayed a worse metabolic profile than NAR women, including higher BMI, visceral adipose tissue, fasting insulin levels, lean body mass, and alanine aminotransferase (ALT), a marker of impaired liver function. After adjustment for BMI, only ALT remained significantly different, while lean body mass (P = 0.08) and visceral adipose tissue (P = 0.07) remained marginally higher. Correlation analysis of all subjects demonstrated that fasting ASP levels correlated positively with albumin and VO(2 peak) and this association remained significant after adjustments for the effect of BMI. In addition, the percentage maximal change in ASP levels during the EHC was positively associated with BMI, lean body mass, visceral adipose tissue, fasting insulin, HOMA, TEE, RMR, ALT and AST. Overall these results suggest that an elevated ASP response during the EHC is associated with metabolic disturbances in overweight and obese postmenopausal women.

Molecular analysis of Cypriot patients with Glutaric aciduria type I: identification of two novel mutations.

PubMed

Georgiou, Theodoros; Nicolaidou, Paola; Hadjichristou, Anastasia; Ioannou, Rodothea; Dionysiou, Maria; Siama, Elli; Chappa, Georgia; Anastasiadou, Violetta; Drousiotou, Anthi

2014-09-01

The purpose of this study was to identify the mutations in the glutaryl-CoA dehydrogenase gene (GCDH) in ten Cypriot patients with Glutaric aciduria type I (GAI). Molecular analysis of the GCDH gene was performed by direct sequencing of the patients' genomic DNA. In silico tools were applied to predict the effect of the novel variants on the structure and function of the protein. All disease alleles were characterized (mutation detection rate 100%). Five missense mutations were identified: c.192G>T (p.Glu64Asp) and c.803G>T (p.Gly268Val), which are novel, and three previously described mutations, c.1123T>C (p.Cys375Arg), c.1204C>T (p.Arg402Trp) and c.1286C>T (p.Thr429Met). Two novel mutations, p.Glu64Asp and p.Gly268Val, account for the majority of disease alleles (76.5%) in Cypriot patients with Glutaric aciduria type I. A founder effect for the p.Glu64Asp and the p.Gly268Val can be suggested based on the place of origin of the carriers of these mutations. Identification of the causative mutations of GAI in Cypriot patients will facilitate carrier detection as well as post- and pre-natal diagnosis. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

An analytical SMASH procedure (ASP) for sensitivity-encoded MRI.

PubMed

Lee, R F; Westgate, C R; Weiss, R G; Bottomley, P A

2000-05-01

The simultaneous acquisition of spatial harmonics (SMASH) method of imaging with detector arrays can reduce the number of phase-encoding steps, and MRI scan time several-fold. The original approach utilized numerical gradient-descent fitting with the coil sensitivity profiles to create a set of composite spatial harmonics to replace the phase-encoding steps. Here, an analytical approach for generating the harmonics is presented. A transform is derived to project the harmonics onto a set of sensitivity profiles. A sequence of Fourier, Hilbert, and inverse Fourier transform is then applied to analytically eliminate spatially dependent phase errors from the different coils while fully preserving the spatial-encoding. By combining the transform and phase correction, the original numerical image reconstruction method can be replaced by an analytical SMASH procedure (ASP). The approach also allows simulation of SMASH imaging, revealing a criterion for the ratio of the detector sensitivity profile width to the detector spacing that produces optimal harmonic generation. When detector geometry is suboptimal, a group of quasi-harmonics arises, which can be corrected and restored to pure harmonics. The simulation also reveals high-order harmonic modulation effects, and a demodulation procedure is presented that enables application of ASP to a large numbers of detectors. The method is demonstrated on a phantom and humans using a standard 4-channel phased-array MRI system. Copyright 2000 Wiley-Liss, Inc.

Fibrinogen variant B[beta]D432A has normal polymerization but does not bind knob 'B'

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

Bowley, Sheryl R.; Lord, Susan T.; UNC)

2009-10-23

Fibrinogen residue B{beta}432Asp is part of hole 'b' that interacts with knob 'B,' whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed B{beta}D432A has normal polymerization, we hypothesized that B{beta}432Asp is not critical for knob 'B' binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from B{beta}D432A. Surprisingly, the structure (rfD-B{beta}D432A+GH) showed the peptide GHRP was not bound to hole 'b.' We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking.more » The turbidity and the rate of - dimer formation for B{beta}D432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of B{beta}D432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than 'B:b' interactions. We conclude that hole 'b' and 'B:b' knob-hole binding per se have no influence on fibrin polymerization.« less

Compliance with the Healthy Eating Standards in YMCA afterschool programs

PubMed Central

Beets, Michael W.; Weaver, R. Glenn; Turner-McGrievy, Brie; Beighle, Aaron; Moore, Justin B.; Webster, Collin; Khan, Mahmud; Saunders, Ruth

2016-01-01

Objective In 2011, the YMCA of the USA adopted Healthy Eating (HE) Standards for all their afterschool programs (ASPs). The extent to which YMCA-ASPs comply with the standards is unknown. Methods Twenty ASPs from all YMCA-ASPs across SC (N=102) were invited to participate. Direct observation of the foods/beverages served and staff behaviors were collected on four non-consecutive days/ASP. Results One ASP did not serve a snack. Of the remaining, a total of 26% ASPs served a fruit/vegetable and 32% served water every day; 26% served sugar-sweetened beverages, 47% served sugar-added foods, and only 11% served whole grains, when grains were served. Staff were sitting with the children (65%) or verbally promoting healthy eating (15%) on at least one observation day. Staff where drinking non-approved drinks (25%) or foods (45%) on at least one observation day. No ASPs served snacks family-style every day. Conclusions/Implications Additional efforts are required to assist YMCA-operated ASPs in achieving these important nutrition standards. PMID:27372234

Combined effects of ankylosing spondylitis-associated ERAP1 polymorphisms outside the catalytic and peptide-binding sites on the processing of natural HLA-B27 ligands.

PubMed

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A

2014-02-14

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis.

Combined Effects of Ankylosing Spondylitis-associated ERAP1 Polymorphisms Outside the Catalytic and Peptide-binding Sites on the Processing of Natural HLA-B27 Ligands*

PubMed Central

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A.

2014-01-01

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis. PMID:24352655

The modeling and design of the Annular Suspension and Pointing System /ASPS/. [for Space Shuttle

NASA Technical Reports Server (NTRS)

Kuo, B. C.; Lin, W. C. W.

1979-01-01

The Annular Suspension and Pointing System (ASPS) is a payload auxiliary pointing device of the Space Shuttle. The ASPS is comprised of two major subassemblies, a vernier and a coarse pointing subsystem. The three functions provided by the ASPS are related to the pointing of the payload, centering the payload in the magnetic actuator assembly, and tracking the payload mounting plate and shuttle motions by the coarse gimbals. The equations of motion of a simplified planar model of the ASPS are derived. Attention is given to a state diagram of the dynamics of the ASPS with position-plus-rate controller, the nonlinear spring characteristic for the wire-cable torque of the ASPS, the design of the analog ASPS through decoupling and pole placement, and the time response of different components of the continuous control system.

ASP archiving solution of regional HUSpacs.

PubMed

Pohjonen, Hanna; Kauppinen, Tomi; Ahovuo, Juhani

2004-09-01

The application service provider (ASP) model is not novel, but widely used in several non-health care-related business areas. In this article, ASP is described as a potential solution for long-term and back-up archiving of the picture archiving and communication system (PACS) of the Hospital District of Helsinki and Uusimaa (HUS). HUSpacs is a regional PACS for 21 HUS hospitals serving altogether 1.4 million citizens. The ultimate goal of this study was to define the specifications for the ASP archiving service and to compare different commercial options for archiving solutions (costs derived by unofficial requests for proposal): in-house PACS components, the regional ASP concept and the hospital-based ASP concept. In conclusion, the large scale of the HUS installation enables a cost-effective regional ASP archiving, resulting in a four to five times more economical solution than hospital-based ASP.

The ASP3 locus in Saccharomyces cerevisiae originated by horizontal gene transfer from Wickerhamomyces.

PubMed

League, Garrett P; Slot, Jason C; Rokas, Antonis

2012-11-01

The asparagine degradation pathway in the S288c laboratory strain of Saccharomyces cerevisiae is comprised of genes located at two separate loci. ASP1 is located on chromosome IV and encodes for cytosolic l-asparaginase I, whereas ASP3 contains a gene cluster located on chromosome XII comprised of four identical genes, ASP3-1, ASP3-2, ASP3-3, and ASP3-4, which encode for cell wall-associated l-asparaginase II. Interestingly, the ASP3 locus appears to be only present, in variable copy number, in S. cerevisiae strains isolated from laboratory or industrial environments and is completely absent from the genomes of 128 diverse fungal species. Investigation of the evolutionary history of ASP3 across these 128 genomes as well as across the genomes of 43 S. cerevisiae strains shows that ASP3 likely arose in a S. cerevisiae strain via horizontal gene transfer (HGT) from, or a close relative of, the wine yeast Wickerhamomyces anomalus, which co-occurs with S. cerevisiae in several biotechnological processes. Thus, because the ASP3 present in the S288c laboratory strain of S. cerevisiae is induced in response to nitrogen starvation, its acquisition may have aided yeast adaptation to artificial environments. Our finding that the ASP3 locus in S. cerevisiae originated via HGT further highlights the importance of gene sharing between yeasts in the evolution of their remarkable metabolic diversity. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Oral administration of D-aspartate, but not L-aspartate, depresses rectal temperature and alters plasma metabolites in chicks.

PubMed

Erwan, Edi; Chowdhury, Vishwajit Sur; Nagasawa, Mao; Goda, Ryosei; Otsuka, Tsuyoshi; Yasuo, Shinobu; Furuse, Mitsuhiro

2014-07-25

L-Aspartate (L-Asp) and D-aspartate (D-Asp) are physiologically important amino acids in mammals and birds. However, the functions of these amino acids have not yet been fully understood. In this study, we therefore examined the effects of L-Asp and D-Asp in terms of regulating body temperature, plasma metabolites and catecholamines in chicks. Chicks were first orally administered with different doses (0, 3.75, 7.5 and 15 mmol/kg body weight) of L- or D-Asp to monitor the effects of these amino acids on rectal temperature during 120 min of the experimental period. Oral administration of D-Asp, but not of L-Asp, linearly decreased the rectal temperature in chicks. Importantly, orally administered D-Asp led to a significant reduction in body temperature in chicks even under high ambient temperature (HT) conditions. However, centrally administered D-Asp did not significantly influence the body temperature in chicks. As for plasma metabolites and catecholamines, orally administered D-Asp led to decreased triacylglycerol and uric acid concentrations and increased glucose and chlorine concentrations but did not alter plasma catecholamines. These results suggest that oral administration of D-Asp may play a potent role in reducing body temperature under both normal and HT conditions. The alteration of plasma metabolites further indicates that D-Asp may contribute to the regulation of metabolic activity in chicks. Copyright © 2014 Elsevier Inc. All rights reserved.

Beta.-glucosidase coding sequences and protein from orpinomyces PC-2

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong; Ximenes, Eduardo A.

2001-02-06

Provided is a novel .beta.-glucosidase from Orpinomyces sp. PC2, nucleotide sequences encoding the mature protein and the precursor protein, and methods for recombinant production of this .beta.-glucosidase.

Effects of long-term administration of aspartame on biochemical indices, lipid profile and redox status of cellular system of male rats.

PubMed

Adaramoye, Oluwatosin A; Akanni, Olubukola O

2016-01-01

Aspartame (N-L-α-aspartyl-L-phenylalanine-1-methyl ester) (ASP) is a synthetic sweetener used in foods and its safety remains controversial. The study was designed to investigate the effects of long-term administration of aspartame on redox status, lipid profile and biochemical indices in tissues of male Wistar rats. Rats were assigned into four groups and given distilled water (control), aspartame at doses of 15 mg/kg (ASP 1), 35 mg/kg (ASP 2) and 70 mg/kg (ASP 3) daily by oral gavage for consecutive 9 weeks. Administration of ASP 2 and ASP 3 significantly increased the weight of liver and brain, and relative weight of liver of rats. Lipid peroxidation products significantly increased in the kidney, liver and brain of rats at all doses of ASP with concomitant depletion of antioxidant parameters, viz. glutathione-s-transferase, glutathione peroxidase, superoxide dismutase, catalase and reduced glutathione. Furthermore, ASP 2 and ASP 3 significantly increased the levels of gamma glutamyl transferase by 70% and 85%; alanine aminotransferase by 66% and 117%; aspartate aminotransferase by 21% and 48%; urea by 72% and 58% and conjugated bilirubin by 63% and 64%, respectively. Also, ASP 2 and ASP 3 significantly increased the levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol in the rats. Histological findings showed that ASP 2 and ASP 3 caused cyto-architectural changes such as degeneration, monocytes infiltration and necrotic lesions in brain, kidney and liver of rats. Aspartame may induce redox and lipid imbalance in rats via mechanism that involves oxidative stress and depletion of glutathione-dependent system.

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

PubMed

Chou, W Y; Chang, H P; Huang, C H; Kuo, C C; Tong, L; Chang, G G

2000-02-01

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

eNOS Glu298Asp polymorphism and hypertension in a cohort study in Japanese.

PubMed

Kishimoto, Takuji; Misawa, Yumiko; Kaetu, Akihiko; Nagai, Maria; Osaki, Yoneatsu; Okamoto, Mikizoh; Yoshida, Soiti; Kurosawa, Yoichi; Fukumoto, Soji

2004-11-01

Some recent case-control association studies have suggested negative and positive relationship between Glu298Asp (the substitution of aspartic acid for glutamic acid at amino acid position 298) polymorphism of the endothelial nitric oxide synthase (eNOS) gene and hypertension. To investigate whether the Glu298Asp polymorphism of the eNOS gene affects the incidence of hypertension, a retrospective cohort study was performed. The baseline data among Japanese workers in Shimane Prefecture, Japan, were obtained at regular health examination in 1992, and a retrospective cohort study was performed to analyze the influence of Glu298Asp polymorphism on the incidence of hypertension in 1998. The incidences of Glu298Glu, Glu298Asp, and Asp298Asp genotypes in the subjects were 86.4%, 12.6% and 1.1%, respectively. The risk ratios of Glu298Asp and Asp298Asp against Glu298Glu for the incidence of hypertension by single variance analysis were 0.830 in total subjects [95% confidence interval (CI) 0.474-1.452], 0.596 in subjects 20-39 years old (95% CI; 0.207-1.717), and 0.915 in subjects 40-59 years old (95% CI; 0.464-1.805). The risk ratios of Glu298Asp and Asp298Asp against Glu298Glu for the incidence of hypertension by multiple variance analysis adjusted for sex, BMI, serum total cholesterol, serum high-density lipoprotein (HDL) cholesterol, fasting glucose, cigarette smoking, drinking habits, eating habits, and exercise in 1992 were 0.750 in total subjects (95% CI; 0.421-1.335), 0.505 in subjects 20-39 years old (95% CI; 0.170-1.496), and 0.873 in subjects 40-59 years old (95% CI; 0.434-1.757). These results suggested no association between the Glu298Asp gene polymorphism and the incidence of hypertension in this selected population.

A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis.

PubMed

Huang, Kai-Fa; Wang, Yu-Ruei; Chang, En-Cheng; Chou, Tsung-Lin; Wang, Andrew H-J

2008-04-01

QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs.

Clinical Utility of Ammonia Concentration as a Diagnostic Test in Monitoring of the Treatment with L-Asparaginase in Children with Acute Lymphoblastic Leukemia

PubMed Central

Czogała, Małgorzata; Balwierz, Walentyna; Sztefko, Krystyna; Rogatko, Iwona

2014-01-01

L-asparaginase (ASP) is an enzyme used as one of the basic regimens in the acute lymphoblastic leukemia (ALL) therapy. Because of the possibility of the enzyme inactivation by antibodies, monitoring of ASP activity is essential. The aim of the study was to examine if plasma concentration of ammonia, a direct product of the reaction catalyzed by ASP, can be used in the assessment of ASP activity. A group of 87 patients with acute lymphoblastic leukemia treated in the Department of Pediatric Oncology and Hematology in Krakow was enrolled to the study. ASP activity and ammonia concentration were measured after ASP administrations during induction. A positive correlation was found between the ammonia concentration and ASP activity (R = 0.44; P < 0.0001) and between the medium values of ammonia concentration and ASP activity (R = 0.56; P < 0.0001). The analysis of ROC curves revealed the moderate accuracy of the ammonia concentration values in the ASP activity assessment. It was also found that the medium value of ammonia concentrations can be useful in identification of the patients with low (<100 IU/L) and undetectable (<30 IU/L) ASP activity. The plasma ammonia concentration may reflect ASP activity and can be useful when a direct measurement of the activity is unavailable. PMID:25157375

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

PubMed Central

Laprevotte, I; Hampe, A; Sherr, C J; Galibert, F

1984-01-01

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing. PMID:6328019

Hb Moscva [β24(B6)Gly→Asp (GGT>GAT), HBB: c.74G>A]: An Unstable Hemoglobin Newly Detected as a De Novo Mutation in a Mauritanian Patient.

PubMed

Ghaber, Sidi M; Trabelsi, Nawel; Salem, Mohamed L; Haddad, Faten; Abba, Aminetou; Darragi, Imen; Abbes, Salem

2018-01-01

Unstable hemoglobins (Hbs) are a group of Hb disorders that could be the origin of chronic hemolytic anemia. Most of these disorders are caused by point mutations taking place in the globin genes and affecting the stability of the Hb molecule. They are inherited as autosomal dominant diseases and described worldwide. Herein we report a new observation of an unstable variant in the Mauritanian population. The patient was a young girl of Mauritanian origin. She presented with chronic hemolytic anemia with an unknown etiology after being referred to several medical centers. Laboratory investigations based on routine analyses, capillary electrophoresis (CE), cation exchange high performance liquid chromatography (HPLC) and DNA sequencing revealed an abnormal unstable Hb known as Hb Moscva [β24(B6)Gly→Asp (GGT>GAT), HBB: c.74G>A] that occurred as a de novo mutation newly detected in an African girl of Mauritanian origin.

Tumor Necrosis Factor α‐Gene Therapy for an Established Murine Melanoma Using RGB (Arg‐Gly‐Asp) Fiber‐mutant Adenovirus Vectors

PubMed Central

Okada, Yuka; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki; Takahashi, Koichi; Mizuno, Nobuyasu; Fujita, Takuya; Yamamoto, Akira; Hayakawa, Takao; Mayumi, Tadanori

2002-01-01

Although adenovirus vectors (Ad) provide high‐level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie‐adenovirus receptor, a primitive Ad‐receptor, was very low in murine and human melanoma cells. We also found that fiber‐mutant Ad containing the Arg‐Gly‐Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting αv‐integrins. In addition, intratumoral injection of RGD fiber‐mutant Ad containing the tumor necrosis factor α gene (AdRGD‐TNFα) revealed dramatic anti‐tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad‐RGD‐TNFα required one‐tenth the dosage of Ad‐TNFα to induce an equal therapeutic effect. These results suggest that αv‐integrin‐targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy. PMID:11985794

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

PubMed

Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

2016-11-01

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

Crystal Structure of Human Nicotinamide Riboside Kinase

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

Khan,J.; Xiang, S.; Tong, L.

2007-01-01

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheetmore » core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.« less

Clinical phenotype and infertility treatment in a male with hypogonadotropic hypogonadism due to mutations Ala129Asp/Arg262Gln of the gonadotropin-releasing hormone receptor.

PubMed

Layman, Lawrence C; Cohen, David P; Xie, Jun; Smith, Gary D

2002-12-01

To characterize the genotype and phenotype of a man with idiopathic hypogonadism with infertility. Molecular analysis and clinical description. Medical school laboratory and reproductive endocrine clinic.A 40-year-old male with idiopathic hypogonadotropic hypogonadism. Denaturing gradient gel electrophoresis analysis and DNA sequencing of the gonadotropin-releasing hormone receptor (GNRHR) gene were performed. The patient was treated with hCG and FSH. GNRHR mutation detection, genotype/phenotype correlation, and testicular response to exogenous gonadotropin therapy. The proband demonstrated compound heterozygosity for Ala129Asp/Arg262Gln GNRHR mutations. He had a complete form of idiopathic hypogonadotropic hypogonadism, with descended testes and severe oligospermia but little response to exogenous gonadotropins. The phenotype of this patient differs from the one other family described with the same mutations. Exogenous gonadotropin therapy may not be as beneficial for increasing sperm concentration in older men with idiopathic hypogonadotropic hypogonadism.

The relationship of Asperger's syndrome to autism: a preliminary EEG coherence study.

PubMed

Duffy, Frank H; Shankardass, Aditi; McAnulty, Gloria B; Als, Heidelise

2013-07-31

It has long been debated whether Asperger's Syndrome (ASP) should be considered part of the Autism Spectrum Disorders (ASD) or whether it constitutes a unique entity. The Diagnostic and Statistical Manual, fourth edition (DSM-IV) differentiated ASP from high functioning autism. However, the new DSM-5 umbrellas ASP within ASD, thus eliminating the ASP diagnosis. To date, no clear biomarkers have reliably distinguished ASP and ASD populations. This study uses EEG coherence, a measure of brain connectivity, to explore possible neurophysiological differences between ASP and ASD. Voluminous coherence data derived from all possible electrode pairs and frequencies were previously reduced by principal components analysis (PCA) to produce a smaller number of unbiased, data-driven coherence factors. In a previous study, these factors significantly and reliably differentiated neurotypical controls from ASD subjects by discriminant function analysis (DFA). These previous DFA rules are now applied to an ASP population to determine if ASP subjects classify as control or ASD subjects. Additionally, a new set of coherence based DFA rules are used to determine whether ASP and ASD subjects can be differentiated from each other. Using prior EEG coherence based DFA rules that successfully classified subjects as either controls or ASD, 96.2% of ASP subjects are classified as ASD. However, when ASP subjects are directly compared to ASD subjects using new DFA rules, 92.3% ASP subjects are identified as separate from the ASD population. By contrast, five randomly selected subsamples of ASD subjects fail to reach significance when compared to the remaining ASD populations. When represented by the discriminant variable, both the ASD and ASD populations are normally distributed. Within a control-ASD dichotomy, an ASP population falls closer to ASD than controls. However, when compared directly with ASD, an ASP population is distinctly separate. The ASP population appears to constitute a neurophysiologically identifiable, normally distributed entity within the higher functioning tail of the ASD population distribution. These results must be replicated with a larger sample given their potentially immense clinical, emotional and financial implications for affected individuals, their families and their caregivers.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

PubMed

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-07-07

To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

The relationship of Asperger’s syndrome to autism: a preliminary EEG coherence study

PubMed Central

2013-01-01

Background It has long been debated whether Asperger’s Syndrome (ASP) should be considered part of the Autism Spectrum Disorders (ASD) or whether it constitutes a unique entity. The Diagnostic and Statistical Manual, fourth edition (DSM-IV) differentiated ASP from high functioning autism. However, the new DSM-5 umbrellas ASP within ASD, thus eliminating the ASP diagnosis. To date, no clear biomarkers have reliably distinguished ASP and ASD populations. This study uses EEG coherence, a measure of brain connectivity, to explore possible neurophysiological differences between ASP and ASD. Methods Voluminous coherence data derived from all possible electrode pairs and frequencies were previously reduced by principal components analysis (PCA) to produce a smaller number of unbiased, data-driven coherence factors. In a previous study, these factors significantly and reliably differentiated neurotypical controls from ASD subjects by discriminant function analysis (DFA). These previous DFA rules are now applied to an ASP population to determine if ASP subjects classify as control or ASD subjects. Additionally, a new set of coherence based DFA rules are used to determine whether ASP and ASD subjects can be differentiated from each other. Results Using prior EEG coherence based DFA rules that successfully classified subjects as either controls or ASD, 96.2% of ASP subjects are classified as ASD. However, when ASP subjects are directly compared to ASD subjects using new DFA rules, 92.3% ASP subjects are identified as separate from the ASD population. By contrast, five randomly selected subsamples of ASD subjects fail to reach significance when compared to the remaining ASD populations. When represented by the discriminant variable, both the ASD and ASD populations are normally distributed. Conclusions Within a control-ASD dichotomy, an ASP population falls closer to ASD than controls. However, when compared directly with ASD, an ASP population is distinctly separate. The ASP population appears to constitute a neurophysiologically identifiable, normally distributed entity within the higher functioning tail of the ASD population distribution. These results must be replicated with a larger sample given their potentially immense clinical, emotional and financial implications for affected individuals, their families and their caregivers. PMID:23902729

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

PubMed Central

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-01-01

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori. PMID:27433095

Intriguing olfactory proteins from the yellow fever mosquito, Aedes aegypti

NASA Astrophysics Data System (ADS)

Ishida, Yuko; Chen, Angela M.; Tsuruda, Jennifer M.; Cornel, Anthon J.; Debboun, Mustapha; Leal, Walter S.

2004-09-01

Four antennae-specific proteins (AaegOBP1, AaegOBP2, AaegOBP3, and AaegASP1) were isolated from the yellow fever mosquito, Aedes aegypti and their full-length cDNAs were cloned. RT-PCR indicated that they are expressed in female and, to a lesser extent, in male antennae, but not in control tissues (legs). AaegOBP1 and AaegOBP3 showed significant similarity to previously identified mosquito odorant-binding proteins (OBPs) in cysteine spacing pattern and sequence. Two of the isolated proteins have a total of eight cysteine residues. The similarity of the spacing pattern of the cysteine residues and amino acid sequence to those of previously identified olfactory proteins suggests that one of the cysteine-rich proteins (AaegOBP2) is an OBP. The other (AaegASP1) did not belong to any group of known OBPs. Structural analyses indicate that six of the cysteine residues in AaegOBP2 are linked in a similar pattern to the previously known cysteine pairing in OBPs, i.e., Cys-24 Cys-55, Cys-51 Cys-104, Cys-95 Cys-113. The additional disulfide bridge, Cys-38 Cys-125, knits the extended C-terminal segment of the protein to a predicted α2-helix. As indicated by circular dichroism (CD) spectra, the extra rigidity seems to prevent the predicted formation of a C-terminal α-helix at low pH.

Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

PubMed

Valliere-Douglass, John F; Lewis, Patsy; Salas-Solano, Oscar; Jiang, Shan

2015-02-01

We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Conformational analysis of the N-terminal sequence Met1 Val60 of the tyrosine hydroxylase

NASA Astrophysics Data System (ADS)

Alieva, Irada N.; Mustafayeva, Narmina N.; Gojayev, Niftali M.

2006-03-01

Molecular mechanics method and molecular dynamics (MD) simulation techniques are used to study the behavior and the effect of the amino acids substitution on structure and molecular dynamics of the specific portion of Met1-Val60 amino acid residues from N-terminal regulatory domain of the tyrosine hydroxylase (TH) and its mutants in which the positively charged arginine residues at positions 37 and 38 were replaced by electrically neutral Gly and negatively charged Glu, and serine residue at position 40 was replaced by Ala or Asp residue. Our study allowed us to make the following conclusions: (i) the higher conformational flexibility of the Met1-Arg16 sequence is revealed in comparision to other part of the N-terminus; (ii) the stretch of amino acid residues Met30-Ser40 within the N-terminus forms β-turn so that two α-helices (residues 16-29 and residues 41-60) are paralel one another; (ii) the significant differences that are observed for the Arg37→Gly37, Arg37-Arg38→Glu37-Glu38 mutant segments indicates that the positive charge of the Arg37 and Arg38 residues is one of the main factor that maintains the characteristic of the turn; (ii) no major conformational changes are observed between Ser40→Ala40, and Ser40→Asp40 mutant segments.

Compliance With the Healthy Eating Standards in YMCA After-School Programs.

PubMed

Beets, Michael W; Weaver, R Glenn; Turner-McGrievy, Gabrielle; Beighle, Aaron; Moore, Justin B; Webster, Collin; Khan, Mahmud; Saunders, Ruth

2016-09-01

In 2011, the YMCA of the US adopted Healthy Eating standards for all of their after-school programs (ASPs). The extent to which YMCA ASPs comply with the standards is unknown. Twenty ASPs from all YMCA ASPs across South Carolina (N = 102) were invited to participate. Direct observation of the food and beverages served and staff behaviors were collected on 4 nonconsecutive days per ASP. One ASP did not serve a snack. Of the remaining ASPs, a total of 26% served a fruit or vegetable and 32% served water every day; 26% served sugar-sweetened beverages, 47% served sugar-added foods, and only 11% served whole grains when grains were served. Staff members sat with the children (65%) or verbally promoted healthy eating (15%) on at least 1 observation day. Staff drank non-approved drinks (25%) or foods (45%) on at least 1 observation day. No ASPs served snacks family-style every day. Additional efforts are required to assist YMCA-operated ASPs in achieving these important nutrition standards. Copyright © 2016 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors

DOE PAGES

Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; ...

2015-05-20

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distalmore » stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs.

PubMed

Hopper, A K; Schultz, L D; Shapiro, R A

1980-03-01

By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code.

PubMed

Fischer, Frédéric; Huot, Jonathan L; Lorber, Bernard; Diss, Guillaume; Hendrickson, Tamara L; Becker, Hubert D; Lapointe, Jacques; Kern, Daniel

2012-06-01

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

Characterizing the Specificity and Co-operation of Aminopeptidases in the Cytosol and ER During MHC Class I antigen Presentation1

PubMed Central

Hearn, Arron; York, Ian A.; Bishop, Courtney; Rock, Kenneth L.

2010-01-01

Many MHC class I binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can be subsequently trimmed by aminopeptidases in the cytosol and/or the ER to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for antigen presentation is not well defined. Here we investigate this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of antigen presentation. In many cases MHC class I binding peptides are produced through sequential trimming in both the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Since N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented. PMID:20351195

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript.

PubMed Central

Veldman, G M; Klootwijk, J; van Heerikhuizen, H; Planta, R J

1981-01-01

We have determined the nucleotide sequence of part of a cloned yeast ribosomal RNA operon extending from the 5.8S RNA gene downstream into the 5' -terminal region of the 26S RNA gene. We mapped the pertinent processing sites, viz. the 5' end of 26S rRNA and the 3'ends of 5.8S rRNA and its immediate precursor, 7S RNA. At the 3' end of 7S RNA we find the sequence UCGUUU which is very similar to the type I consensus sequence UCAUUA/U present at the 3' ends of 17S, 5.8S and 26S rRNA as well as 18S precursor rRNA in yeast. At the 5' end of the 26S RNA gene we find a sequence of thirteen nucleotides which is homologous to the type II sequence present at the 5' termini of both the 17S and the 5.8S RNA gene. These findings further support the suggestion put forward earlier (G.M. Veldman et al. (1980) Nucl. Acids Res. 8, 2907-2920) that both consensus sequences are involved in the recognition of precursor rRNA by the processing nuclease(s). We discuss a model for the processing of yeast rRNA in which a processing enzyme sequentially recognizes several combinations of a type I and a type II consensus sequence. We also describe the existence of a significant base complementarity between sequences in the 5' -terminal region of 26S rRNA and the 3' -terminal region of 5.8S rRNA. We suggest that base pairing between these sequences contributes to the binding between 5.8S and 26S rRNA. Images PMID:7312619

[Experimental study on aging effect of Angelica sinensis polysaccharides combined with cytarabine on human leukemia KG1alpha cell lines].

PubMed

Xu, Chun-Yan; Geng, Shan; Liu, Jun; Zhu, Jia-Hong; Zhang, Xian-Ping; Jiang, Rong; Wang, Ya-Ping

2014-04-01

The latest findings of our laboratory showed that Angelica sinensis polysaccharide (ASP) showed a definite effect in regulating the aging of hematopoietic stem cells. Leukemia is a type of malignant hematopoietic tumor in hematopoietic stem cells. There have been no relevant reports about ASP's effect in regulating the aging of leukemia cells. In this study, human acute myeloid leukemia (AML) KG1alpha cell lines in logarithmic growth phase were taken as the study object, and were divided into the ASP group, the cytarabine (Ara-C) group, the ASP + Ara-C group and the control group. The groups were respectively treated with different concentration of ASP, Ara-C and ASP + Ara-C for different periods, with the aim to study the effect of ASP combined with Ara-C in regulating the aging of human acute myeloid leukemia KG1alpha cell lines and its relevant mechanism. The results showed that ASP, Ara-C and ASP + Ara-C could obviously inhibit KG1alpha cell proliferation in vitro, block the cells in G0/G1 phase. The cells showed the aging morphological feature. The percentage of positive stained aging cells was dramatically increased, and could significantly up-regulate the expression of aging-related proteins P16 and RB, which were more obvious in the ASP + Ara-C group. In conclusion, the aging mechanism of KG1alpha cell induced by ASP and Ara-C may be related to the regulation of the expression of aging-related proteins, suggesting that the combined administration of ASP and anticancer drugs plays a better role in the treatment of leukemia .

Structural Effect of the Asp345a Insertion in Penicillin-Binding Protein 2 from Penicillin-Resistant Strains of Neisseria gonorrhoeae

PubMed Central

2015-01-01

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c−β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain. PMID:25403720

Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

PubMed

Fedarovich, Alena; Cook, Edward; Tomberg, Joshua; Nicholas, Robert A; Davies, Christopher

2014-12-09

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the β2c-β2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the β-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the β-lactam ring during acylation because of the negative charge of its side chain.

Education and Communication in an Interprofessional Antimicrobial Stewardship Program.

PubMed

Foral, Pamela A; Anthone, Jennifer M; Destache, Christopher J; Vivekanandan, Renuga; Preheim, Laurel C; Gorby, Gary L; Horne, John M; Dobronski, Leo A; Syed, Javeria J; Mindru, Cezarina; Ali, Mir A; Ali, Karim F; Neemann, Kari A; Bittner, Marvin J

2016-09-01

Interprofessional education/interprofessional practice (IPE/IPP) is an essential component in medical education and training. A collaborative interprofessional team environment ensures optimal patient-centered care. To describe the implementation of 2 interprofessional antimicrobial stewardship program (ASP) teams using IPE/IPP and to assess the acceptance rate by the primary medical and surgical teams of ASP recommendations for antimicrobial interventions. A business plan for the ASP was approved at 2 academic medical centers used for the present study. During a 3-year study period, 2 interprofessional ASP teams included an attending physician specializing in infectious disease (ID), an ID physician fellow, an ASP pharmacist, physician residents, medical students, pharmacy residents, and pharmacy students. Educational seminars were presented for all adult-admitting physicians to discuss the need for the ASP and the prospective audit and feedback process. Cases were presented for discussion during ASP/ID rounds and recommendations were agreed upon by the ASP team. A motivational interviewing face-to-face technique was frequently used to convey the ASP team recommendation to the primary medical or surgical team in a noncoercive and educational manner. The ASP team recommendations for ASP interventions were documented in the medical records. The overall acceptance rate of recommendations by the primary medical and surgical teams were greater than 90% (2051 of 2266). The most frequent interventions provided were streamline therapy (601), route of administration change (452), bug-drug mismatch (190), and discontinuation of therapy (179). Route of administration change was also the most frequently accepted intervention (96%). The motivational face-to-face communication technique was particularly useful in conveying ASP team member recommendations to the primary medical or surgical teams. Communicating recommendations as a multidisciplinary team in an educational manner seems to have resulted in to greater acceptance of recommendations.

Experimental and Theoretical Investigations of Infrared Multiple Photon Dissociation Spectra of Aspartic Acid Complexes with Zn2+ and Cd2.

PubMed

Boles, Georgia C; Hightower, Randy L; Coates, Rebecca A; McNary, Christopher P; Berden, Giel; Oomens, Jos; Armentrout, P B

2018-04-12

Complexes of aspartic acid (Asp) cationized with Zn 2+ : Zn(Asp-H) + , Zn(Asp-H) + (ACN) where ACN = acetonitrile, and Zn(Asp-H) + (Asp); as well as with Cd 2+ , CdCl + (Asp), were examined by infrared multiple photon dissociation (IRMPD) action spectroscopy using light generated from a free electron laser. A series of low-energy conformers for each complex was found using quantum chemical calculations to identify the structures formed experimentally. The main binding motif observed for the heavy-metal complex, CdCl + (Asp)[N,CO,CO s ], is a charge-solvated, tridentate structure, where the metal center binds to the backbone amino group and carbonyl oxygens of the backbone and side-chain carboxylic acids. Likewise, the deprotonated Zn(Asp-H) + (ACN) and Zn(Asp-H) + (Asp) complexes show comparable [N,CO - ,CO s ](ACN) and [N,CO - ,CO s ][N,CO,CO s ] coordinations, respectively. Interestingly, there was only minor spectral evidence for the analogous Zn(Asp-H) + [N,CO - ,CO s ] binding motif, even though this species is predicted to be the lowest-energy conformer. Instead, rearrangement and partial dissociation of the amino acid are observed, as spectral features most consistent with the experimental spectrum are exhibited by a four-coordinate Zn(Asp-NH 4 ) + [CO 2 - ,CO s ](NH 3 ) complex. Analysis of the mechanistic pathway leading from the predicted lowest-energy conformer to the isobaric deaminated complex is explored theoretically. Further, comparison of the current work to that of Zn 2+ and Cd 2+ complexes of asparagine (Asn) allows additional conclusions regarding populated conformers and effects of carboxamide versus carboxylic acid binding to be drawn.

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

PubMed

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

2016-12-01

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

The L76V Drug Resistance Mutation Decreases the Dimer Stability and Rate of Autoprocessing of HIV-1 Protease by Reducing Internal Hydrophobic Contacts

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

Louis, John M.; Zhang, Ying; Sayer, Jane M.

2011-09-06

The mature HIV-1 protease (PR) bearing the L76V drug resistance mutation (PR{sub L76V}) is significantly less stable, with a >7-fold higher dimer dissociation constant (K{sub d}) of 71 {+-} 24 nM and twice the sensitivity to urea denaturation (UC{sub 50} = 0.85 M) relative to those of PR. Differential scanning calorimetry showed decreases in T{sub m} of 12 C for PR{sub L76V} in the absence of inhibitors and 5-7 C in the presence of inhibitors darunavir (DRV), saquinavir (SQV), and lopinavir (LPV), relative to that of PR. Isothermal titration calorimetry gave a ligand dissociation constant of 0.8 nM for DRV,more » {approx}160-fold higher than that of PR, consistent with DRV resistance. Crystal structures of PR{sub L76V} in complexes with DRV and SQV were determined at resolutions of 1.45-1.46 {angstrom}. Compared to the corresponding PR complexes, the mutated Val76 lacks hydrophobic interactions with Asp30, Lys45, Ile47, and Thr74 and exhibits closer interactions with Val32 and Val56. The bound DRV lacks one hydrogen bond with the main chain of Asp30 in PR{sub L76V} relative to PR, possibly accounting for the resistance to DRV. SQV shows slightly improved polar interactions with PR{sub L76V} compared to those with PR. Although the L76V mutation significantly slows the N-terminal autoprocessing of the precursor TFR-PR{sub L76V} to give rise to the mature PR{sub L76V}, the coselected M46I mutation counteracts the effect by enhancing this rate but renders the TFR-PRM46I/L76V precursor less responsive to inhibition by 6 {micro}M LPV while preserving inhibition by SQV and DRV. The correlation of lowered stability, higher K{sub d}, and impaired autoprocessing with reduced internal hydrophobic contacts suggests a novel molecular mechanism for drug resistance.« less

Crypteins derived from the mouse neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice.

PubMed

Kotlinska, Jolanta H; Gibula-Bruzda, Ewa; Suder, Piotr; Wasielak, Magdalena; Bray, Lauriane; Raoof, Hana; Bodzon-Kulakowska, Anna; Silberring, Jerzy

2012-07-01

NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Otto, H.; Marti, T.; Holz, M.

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-((3-cholamidopropyl)dimethylammonio)-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting amore » role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base.« less

Structural basis of potassium activation in plant asparaginases.

PubMed

Ajewole, Ebenezer; Santamaria-Kisiel, Liliana; Pajak, Agnieszka; Jaskolski, Mariusz; Marsolais, Frédéric

2018-04-01

l-asparaginases (EC 3.5.1.1) play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium-dependent (PvAspG1) and potassium-independent (PvAspG-T2) l-asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser-118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG-T2. Conversely, the PvAspG1-S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG-T2-I117S mutant had a similar half-maximal effective concentration (EC 50 ) value for potassium activation, between 0.1 and 0.3 mm. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser-118 influences the ionization of specific functional groups in the enzyme-substrate complex. Together, the results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant l-asparaginases. ENZYME: l-Asparaginase (EC 3.5.1.1). © 2018 Federation of European Biochemical Societies.

Controversies in Antimicrobial Stewardship: Focus on New Rapid Diagnostic Technologies and Antimicrobials

PubMed Central

Wenzler, Eric; Wong, Jordan R.; Goff, Debra A.; Jankowski, Christopher A.; Bauer, Karri A.

2016-01-01

Antimicrobial stewardship programs (ASPs) are challenged with ensuring appropriate antimicrobial use while minimizing expenditures. ASPs have consistently demonstrated improved patient outcomes and significant cost reductions but are continually required to justify the costs of their existence and interventions due to the silo mentality often adopted by hospital administrators. As new technologies and antimicrobials emerge, ASPs are in a constant tug-of-war between providing optimal clinical outcomes and ensuring cost containment. Additionally, robust data on cost-effectiveness of new rapid diagnostic technologies and antimicrobials with subsequent ASP interventions to provide justification are lacking. As the implementation of an ASP will soon be mandatory for acute care hospitals in the United States, ASPs must find ways to justify novel interventions to align themselves with healthcare administrators. This review provides a framework for the justification of implementing a rapid diagnostic test or adding a new antimicrobial to formulary with ASP intervention, reviews approaches to demonstrating cost-effectiveness, and proposes methods for which ASPs may reduce healthcare expenditures via alternative tactics. PMID:27025521

Influence of the magnesium aspartate hydrochloride administration to the maternal circuit on the aspartate concentration of the fetal circuit under in vitro perfusion of human placenta.

PubMed

Malek, A; Leiser, R

2009-01-01

Magnesium aspartate hydrochloride (Magnesiocard, Mg-Asp-HCl) is proposed as a substitute of magnesium sulfate for the treatment of preeclampsia and premature labor. After an i.v. administration of a dose equivalent to that used in the treatment of preeclampsia to nonpregnant volunteers, a 10-fold increase of aspartic acid (Asp) over the physiological level was observed. Animal experiments have demonstrated that highly increased fetal levels of acidic amino acids such as Asp could be associated with neurotoxic damage in the fetal brain. The influence of such an elevation of Asp concentration in the maternal circuit on the fetal level, using the in vitro perfusion model of human placenta, was investigated. After a control phase (2h), a therapeutic dose of Mg combined with Asp (Magnesiocard, Mg-Asp-HCl) was applied to the maternal circuit approaching 10 times the physiological level of Asp. The administration was performed in two different phases simulating either a peak of maximum concentration (bolus application, 2h) or a steady state level (initially added, 4h). In four experiments, during experimental phases (6h) a slow increase in concentration in the fetal circuit was seen for Mg, AIB (alpha-aminoisobutyric acid, artificial amino acid) and creatinine confirming previous observations. In contrast, no net transfer of Asp across the placenta was seen. A continuous decrease in the concentration of Asp on both maternal and fetal side suggests active uptake and metabolization by the placenta. Viability control parameters remained stable indicating the absence of an effect on placental metabolism, permeability and morphology. Elevation of Asp concentration up to 10 times the physiological level by the administration of Mg-Asp-HCl to the maternal circuit under in vitro perfusion conditions of human placenta has no influence on the fetal level of Asp suggesting no transfer of Asp from the maternal to fetal compartment. Therefore, the administration of Mg-Asp-HCl to preeclamptic patients would be beneficial for the patients without any impact on placental or fetal physiology.

L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain.

PubMed

Krintel, Christian; Frydenvang, Karla; Ceravalls de Rabassa, Anna; Kaern, Anne M; Gajhede, Michael; Pickering, Darryl S; Kastrup, Jette S

2014-05-01

In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because L-Glu is difficult to displace, despite extensive dialysis. Here, we show that L-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mM) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mM), and we use differential scanning fluorimetry to show that L-Asp is able to stabilize the isolated GluA2 LBD. We also show that L-Asp can replace L-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mM L-Asp. Surprisingly, with 7.5 mM L-Asp, the GluA2 LBD crystallized as a mixed dimer, with L-Glu being present in one subunit, and neither L-Asp nor L-Glu being present in the other subunit. Thus, residual L-Glu is retained from the expression medium. On the other hand, only L-Asp was found at the binding site when 50 or 250 mM L-Asp was used for crystallization. The binding mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taking our findings together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mM L-Asp), 4O3C (50 mM L-Asp), and 4O3A (250 mM L-Asp). © 2014 FEBS.

Determination of ASPS performance for large payloads in the shuttle orbiter disturbance environment. [digital simulation

NASA Technical Reports Server (NTRS)

Keckler, C. R.; Kibler, K. S.; Powell, L. F.

1979-01-01

A high fidelity simulation of the annular suspension and pointing system (ASPS), its payload, and the shuttle orbiter was used to define the worst case orientations of the ASPS and its payload for the various vehicle disturbances, and to determine the performance capability of the ASPS under these conditions. The most demanding and largest proposed payload, the Solar Optical Telescope was selected for study. It was found that, in all cases, the ASPS more than satisfied the payload's requirements. It is concluded that, to satisfy facility class payload requirements, the ASPS or a shuttle orbiter free-drift mode (control system off) should be utilized.

Characterization of chicken antisera raised against Aspergillus spp. by enzyme linked immunosorbent assay.

PubMed

Chen, F S; Chen, J W; Zhao, S; Gan, Z B; Luo, X C; Zhou, Q

2000-10-01

Enzyme linked immunosorbent assays of three Aspergillus species have been developed. Laying hens were immunized with the exoantigens from Asp. flavus, Asp. ochreaus and Asp. versicolor. All test chickens except for one produced antisera raised against the exoantigens. The antisera production process and ELISA titer were analysed. Fourteen days after the first injection, the antisera began to produce largely, on the 35th day reached to the peak, and maintained a stable level until the 42nd day. The maximum ELISA titer of the antisera to the exoantigens from Asp. flavus, Asp. ochreaus and Asp. versicolor was 1:8,000, 1:10,000 and 1:10,000, respectively. The cross-reactivities of antisera were determined with seventeen species of Aspergillus, ten species of fungi from other genera and the buffer-extracts of grain. The antisera did not cross-react with the exoantigens from other genera and the buffer-extracts of grain. The antiserum to exoantigen from Asp. ochreaus was species-specific, whereas the antisera against Asp. flavus and Asp. versicolor tended to cross-react with other Aspergillus species to varying degrees. The results suggest that exoantigens immunoassays can be developed to indentify and detect Aspergillus genus in grains.

Effects of aspartic acid and potassium chloride on arginine kinase from shrimp.

PubMed

Tang, Hong-min; Yang, Yin-ye; Zhang, Song-fu

2006-12-15

The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding.

Comparison of subcutaneous soluble human insulin and insulin analogues (AspB9, GluB27; AspB10; AspB28) on meal-related plasma glucose excursions in type I diabetic subjects.

PubMed

Kang, S; Creagh, F M; Peters, J R; Brange, J; Vølund, A; Owens, D R

1991-07-01

To compare postprandial glucose excursions and plasma free insulin-analogue levels after subcutaneous injection of three novel human insulin analogues (AspB10; AspB9, GluB27; and AspB28) with those after injection of soluble human insulin (Actrapid HM U-100). Six male subjects with insulin-dependent diabetes, at least 1 wk apart and after an overnight fast and basal insulin infusion, received 72 nmol (approximately 12 U) s.c. of soluble human insulin 30 min before, or 72 nmol of each of the three analogues immediately before, a standard 500-kcal meal. Mean basal glucoses were similar on the 4 study days. Compared to human insulin (6.3 +/- 0.8 mM), mean +/- SE peak incremental glucose rises were similar after analogues AspB10 (5.4 +/- 0.8 mM) and AspB9, GluB27 (5.4 +/- 0.7 mM) and significantly lower after analogue AspB28 (3.6 +/- 1.2 mM, P less than 0.02). Relative to soluble human insulin (100% +/- SE21), incremental areas under the glucose curve between 0 and 240 min were 79% +/- 34 (AspB10, NS), 70% +/- 29 (AspB9, GluB27, NS), and 43% +/- 23 (AspB28, P less than 0.02). Basal plasma free insulin levels were similar on the 4 study days. Plasma free insulin-analogue levels rose rapidly to peak 30 min after injection at 308 +/- 44 pM (AspB10); 1231 +/- 190 pM (AspB9, GluB27) and 414 +/- 42 pM (AspB28) and were significantly higher than corresponding (i.e., 30 min postmeal) plasma free insulin levels of 157 +/- 15 pM (P less than 0.02 in each case). Plasma profiles of the insulin analogues were more physiological than that of human insulin after subcutaneous injection. All three analogues given immediately before the meal are at least as effective as soluble human insulin given 30 min earlier. These analogues are promising potential candidates for short-acting insulins of the future.

MPEG-4 ASP SoC receiver with novel image enhancement techniques for DAB networks

NASA Astrophysics Data System (ADS)

Barreto, D.; Quintana, A.; García, L.; Callicó, G. M.; Núñez, A.

2007-05-01

This paper presents a system for real-time video reception in low-power mobile devices using Digital Audio Broadcast (DAB) technology for transmission. A demo receiver terminal is designed into a FPGA platform using the Advanced Simple Profile (ASP) MPEG-4 standard for video decoding. In order to keep the demanding DAB requirements, the bandwidth of the encoded sequence must be drastically reduced. In this sense, prior to the MPEG-4 coding stage, a pre-processing stage is performed. It is firstly composed by a segmentation phase according to motion and texture based on the Principal Component Analysis (PCA) of the input video sequence, and secondly by a down-sampling phase, which depends on the segmentation results. As a result of the segmentation task, a set of texture and motion maps are obtained. These motion and texture maps are also included into the bit-stream as user data side-information and are therefore known to the receiver. For all bit-rates, the whole encoder/decoder system proposed in this paper exhibits higher image visual quality than the alternative encoding/decoding method, assuming equal image sizes. A complete analysis of both techniques has also been performed to provide the optimum motion and texture maps for the global system, which has been finally validated for a variety of video sequences. Additionally, an optimal HW/SW partition for the MPEG-4 decoder has been studied and implemented over a Programmable Logic Device with an embedded ARM9 processor. Simulation results show that a throughput of 15 QCIF frames per second can be achieved with low area and low power implementation.

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry.

PubMed

Gurevich-Messina, Juan M; Giudicessi, Silvana L; Martínez-Ceron, María C; Acosta, Gerardo; Erra-Balsells, Rosa; Cascone, Osvaldo; Albericio, Fernando; Camperi, Silvia A

2015-01-01

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of 'one-bead-one-peptide' combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4-hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc-Asp[2-phenylisopropyl (OPp)]-OH to Ala-Gly-oxymethylbenzamide-ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N-terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N-Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one-bead-one-cyclic depsipeptide libraries that can be easily open for its sequencing by matrix-assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Deep Sequencing Reveals Spatially Distributed Distinct Hot Spot Mutations in DICER1-Related Multinodular Goiter.

PubMed

de Kock, Leanne; Bah, Ismaël; Revil, Timothée; Bérubé, Pierre; Wu, Mona K; Sabbaghian, Nelly; Priest, John R; Ragoussis, Jiannis; Foulkes, William D

2016-10-01

Nontoxic multinodular goiter (MNG) occurs frequently, but its genetic etiology is not well established. Familial MNG and MNG occurring with ovarian Sertoli-Leydig cell tumor are associated with germline DICER1 mutations. We recently identified second somatic DICER1 ribonuclease (RNase) IIIb mutations in two MNGs. The objective of the study was to investigate the occurrence of somatic DICER1 mutations and mutational clonality in MNG. MNGs from 15 patients (10 with and five without germline DICER1 mutations) were selected based on tissue availability. Core biopsies/scrapings (n = 70) were obtained, sampling areas of follicular hyperplasia, hyperplasia within colloid pools, unremarkable thyroid parenchyma, and areas of thyroid parenchyma, not classified. After capture with a Fluidigm access array, the coding sequence of DICER1 was deep sequenced using DNA from each core/scraping. All germline DICER1-mutated cases were found to harbor at least one RNase III mutation. Specifically, we identified 12 individually distinct DICER1 RNase IIIb hot spot mutations in 32 of the follicular hyperplasia or hyperplasia within colloid pools cores/scrapings. These mutations are predicted to affect the metal-ion binding residues at positions p.Glu1705, p.Asp1709, p.Gly1809, p.Asp1810, and p.Glu1813. Somatic RNase IIIb mutations were identified in the 10 DICER1 germline mutated MNGs as follows: two cases contained one somatic mutation, five cases contained two mutations, and three cases contained three distinct somatic hot spot mutations. No RNase IIIb mutations were identified in the MNGs from individuals without germline DICER1 mutations. This study demonstrates that nodules within MNG occurring in DICER1 syndrome are associated with spatially distributed somatic DICER1 RNase IIIb mutations.

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

PubMed

Strauss, E G; Levinson, R; Rice, C M; Dalrymple, J; Strauss, J H

1988-05-01

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

BEST1 sequence variants in Italian patients with vitelliform macular dystrophy

PubMed Central

Sodi, Andrea; Passerini, Ilaria; Caputo, Roberto; Bacci, Giacomo Maria; Bodoj, Mirela; Torricelli, Francesca; Menchini, Ugo

2012-01-01

Purpose To analyze the spectrum of sequence variants in the BEST1 gene in a group of Italian patients affected by Best vitelliform macular dystrophy (VMD). Methods Thirty Italian patients with a diagnosis of VMD and 20 clinically healthy relatives were recruited. They belonged to 19 Italian families predominantly originating from central Italy. They received a standard ophthalmologic examination, OCT scan, and electrophysiological tests (ERG and EOG). Fluorescein and ICG angiographies and fundus autofluorescence imaging were performed in selected cases. DNA samples were analyzed for sequence variants of the BEST1 gene by direct sequencing techniques. Results Nine missense variants and one deletion were found in the affected patients; each patient carried one mutation. Five variants [c.73C>T (p.Arg25Trp), c.652C>T (p.Arg218Cys), c.652C>G (p.Arg218Gly), c.728C>T (p.Ala243Val), c.893T>C (p.Phe298Ser)] have already been described in literature while another five variants [c.217A>C (p.Ile73Leu), c.239T>G (p.Phe80Cys), c.883_885del (p.Ile295del), c.907G>A (p.Asp303Asn), c.911A>G (p.Asp304Gly)] had not previously been reported. Affected patients, sometimes even from the same family, occasionally showed variable phenotypes. One heterozygous variant was also found in five clinically healthy relatives with normal fundus, visual acuity and ERG but with abnormal EOG. Conclusions Ten variants in the BEST1 gene were detected in a group of individuals with clinically apparent VMD, and in some clinically normal individuals with an abnormal EOG. The high prevalence of novel variants and the frequent report of a specific variant (p.Arg25Trp) that has rarely been described in other ethnic groups suggests a distribution of BEST1 variants peculiar to Italian VMD patients. PMID:23213274

Xeroderma pigmentosum group D polymorphisms and esophageal cancer susceptibility: a meta-analysis based on case-control studies.

PubMed

Yang, Rong; Zhang, Chong; Malik, Armah; Shen, Zhi-Da; Hu, Jian; Wu, Yi-He

2014-11-28

To clarify the effects of the xeroderma pigmentosum group D (XPD) Asp312Asn and Lys751Gln gene polymorphisms on the risk of esophageal cancer (EC). A computerised literature search was conducted to identify the relevant studies from the PUBMED and EMBASE databases, reviews, and reference lists of relevant articles. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the associations between the XPD Asp312Asn and/or Lys751Gln polymorphisms and EC susceptibility. Statistical analyses were performed using the software Stata 12.0. A fixed or random effects model was selected based on a heterogeneity test. Publication bias was estimated using funnel plots and Egger's linear regression method. Subgroup analyses were performed based on histological type and ethnicity. Thirteen case-control studies with a total of 10 comparisons for the Asp312Asn polymorphism, including 2373 cases and 3175 controls, and 15 comparisons for the Lys751Gln polymorphism, including 3226 cases and 5237 controls, were recruited for the meta-analysis. In terms of the XPD Asp312Asn polymorphism, significantly increased EC risks were identified in the Asp/Asn vs Asp/Asp comparison (OR = 1.17, 95%CI: 1.02-1.33, P = 0.03) and in the dominant-model comparison (Asn/Asn+Asp/Asn vs Asp/Asp: OR = 1.18, 95%CI: 1.04-1.34, P = 0.01). However, no significant associations were found in the Asn/Asn vs Asp/Asp comparison (OR = 1.30, 95%CI: 1.00-1.70, P = 0.05) or in the recessive-model comparison (Asn/Asn vs Asp/Asn + Asp/Asp: OR = 1.17, 95%CI: 0.91-1.50, P = 0.22). In terms of the XPD Lys751Gln polymorphism, a significant association with EC susceptibility was found under the recessive model (Gln/Gln vs Lys/Gln+Lys/Lys: OR = 1.21, 95%CI: 1.02-1.43, P = 0.03). However, no associations were identified in the other comparisons (co-dominant model: Lys/Gln vs Lys/Lys: OR = 1.11, 95%CI: 0.94-1.31, P = 0.20; Gln/Gln vs Lys/Lys: OR = 1.31, 95%CI: 0.98-1.75, P = 0.07; dominant model: OR = 1.14, 95%CI: 0.96-1.35, P = 0.14). The results of this meta-analysis suggest that the XPD Asp312Asn and Lys751Gln gene polymorphisms are associated with a significantly increased risk for EC.

Ingested d-Aspartate Facilitates the Functional Connectivity and Modifies Dendritic Spine Morphology in Rat Hippocampus.

PubMed

Kitamura, Akihiko; Hojo, Yasushi; Ikeda, Muneki; Karakawa, Sachise; Kuwahara, Tomomi; Kim, Jonghyuk; Soma, Mika; Kawato, Suguru; Tsurugizawa, Tomokazu

2018-05-30

d-Aspartate (d-Asp), the stereoisomer of l-aspartate, has a role in memory function in rodents. However, the mechanism of the effect of d-Asp has not been fully understood. In this study, we hypothesized that ingested d-Asp directly reaches the hippocampal tissues via the blood circulation and modifies the functional connectivity between hippocampus and other regions through spinogenesis in hippocampal CA1 neurons. The spinogenesis induced by the application of d-Asp was investigated using rat acute hippocampal slices. The density of CA1 spines was increased following 21 and 100 μM d-Asp application. The nongenomic spine increase pathway involved LIM kinase. In parallel to the acute slice study, brain activation was investigated in awake rats using functional MRI following the intragastric administration of 5 mM d-Asp. Furthermore, the concentration of d-Asp in the blood serum and hippocampus was significantly increased 15 min after intragastric administration of d-Asp. A functional connectivity by awake rat fMRI demonstrated increased slow-frequency synchronization in the hippocampus and other regions, including the somatosensory cortex, striatum, and the nucleus accumbens, 10-20 min after the start of d-Asp administration. These results suggest that ingested d-Asp reaches the brain through the blood circulation and modulates hippocampal neural networks through the modulation of spines.

The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

PubMed

Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

2016-09-14

Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.

C3 Polymorphism Influences Circulating Levels of C3, ASP and Lipids in Schizophrenic Patients.

PubMed

Nsaiba, Mohamed Jalloul; Lapointe, Marc; Mabrouk, Hajer; Douki, Wahiba; Gaha, Lotfi; Pérusse, Louis; Bouchard, Claude; Jrad, Besma Bel Hadj; Cianflone, Katherine

2015-05-01

Excessive activation of complement is associated with many diseases including schizophrenia. Investigation of C3 polymorphisms, circulating C3, cleavage product ASP/C3adesArg, and lipid metabolism. Cross-sectional analysis. C3 genotyping (CC vs GG for R102L) was performed on 434 Tunisian people consisting of 272 schizophrenic (SZ) patients and 162 control subjects. In a age- and gender-matched subgroups of the three genotypes (131 SZ and 112 NOR), plasma triglycerides, total cholesterol (C), LDL-C, HDL-C, ASP, and complement C3 were measured. C3 gene polymorphism influences BMI and plasma C3, ASP, triglyceride, total cholesterol, LDL-C and HDL-C among SZ patients (p < 0.05-0.0001), with increasing values demonstrated from CC (common form) to CG (heterozygote form) to GG (rare homozygote) forms. Significant correlations between plasma C3 and BMI, triglyceride, HDL-C and ASP (p < 0.05-0.0001) were observed, while ASP correlated with BMI and LDL-C (p = 0.005, p = 0.001, respectively) in SZ patients. Further, proportional conversion of C3 to ASP (%ASP/C3) also increased (p < 0.0001, GG>CG>CC). C3 polymorphisms and plasma C3, ASP and %ASP/C3 correlated with lipid parameters in this SZ population, suggesting that factors predisposing patients to schizophrenia are permissive for complement pathway activation and dyslipidemic influences.

Additive antithrombotic effect of ASP6537, a selective cyclooxygenase (COX)-1 inhibitor, in combination with clopidogrel in guinea pigs.

PubMed

Sakata, Chinatsu; Suzuki, Ken-Ichi; Morita, Yoshiaki; Kawasaki, Tomihisa

2017-03-05

Clopidogrel (Plavix ® , Sanofi-Aventis), the adenosine diphosphate P2Y 12 receptor antagonist, is reported to be effective in the prevention of cardiovascular events and is often used in combination with aspirin, particularly in high-risk patients. ASP6537 is a reversible cyclooxygenase (COX)-1 inhibitor that is under investigation as an anti-platelet agent. First, we investigated the reversibility of the antiplatelet effect of ASP6537 and its interaction with ibuprofen to compare the usability of ASP6537 with that of aspirin. We then evaluated the antithrombotic effect of ASP6537 in combination with clopidogrel using a FeCl 3 -induced thrombosis model in guinea pigs. ASP6537 exerted reversible antiplatelet activity, and no pharmacodynamic interaction with ibuprofen was noted. When administered as monotherapy, ASP6537 exerted a significant antithrombotic effect at ≥3mg/kg, while aspirin inhibited thrombosis at 100mg/kg. ASP6537 exerted significant additive effects in combination with clopidogrel, and the minimum antithrombotic dose was reduced by concomitant administration of clopidogrel. Our study showed that ASP6537 did not interact with ibuprofen and has clear additive effects in combination with clopidogrel. ASP6537 may therefore represent a promising antiplatelet agent for use in clinical settings in combination with clopidogrel. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanism research on starting residual oil migration in ASP flooding with different Alkali concentration

NASA Astrophysics Data System (ADS)

Xia, Huifen; Pan, Junliang; Niu, Lijuan; Xu, Tianhan

2018-02-01

The results illustrate that under the condition of the same viscosity of ASP system, oil displacement efficiency is different while the ASP system with different alkali concentration has the same order of magnitude as the interfacial tension of oil. In this paper, the microscopic simulation visual model is used to study the mechanism of starting migration of residual oil by doing ASP flooding experiments with different alkali concentration. The results indicate that the migration of residual oil is different from that in the ASP systems with different alkali concentration. ASP system with high alkali concentration can start the migration by means of emulsifying residual oil into oil droplets and oil threads, on this account, increasing the alkali concentration can make the recovery degree of ASP system higher, which will finally be beneficial to the oil recovery.

BIOPEP database and other programs for processing bioactive peptide sequences.

PubMed

Minkiewicz, Piotr; Dziuba, Jerzy; Iwaniak, Anna; Dziuba, Marta; Darewicz, Małgorzata

2008-01-01

This review presents the potential for application of computational tools in peptide science based on a sample BIOPEP database and program as well as other programs and databases available via the World Wide Web. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of protein fragments, calculation of quantitative descriptors as measures of the value of proteins as potential precursors of bioactive peptides, and prediction of bonds susceptible to hydrolysis by endopeptidases in a protein chain. Other bioactive and allergenic peptide sequence databases are also presented. Programs enabling the construction of binary and multiple alignments between peptide sequences, the construction of sequence motifs attributed to a given type of bioactivity, searching for potential precursors of bioactive peptides, and the prediction of sites susceptible to proteolytic cleavage in protein chains are available via the Internet as are other approaches concerning secondary structure prediction and calculation of physicochemical features based on amino acid sequence. Programs for prediction of allergenic and toxic properties have also been developed. This review explores the possibilities of cooperation between various programs.

Low temperature effects on nitrification and nitrifier community structure in V-ASP for decentralized wastewater treatment and its improvement by bio-augmentation.

PubMed

Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke

2018-03-01

The vegetation-activated sludge process (V-ASP) has been proved to be an environment-friendly decentralized wastewater treatment system with extra esthetic function and less footprint. However, the effects of low temperature on the treatment performance of V-ASP and related improvement methods are rarely investigated, up to now. In this work, the effect of low temperature on nitrification in V-ASP was comprehensively investigated from overall nitrification performance, substrate utilization kinetics, functional enzymatic activities, and microbial community structure shift by comparison with conventional ASP. Bio-augmentation methods in terms of single-time nitrifier-enriched biomass dosage were employed to improve nitrification efficiency in bench- and full-scale systems. The experiment results demonstrated that the NH 4 + -N removal efficiency in V-ASP system decreased when the operational temperature decreased from 30 to 15 °C, and the decreasing extent was rather smaller compared to ASP, as well as ammonium and nitrite oxidation rates and enzymatic activities, which indicated the V-ASP system possesses high resistance to low temperature. With direct dosage of 1.6 mg nitrifier/gSS sludge, the nitrification efficiency in V-ASP was enhanced dramatically from below 50% to above 90%, implying that bio-augmentation was effective for V-ASP whose enzymatic activities and microbial communities were both also improved. The feasibility and effectiveness of bio-augmentation was further confirmed in a full-scale V-ASP system after a long-term experiment which is instructive for the practical application.

Fidelity in After-School Program Intervention Research: A Systematic Review

ERIC Educational Resources Information Center

Maynard, Brandy R.; Peters, Kristen E.; Vaughn, Michael G.; Sarteschi, Christine M.

2014-01-01

Over the past 2 decades, the number of after-school programs (ASP) and the number of students attending ASPs has markedly increased. Although several reviews and meta-analyses have examined the outcomes of ASPs, ASP intervention study reviews have not specifically examined intervention fidelity. Establishing intervention fidelity is critically…

The Development of After-School Program Educators through University-Community Partnerships

ERIC Educational Resources Information Center

Mahoney, Joseph L.; Levine, Mark D.; Hinga, Briana

2010-01-01

Participation in after-school programs (ASPs) "can" positively affect the development of young people. However, "whether" ASPs are beneficial depends on program quality. Although many factors influence the quality of a program, the competencies of adult staff who lead ASPs are a critical determinant. Unfortunately, ASP staff…

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands.

PubMed

Auvynet, Constance; Seddiki, Nabila; Dunia, Irene; Nicolas, Pierre; Amiche, Mohamed; Lacombe, Claire

2006-01-01

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.

Common variants of the vitamin D binding protein gene and adverse health outcomes

PubMed Central

Malik, Suneil; Fu, Lei; Juras, David James; Karmali, Mohamed; Wong, Betty Y. L.; Gozdzik, Agnes

2013-01-01

The vitamin D binding protein (DBP) is the major plasma carrier for vitamin D and its metabolites, but it is also an actin scavenger, and is the precursor to the immunomodulatory protein, Gc-MAF. Two missense variants of the DBP gene – rs7041 encoding Asp432Glu and rs4588 encoding Thr436Lys – change the amino acid sequence and alter the protein function. They are common enough to generate population-wide constitutive differences in vitamin D status, based on assay of the serum metabolite, 25-hydroxyvitamin D (25OHD). Whether these variants also influence the role of vitamin D in an immunologic milieu is not known. However, the issue is relevant, given the immunomodulatory effects of DBP and the role of protracted innate immune-related inflammation in response to tissue injury or repeated infection. Indeed, DBP and vitamin D may jointly or independently contribute to a variety of adverse health outcomes unrelated to classical notions of their function in bone and mineral metabolism. This review summarizes the reports to date of associations between DBP variants, and various chronic and infectious diseases. The available information leads us to conclude that DBP variants are a significant and common genetic factor in some common disorders, and therefore, are worthy of closer attention. In view of the heightened interest in vitamin D as a public health target, well-designed studies that look simultaneously at vitamin D and its carrier in relation to genotypes and adverse health outcome should be encouraged. PMID:23427793

America’s Holy War

DTIC Science & Technology

2006-04-14

Journal. 24 April 2002. http://www.mepc.org/public_asp/journal/ vol10 /0306_alqaeda.asp accessed on 21 Sept 05. 21 “Translation of April 24, 2002 al...Qaeda document”. Middle East Policy Council Journal. 24 April 2002. http://www.mepc.org/public_asp/journal/ vol10 /0306_alqaeda.asp accessed on 21 Sept... vol10 /0306_alqaeda.asp accessed on 21 Sept 05. 23 “Osama bin Laden’s Speech on the Eve of the 2004 US Elections”. The Middle East Media Research

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

PubMed

de Moura, Andréa A; Kayano, Anderson M; Oliveira, George A; Setúbal, Sulamita S; Ribeiro, João G; Barros, Neuza B; Nicolete, Roberto; Moura, Laura A; Fuly, Andre L; Nomizo, Auro; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M; Calderon, Leonardo A

2014-01-01

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep groundwater.

PubMed

Eydal, Hallgerd S C; Jägevall, Sara; Hermansson, Malte; Pedersen, Karsten

2009-10-01

Viruses were earlier found to be 10-fold more abundant than prokaryotes in deep granitic groundwater at the Aspö Hard Rock Laboratory (HRL). Using a most probable number (MPN) method, 8-30 000 cells of sulphate-reducing bacteria per ml were found in groundwater from seven boreholes at the Aspö HRL. The content of lytic phages infecting the indigenous bacterium Desulfovibrio aespoeensis in Aspö groundwater was analysed using the MPN technique for phages. In four of 10 boreholes, 0.2-80 phages per ml were found at depths of 342-450 m. Isolates of lytic phages were made from five cultures. Using transmission electron microscopy, these were characterized and found to be in the Podoviridae morphology group. The isolated phages were further analysed regarding host range and were found not to infect five other species of Desulfovibrio or 10 Desulfovibrio isolates with up to 99.9% 16S rRNA gene sequence identity to D. aespoeensis. To further analyse phage-host interactions, using a direct count method, growth of the phages and their host was followed in batch cultures, and the viral burst size was calculated to be approximately 170 phages per lytic event, after a latent period of approximately 70 h. When surviving cells from infected D. aespoeensis batch cultures were inoculated into new cultures and reinfected, immunity to the phages was found. The parasite-prey system found implies that viruses are important for microbial ecosystem diversity and activity, and for microbial numbers in deep subsurface groundwater.

Synthesis of mixed MOR/KOR efficacy cyclic opioid peptide analogs with antinociceptive activity after systemic administration.

PubMed

Perlikowska, Renata; Piekielna, Justyna; Gentilucci, Luca; De Marco, Rossella; Cerlesi, Maria Camilla; Calo, Girolamo; Artali, Roberto; Tömböly, Csaba; Kluczyk, Alicja; Janecka, Anna

2016-02-15

Cyclic pentapeptide Tyr-c[D-Lys-Phe-Phe-Asp]NH2, based on the structure of endomorphin-2 (EM-2), which shows high affinity to the μ-opioid receptor (MOR) and a very strong antinociceptive activity in mice was used as a parent compound for the structure-activity relationship studies. In this report we synthesized analogs of a general sequence Dmt-c[D-Lys-Xaa-Yaa-Asp]NH2, with D-1- or D-2-naphthyl-3-alanine (D-1-Nal or D-2-Nal) in positions 3 or 4. In our earlier papers we have indicated that replacing a phenylalanine residue by the more extended aromatic system of naphthylalanines may result in increased bioactivities of linear analogs. The data obtained here showed that only cyclopeptides modified in position 4 retained the sub-nanomolar MOR and nanomolar κ-opioid receptor (KOR) affinity, similar but not better than that of a parent cyclopeptide. In the in vivo mouse hot-plate test, the most potent analog, Dmt-c[D-Lys-Phe-D-1-Nal-Asp]NH2, exhibited higher than EM-2 but slightly lower than the cyclic parent peptide antinociceptive activity after peripheral (ip) and also central administration (icv). Conformational analyses in a biomimetic environment and molecular docking studies disclosed the structural determinants responsible for the different pharmacological profiles of position 3- versus position 4-modified analogs. Copyright © 2015. Published by Elsevier Masson SAS.

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

PubMed

Varela, M F; Brooker, R J; Wilson, T H

1997-09-01

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

Fibrinogen Bastia (gamma 318 Asp-->Tyr) a novel abnormal fibrinogen characterized by defective fibrin polymerization.

PubMed

Lounes, K C; Soria, C; Valognes, A; Turchini, M F; Soria, J; Koopman, J

1999-12-01

A new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the gamma-chain since by SDS-PAGE performed according to the method of Laemmli two gamma-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia gamma-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the gamma-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G-->T) in the exon VIII of the gamma chain gene, resulting in the amino acid substitution 318 Asp (GAC)-->Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the gamma-chain.

Analysis of the binding sites of vitamin D 1α-hydroxylase (CYP27B1) and vitamin D 24-hydroxylase (CYP24A1) for the design of selective CYP24A1 inhibitors: Homology modelling, molecular dynamics simulations and identification of key binding requirements.

PubMed

Taban, Ismail M; Zhu, Jinge; DeLuca, Hector F; Simons, Claire

2017-10-15

A homology model of human CYP27B1 was built using MOE and was further optimised by molecular dynamics simulations of the hCYP27B1 homology model and a hCYP27B1-SDZ-88357 complex. Docking results from the hCYP27B1-SDZ-88357 complex showed amino acids Arg107, Asn387 and Asp320 have an important role in binding interaction, with Asp320 part of the important acid-alcohol pair situated in the I-helix with the conserved sequence (A/G) GX (E/D) (T/S), which assumes an essential role in the binding of an oxygen molecule for catalysis. Additional docking experiments with selective hCYP27B1 or hCYP24A1 inhibitors using both the hCYP27B1 model and a triple mutant hCYP24A1 model provided further support for the importance of H-bonding interactions with the three identified active site amino acids. To confirm the role of Arg107, Asn387 and Asp320 in the active site of hCYP27B1 compounds were designed that would form H-bonding interactions, as determined from docking experiments with the hCYP27B1 model. Subsequent synthesis and CYP24A1 and CYP27B1 enzyme assays of the designed compounds 1a and 1b showed a∼5-fold selectivity for CYP27B1 confirming the importance of Asp320 in particular and also Asn387 and Arg107 as important amino acids for CYP27B1 inhibitory activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mapping of the human cone transducin {alpha} subunit (GNAT2) gene to 1p13 and mutation analysis in patients with Stargardt`s disease

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

Magovcevic, I.; Weremowicz, S.; Morton, C.C.

Transducin {alpha} subunits are members of a large family of G-proteins and play an important role in phototransduction in rod and cone photoreceptors. We report the localization of the human cone {alpha} transducin (GNAT2) gene using fluorescence in situ hybridization (FISH) on chromosome 1 in band p13. The recent assignment of a gene for Stargardt`s disease to the same chromosomal region by linkage analysis prompted us to investigate the possible role of GNAT2 in the pathogenesis of this disease. Stargardt`s disease is characterized by degeneration in late childhood or early adulthood of the macula of the retina, a region richmore » in cones. We screened patients with Stargardt`s disease, with or without peripheral cone involvement as monitored by the full-field ERG, for mutations in this gene. We investigated 66 unrelated patients including 22 with peripheral cone dysfunction for mutations in the coding region of the GNAT2 gene using polymerase chain reaction-single strand conformation polymorphism analysis (SSCP) and direct sequencing. One patient (034-16) was heterozygous for a silent change in exon VI, Asp238Asp (GAT to GAC). Two patients, one (035-005) with peripheral cone involvement and one (071-001) without peripheral cone involvement, were heterozygous for the missense change Val124Met (GTG to ATG) in exon IV. A subsequent screen of 96 unrelated, unaffected controls revealed one individual (N10) who was also heterozygous for the Val124Met alteration. We concluded that Asp238Asp and Val124Met are rare variants not causing Stargardt`s disease. Hence, no disease-specific mutations were found indicating that GNAT2 is probably not involved in the pathogenesis of most cases of Stargardt`s disease.« less

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.

PubMed

Mayrhofer, Severine; Pöggeler, Stefanie

2005-04-01

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.

DETERMINATION OF C1 AND Cx CELLULOLYTIC ACTIVITIES IN ENZYME PREPARATIONS OF MOLD FUNGI (Opredelenie C1 i Cx Tsellyuloliticheskikh Aktivnostei v Fermentnykh Preparatakh iz Plesnevykh Gribov),

DTIC Science & Technology

Trichotecium roseum, Aspergillus awamory, Asp. niger , Asp. flavus. Differences in the distribution of C1 - and Cx - activities in the preparations of various strains of the same fungus (Asp. awamory, Asp. oryzae) are shown. (Author)

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

Metz, G.; Siebert, F.; Engelhard, M.

Solid state {sup 13}C nuclear magnetic resonance measurements of bacteriorhodopsin labeled with (4-{sup 13}C)Asp show that resonances of single amino acids can be resolved. In order to assign and characterize the resonances of specific Asp residues, three different approaches were used. (1) Determination of the chemical shift anisotropy from side-band intensities provides information about the protonation state of Asp residues; (2) relaxation studies and T{sub 1} filtering allow one to discriminate between resonances with different mobility; (3) a comparison of the spectra of light- and dark-adapted bacteriorhodopsin provides evidence for resonances from aspartic acid residues in close neighborhood of themore » chromophore. In agreement with other investigations, four resonances are assigned to internal residues. Two of them are protonated in the ground state up to pH 10 (Asp{sub 96} and Asp{sub 115}). All other detected resonance, including Asp{sub 85} and Asp{sub 212}, are due to deprotonated aspartic acid. Two lines due to the two internal deprotonated groups change upon dark and light adaptation, whereas the protonated Asp residues are unaffected.« less

Biodegradation of phenanthrene in bioaugmented microcosm by consortium ASP developed from coastal sediment of Alang-Sosiya ship breaking yard.

PubMed

Patel, Vilas; Patel, Janki; Madamwar, Datta

2013-09-15

A phenanthrene-degrading bacterial consortium (ASP) was developed using sediment from the Alang-Sosiya shipbreaking yard at Gujarat, India. 16S rRNA gene-based molecular analyses revealed that the bacterial consortium consisted of six bacterial strains: Bacillus sp. ASP1, Pseudomonas sp. ASP2, Stenotrophomonas maltophilia strain ASP3, Staphylococcus sp. ASP4, Geobacillus sp. ASP5 and Alcaligenes sp. ASP6. The consortium was able to degrade 300 ppm of phenanthrene and 1000 ppm of naphthalene within 120 h and 48 h, respectively. Tween 80 showed a positive effect on phenanthrene degradation. The consortium was able to consume maximum phenanthrene at the rate of 46 mg/h/l and degrade phenanthrene in the presence of other petroleum hydrocarbons. A microcosm study was conducted to test the consortium's bioremediation potential. Phenanthrene degradation increased from 61% to 94% in sediment bioaugmented with the consortium. Simultaneously, bacterial counts and dehydrogenase activities also increased in the bioaugmented sediment. These results suggest that microbial consortium bioaugmentation may be a promising technology for bioremediation. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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

2018-04-26

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

The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code

PubMed Central

Fischer, Frédéric; Huot, Jonathan L.; Lorber, Bernard; Diss, Guillaume; Hendrickson, Tamara L.; Becker, Hubert D.; Lapointe, Jacques; Kern, Daniel

2012-01-01

Helicobacter pylori catalyzes Asn-tRNAAsn formation by use of the indirect pathway that involves charging of Asp onto tRNAAsn by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNAAsn binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNAAsn is bound by ND-AspRS which releases the Asp-tRNAAsn product much slower than the cognate Asp-tRNAAsp; this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNAAsn before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn. PMID:22362756

Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response.

PubMed

González-Hernández, Ana; Van Coppernolle, Stefanie; Borloo, Jimmy; Van Meulder, Frederik; Paerewijck, Oonagh; Peelaers, Iris; Leclercq, Georges; Claerebout, Edwin; Geldhof, Peter

2016-07-11

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity.

Racemization at the Asp 58 residue in αA-crystallin from the lens of high myopic cataract patients.

PubMed

Zhu, Xiang-Jia; Zhang, Ke-Ke; He, Wen-Wen; Du, Yu; Hooi, Michelle; Lu, Yi

2018-02-01

Post-translational modifications in lens proteins are key causal factors in cataract. As the most abundant post-translational modification in the lens, racemization may be closely related to the pathogenesis of cataract. Racemization of αA-crystallin, a crucial structural and heat shock protein in the human lens, could significantly influence its structure and function. In previous studies, elevated racemization from l-Asp 58 to d-isoAsp58 in αA-crystallin has been found in age-related cataract (ARC) lenses compared to normal aged human lenses. However, the role of racemization in high myopic cataract (HMC), which is characterized by an early onset of nuclear cataract, remains unknown. In the current study, apparently different from ARC, significantly increased racemization from l-Asp 58 to d-Asp 58 in αA-crystallin was identified in HMC lenses. The average racemization rates for each Asp isoform were calculated in ARC and HMC group. In ARC patients, the conversion of l-Asp 58 to d-isoAsp 58, up to 31.89%, accounted for the main proportion in racemization, which was in accordance with the previous studies. However, in HMC lenses, the conversion of l-Asp 58 to d-Asp 58, as high as 35.44%, accounted for the largest proportion of racemization in αA-crystallin. The different trend in the conversion of αA-crystallin by racemization, especially the elevated level of d-Asp 58 in HMC lenses, might prompt early cataractogenesis and a possible explanation of distinct phenotypes of cataract in HMC. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Fungal extracellular polysaccharides in house dust as a marker for exposure to fungi: relations with culturable fungi, reported home dampness, and respiratory symptoms.

PubMed

Douwes, J; van der Sluis, B; Doekes, G; van Leusden, F; Wijnands, L; van Strien, R; Verhoeff, A; Brunekreef, B

1999-03-01

Epidemiologic studies have demonstrated an association between indoor fungal growth and respiratory symptoms. However, in only a few studies was fungal exposure actually measured. The purpose of this study was to evaluate the measurement by enzyme immunoassay of extracellular polysaccharides of Aspergillus and Penicillium species (EPS-Asp/Pen ) in house dust as a marker for fungal exposure and to study the relations between EPS-Asp/Pen levels and home dampness and respiratory symptoms in children. Extracts of house dust samples from bedroom and living room floors and mattresses from homes of 31 children with chronic respiratory symptoms and 29 children with no chronic respiratory symptoms were analyzed for EPS-Asp/Pen. EPS-Asp/Pen were readily detectable (40 to 46,513 nanogram equivalent/g dust) in 161 house dust extracts, with highest concentrations in living room floor dust. EPS-Asp/Pen levels were 2 to 3 times higher on carpeted floors than on smooth floors. EPS-Asp/Pen were significantly correlated with total culturable fungi (r = 0.3 to 0.5) and with house dust mite allergens (r = 0.3 to 0.5). EPS-Asp/Pen levels in living room floor dust were positively associated with occupant-reported home dampness. This was not observed for EPS-Asp/Pen in bedroom floor and mattress dust. EPS-Asp/Pen levels in living room floor dust were positively associated with respiratory symptoms. EPS-Asp/Pen in bedroom floor and mattress dust showed a reversed association with respiratory symptoms, possibly because of allergen-avoidance measures taken in the bedroom. The enzyme immunoassay for fungal EPS-Asp/Pen may be a useful method for exposure assessment of indoor fungi.

Correlation between cervical lordosis and adjacent segment pathology after anterior cervical spinal surgery.

PubMed

Lee, Soo Eon; Jahng, Tae-Ahn; Kim, Hyun Jib

2015-12-01

To evaluate the incidence and risk factors for adjacent segment pathology (ASP) after anterior cervical spinal surgery. Fourteen patients (12 male, mean age 47.1 years) who underwent single-level cervical disk arthroplasty (CDA group) and 28 case-matched patients (24 male, mean age 53.6 years) who underwent single-level anterior cervical discectomy and fusion (ACDF group) were included. Presence of radiologic ASP (RASP) was based on observed changes in anterior osteophytes, disks, and calcification of the anterior longitudinal ligament on lateral radiographs. The mean follow-up period was 43.4 months in the CDA group and 44.6 months in the ACDF group. At final follow-up, ASP was observed in 5 (35.7%) CDA patients and 16 (57.1%) ACDF patients (p = 0.272). The interval between surgery and ASP development was 33.8 months in the CDA group and 16.3 months in the ACDF group (p = 0.046). The ASP risk factor analysis indicated postoperative cervical angle at C3-7 being more lordotic in non-ASP patients in both groups. Restoration of lordosis occurred in the CDA group regardless of the presence of ASP, but heterotopic ossification development was associated with the presence of ASP in the CDA group. And the CDA group had significantly greater clinical improvements than those in the ACDF group when ASP was present. In both CDA and ACDF patients, RASP developed, but CDA was associated with a delay in ASP development. A good clinical outcome was expected in CDA group, even when ASP developed. Restoration of cervical lordosis was an important factor in anterior cervical spine surgery.

Different processing of CAPA and pyrokinin precursors in the giant mealworm beetle Zophobas atratus (Tenebrionidae) and the boll weevil Anthonomus grandis grandis (Curculionidae).

PubMed

Neupert, Susanne; Marciniak, Pawel; Köhler, Rene; Nachman, Ronald J; Suh, Charles P-C; Predel, Reinhard

2018-03-01

Capa and pyrokinin (pk) genes in hexapods share a common evolutionary origin. Using transcriptomics and peptidomics, we analyzed products of these genes in two beetles, the giant mealworm beetle (Zophobas atratus; Tenebrionidae) and the boll weevil (Anthonomus grandis grandis; Curculionidae). Our data revealed that even within Coleoptera, which represents a very well-defined group of insects, highly different evolutionary developments occurred in the neuropeptidergic system. These differences, however, primarily affect the general structure of the precursors and differential processing of mature peptides and, to a lesser degree, the sequences of the active core motifs. With the differential processing of the CAPA-precursor in Z. atratus we found a perfect example of completely different products cleaved from a single neuropeptide precursor in different cells. The CAPA precursor in abdominal ganglia of this species yields primarily periviscerokinins (PVKs) whereas processing of the same precursor in neurosecretory cells of the subesophageal ganglion results in CAPA-tryptoPK and a novel CAPA-PK. Particularly important was the detection of that CAPA-PK which has never been observed in the CNS of insects before. The three different types of CAPA peptides (CAPA-tryptoPK, CAPA-PK, PVK) each represent potential ligands which activate different receptors. In contrast to the processing of the CAPA precursor from Z. atratus, no indications of a differential processing of the CAPA precursor were found in A. g. grandis. These data suggest that rapid evolutionary changes regarding the processing of CAPA precursors were still going on when the different beetle lineages diverged. The sequence of the single known PVK of A. g. grandis occupies a special position within the known PVKs of insects and might serve asa basis to develop lineage-specific peptidomimetics capable of disrupting physiological processes regulated by PVKs. Copyright © 2017 Elsevier Inc. All rights reserved.

Insulin degludec/insulin aspart once daily in Type 2 diabetes: a comparison of simple or stepwise titration algorithms (BOOST® : SIMPLE USE).

PubMed

Park, S W; Bebakar, W M W; Hernandez, P G; Macura, S; Hersløv, M L; de la Rosa, R

2017-02-01

To compare the efficacy and safety of two titration algorithms for insulin degludec/insulin aspart (IDegAsp) administered once daily with metformin in participants with insulin-naïve Type 2 diabetes mellitus. This open-label, parallel-group, 26-week, multicentre, treat-to-target trial, randomly allocated participants (1:1) to two titration arms. The Simple algorithm titrated IDegAsp twice weekly based on a single pre-breakfast self-monitored plasma glucose (SMPG) measurement. The Stepwise algorithm titrated IDegAsp once weekly based on the lowest of three consecutive pre-breakfast SMPG measurements. In both groups, IDegAsp once daily was titrated to pre-breakfast plasma glucose values of 4.0-5.0 mmol/l. Primary endpoint was change from baseline in HbA 1c (%) after 26 weeks. Change in HbA 1c at Week 26 was IDegAsp Simple -14.6 mmol/mol (-1.3%) (to 52.4 mmol/mol; 6.9%) and IDegAsp Stepwise -11.9 mmol/mol (-1.1%) (to 54.7 mmol/mol; 7.2%). The estimated between-group treatment difference was -1.97 mmol/mol [95% confidence interval (CI) -4.1, 0.2] (-0.2%, 95% CI -0.4, 0.02), confirming the non-inferiority of IDegAsp Simple to IDegAsp Stepwise (non-inferiority limit of ≤ 0.4%). Mean reduction in fasting plasma glucose and 8-point SMPG profiles were similar between groups. Rates of confirmed hypoglycaemia were lower for IDegAsp Stepwise [2.1 per patient years of exposure (PYE)] vs. IDegAsp Simple (3.3 PYE) (estimated rate ratio IDegAsp Simple /IDegAsp Stepwise 1.8; 95% CI 1.1, 2.9). Nocturnal hypoglycaemia rates were similar between groups. No severe hypoglycaemic events were reported. In participants with insulin-naïve Type 2 diabetes mellitus, the IDegAsp Simple titration algorithm improved HbA 1c levels as effectively as a Stepwise titration algorithm. Hypoglycaemia rates were lower in the Stepwise arm. © 2016 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.

[Comparison of the Effectiveness and Safety of Combined Chemotherapy with PEG-Asp for Treatment of ALL and T-NHL Patients].

PubMed

Xu, Yan; Wang, Jin; Yang, Nan; Bai, Ju; Zhang, Peng-Yu; Gu, Liu-Fang; Lei, Bo; Liu, Jie; Wang, Fang-Xia; Huang, Bing-Qiao; Zhang, Wang-Gang; He, Ai-Li; Cao, Xing-Mei; Chen, Yin-Xia; Ma, Xiao-Rong

2016-04-01

To explore the effectiveness and safety of combined chemotherapy with pegasparaginase (PEG-Asp) for treatment of patients with acute lymphoblastic leukemia (ALL) and T cell non-Hodgkin's lymphoma (T-NHL) patients. A total of 62 ALL or T-NHL patients were diagnosed and treated in our department and were enrolled in this study. Among them, 22 patients received the combined chemotherapy with PEG-Asp, while the other 40 patients received the standard chemotherapy with L-asparaginase (L-Asp) as the control. Therapeutic effectiveness, adverse effects, duration and expense of hospitalization, treatment-related mortality and survival were evaluated and compared in 2 different groups. In group of combined chemotherapy with PEG-Asp, the overall response rate was 90.91% (20 cases), among them CR rate and PR rate are 77.27% (17 cases) and 13.64% (3 cases), respectively. In the group of standard chemotherapy with L-Asp, the overall response rate was 87.5% (35 cases), among them CR rate and PR rate were 72.5% (29 cases) and 15% (6 cases), respectively. The difference neither between PEG-Asp and L-Asp chemotherapy groups nor between ALL and T-NHL subgroups was significant (P > 0.05). The 6-month and 12-month overall survival rates were not significantly different between the PEG-Asp and L-Asp chemotherapy groups, respectively (P > 0.05). The adverse effects were identified as degree 1-2 according to the WHO criteria of drug toxicity. Neither the adverse effects identified as degree 3-4 nor the treatment-related death were observed. Expect for allergy and hyperglycaemia, the difference of side-effect incidence between the two groups were not significant (P > 0.05). The treatment for all the patients in PEG-Asp chemotherapy group was completed, while the treatment with L-Asp was completed only in 29 cases. Moreover, both average duration and expense of hospitalization after the combined chemotherapy were less than the control. With higher response rate, lower drug toxicity and allergy incidence, the combined chemotherapy with PEG-Asp can replace the standard chemotherapy with L-Asp in the treatment of ALL and T-NHL. The optimization of the combined chemotheropeutic protocols for more cases and long-term survival rates need to further and deeply explorate.

Colloidal aggregation and structural assembly of aspect ratio variant goethite (α-FeOOH) with nC60 fullerene in environmental media.

PubMed

Ghosh, Saikat; Pradhan, Nihar R; Mashayekhi, Hamid; Zhang, Qiu; Pan, Bo; Xing, Baoshan

2016-12-01

Environmental mobility of C 60 fullerene can be significantly affected in the presence of naturally abundant α-FeOOH. However, α-FeOOH vary significantly in sizes, shapes and associated properties that can greatly influence the fate and transport of C 60 fullerene in environmental media. Therefore, colloidal hetero-association between well crystallized low aspect (L Asp ) α-FeOOH and nC 60 fullerene may differ substantially to weakly crystallized high-aspect (H Asp ) counterpart. In contrast to L Asp α-FeOOH, inherent crystal defects and surface charge generation in H Asp α-FeOOH facilitated strong Coulombic attraction and aggregation with fullerene in acidic pH. However, L Asp α-FeOOH demonstrated subtle entropic depletion mediated interaction with fullerene prevalent in hard rods. Humic acid (HA) encapsulation of H Asp α-FeOOH substantially blocked fullerene attachment. Minute enhancement in colloidal stability was detected for HA-coated H Asp α-FeOOH and fullerene mixture to HA-coated H Asp α-FeOOH alone. To investigate the interfacial assembly of α-FeOOH with fullerene "in situ" differential interference contrast (DIC) microscopic investigations were employed. This study showed significantly different interface behavior of the binary mixtures of fullerene and H Asp α-FeOOH NPs, and L Asp particles. On air-water interface, bare H Asp α-FeOOH displayed liquid crystalline packing. However, addition of fullerene to H Asp α-FeOOH suspension at pH5 produced closed-loop polygonal and circular ring structures. Head-to-tail alignment of magnetic dipoles as well as fullerene hydrophobicity facilitated such assembly formation. "Ex situ" AFM investigation revealed further the presence of magnetically derived ring structure which asserts that the formed "in situ" ensembles were not transient, hence, may abate fullerene transport through environmental interfaces. Barring hydrophobicity assisted attachment of fullerene to L Asp α-FeOOHs, the absence of any close-packed structures may unlikely abate fullerene transport as envisaged in case of H Asp α-FeOOH. Thus, aspect ratio variation and associated material properties of naturally abundant α-FeOOH may significantly impact fullerene transport through environmental media. Copyright © 2016 Elsevier Ltd. All rights reserved.

Absorption kinetics and action profiles of subcutaneously administered insulin analogues (AspB9GluB27, AspB10, AspB28) in healthy subjects.

PubMed

Kang, S; Brange, J; Burch, A; Vølund, A; Owens, D R

1991-11-01

The subcutaneous absorption and resulting changes in plasma insulin or analogue, glucose, C-peptide, and blood intermediary metabolite concentrations after subcutaneous bolus injection of three soluble human insulin analogues (AspB9GluB27, monomeric; AspB28, mixture of monomers and dimers; and AspB10, dimeric) and soluble human insulin were evaluated. Fasting healthy male volunteers (n = 7) were studied on five occasions 1 wk apart randomly receiving 0.6 nmol.kg-1 s.c. 125I-labeled AspB10 or soluble human insulin (Novolin R, Novo, Copenhagen); 1st study and 0.6 nmol.kg-1 s.c. 125I-labeled AspB28, AspB9GluB27 or soluble human insulin (2nd study). Residual radioactivity at the injection site was measured over 8 h with frequent venous sampling for plasma immunoreactive insulin or analogue, glucose, C-peptide, and blood intermediary metabolite concentrations. The three analogues were absorbed 2-3 times faster than human insulin. The mean +/- SE time to 50% residual radioactivity was 94 +/- 6 min for AspB10 compared with 184 +/- 10 min for human insulin (P less than 0.001), 83 +/- 8 min for AspB28 (P less than 0.005), and 63 +/- 9 min for AspB9GluB27 (P less than 0.001) compared with 182 +/- 21 min for human insulin. delta Peak plasma insulin analogue levels were significantly higher after each analogue than after human insulin (P less than 0.005). With all three analogues, the mean hypoglycemic nadir occurred earlier at 61-65 min postinjection compared with 201-210 min for the reference human insulins (P less than 0.005). The magnitude of the hypoglycemic nadir was greater after AspB9GluB27 (P less than 0.05) and AspB28 (P less than 0.001) compared with human insulin. There was a significantly faster onset and offset of responses in C-peptide and intermediary metabolite levels after the analogues than after human insulin (P less than 0.05). The rapid absorption and biological actions of these analogues offer potential therapeutic advantages over the current short-acting neutral soluble insulins.

β-secretase inhibitors for Alzheimer's disease: identification using pharmacoinformatics.

PubMed

Islam, Md Ataul; Pillay, Tahir S

2018-02-01

In this study we searched for potential β-site amyloid precursor protein cleaving enzyme1 (BACE1) inhibitors using pharmacoinformatics. A large dataset containing 7155 known BACE1 inhibitors was evaluated for pharmacophore model generation. The final model (R = 0.950, RMSD = 1.094, Q 2  = 0.901, se = 0.332, [Formula: see text] = 0.901, [Formula: see text] = 0.756, sp = 0.468, [Formula: see text] = 0.667) was revealed with the importance of spatial arrangement of hydrogen bond acceptor and donor, hydrophobicity and aromatic ring features. The validated model was then used to search NCI and InterBioscreen databases for promising BACE1 inhibitors. The initial hits from both databases were sorted using a number of criteria and finally three molecules from each database were considered for further validation using molecular docking and molecular dynamics studies. Different protonation states of Asp32 and Asp228 dyad were analysed and best protonated form used for molecular docking study. Observation of the number of binding interactions in the molecular docking study supported the potential of these molecules being promising inhibitors. Values of RMSD, RMSF, Rg in molecular dynamics study and binding energies unquestionably explained that final screened molecules formed stable complexes inside the receptor cavity of BACE1. Hence, it can be concluded that the final screened six compounds may be potential therapeutic agents for Alzheimer's disease.

New features of the delta opioid receptor: conformational properties of deltorphin I analogues.

PubMed

Balboni, G; Marastoni, M; Picone, D; Salvadori, S; Tancredi, T; Temussi, P A; Tomatis, R

1990-06-15

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

The 86-kilodalton antigen from Schistosoma mansoni is a heat-shock protein homologous to yeast HSP-90.

PubMed

Johnson, K S; Wells, K; Bock, J V; Nene, V; Taylor, D W; Cordingley, J S

1989-08-01

We report the sequence of a cDNA clone encoding an 86-kDa polypeptide antigen (p86) from Schistosoma mansoni. Fusion proteins made in Escherichia coli are recognized by human infection sera. The reading frame of this antigen is highly homologous to those of the large heat-shock proteins of Saccharomyces cerevisiae (HSP90) and Drosophila melanogaster (HSP83). mRNA encoding p86 increases in response to heat shock of adult worms, as does HSP70. Comparisons of the sequences of HSP70 and HSP83 homologues show that these two families of heat-shock proteins are not significantly related except for the last four amino acid residues, which are Glu-Glu-Val-Asp in every case. This sequence is not found at the carboxy terminus of any other protein in the current databases.

What Difference Does an Hour Make? Examining the Effects of an After School Program

ERIC Educational Resources Information Center

Farmer-Hinton, Raquel L.; Sass, Daniel A.; Schroeder, Mark

2009-01-01

The use and scope of after-school programs (ASPs) have always varied with the local context. Historically, affluent families used ASPs to provide enrichment for their children. During the Civil Rights and Black Nationalist movements, African Americans used ASPs for cultural and educational activities. In recent times, ASPs have been used for…

Anisotropic Dispersion and Partial Localization of Acoustic Surface Plasmons on an Atomically Stepped Surface: Au(788)

NASA Astrophysics Data System (ADS)

Smerieri, M.; Vattuone, L.; Savio, L.; Langer, T.; Tegenkamp, C.; Pfnür, H.; Silkin, V. M.; Rocca, M.

2014-10-01

Understanding acoustic surface plasmons (ASPs) in the presence of nanosized gratings is necessary for the development of future devices that couple light with ASPs. We show here by experiment and theory that two ASPs exist on Au(788), a vicinal surface with an ordered array of monoatomic steps. The ASPs propagate across the steps as long as their wavelength exceeds the terrace width, thereafter becoming localized. Our investigation identifies, for the first time, ASPs coupled with intersubband transitions involving multiple surface-state subbands.

Effect of peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly on the morphology of the thymus in hypophysectomized young and old birds.

PubMed

Pateyk, A V; Baranchugova, L M; Rusaeva, N S; Obydenko, V I; Kuznik, B I

2013-03-01

Investigations were carried out on chicks of different age. It was found that the most pronounced changes in the morphology of the thymus occurred after neonatal hypophysectomy. These changes are least pronounced in old chicks. Peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly synthesized on the basis of amino acid composition of peptide complexes of the anterior and posterior pituitary lobes administered to hypophysectomized birds regardless of age promoted recovery of the morphological structures of the thymus. The anterior pituitary peptide (Lys-Glu-Asp-Gly) had more pronounced effect on the recovery of thymic structure than posterior pituitary peptide (Ala-Glu-Asp-Gly).

3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering.

PubMed

Daly, Andrew C; Cunniffe, Gráinne M; Sathy, Binulal N; Jeon, Oju; Alsberg, Eben; Kelly, Daniel J

2016-09-01

The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

PubMed

Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

2017-01-01

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Liquid chromatography-high resolution mass spectrometric methods for the surveillance monitoring of cyanotoxins in freshwaters.

PubMed

Bogialli, Sara; Bortolini, Claudio; Di Gangi, Iole Maria; Di Gregorio, Federica Nigro; Lucentini, Luca; Favaro, Gabriella; Pastore, Paolo

2017-08-01

A comprehensive risk management on human exposure to cyanotoxins, whose production is actually unpredictable, is limited by reliable analytical tools for monitoring as many toxic algal metabolites as possible. Two analytical approaches based on a LC-QTOF system for target analysis and suspect screening of cyanotoxins in freshwater were presented. A database with 369 compounds belonging to cyanobacterial metabolites was developed and used for a retrospective data analysis based on high resolution mass spectrometry (HRMS). HRMS fragmentation of the suspect cyanotoxin precursor ions was subsequently performed for correctly identifying the specific variants. Alternatively, an automatic tandem HRMS analysis tailored for cyanotoxins was performed in a single chromatographic run, using the developed database as a preferred precursor ions list. Twenty-five extracts of surface and drinking waters contaminated by cyanobacteria were processed. The identification of seven uncommon microcystins (M(O)R, MC-FR, MSer 7 -YR, D-Asp 3 MSer 7 -LR, MSer 7 -LR, dmAdda-LR and dmAdda-YR) and 6 anabaenopeptins (A, B, F, MM850, MM864, oscyllamide Y) was reported. Several isobaric variants, fully separated by chromatography, were pointed out. The developed methods are proposed to be used by environmental and health agencies for strengthening the surveillance monitoring of cyanotoxins in water. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and characterization of the chicken trypsinogen gene family.

PubMed Central

Wang, K; Gan, L; Lee, I; Hood, L

1995-01-01

Based on genomic Southern hybridizations and cDNA sequence analyses, the chicken trypsinogen gene family can be divided into two multi-member subfamilies, a six-member trypsinogen I subfamily which encodes the cationic trypsin isoenzymes and a three-member trypsinogen II subfamily which encodes the anionic trypsin isoenzymes. The chicken cDNA and genomic clones containing these two subfamilies were isolated and characterized by DNA sequence analysis. The results indicated that the chicken trypsinogen genes encoded a signal peptide of 15 to 16 amino acid residues, an activation peptide of 9 to 10 residues and a trypsin of 223 amino acid residues. The chicken trypsinogens contain all the common catalytic and structural features for trypsins, including the catalytic triad His, Asp and Ser and the six disulphide bonds. The trypsinogen I and II subfamilies share approximately 70% sequence identity at the nucleotide and amino acid level. The sequence comparison among chicken trypsinogen subfamily members and trypsin sequences from other species suggested that the chicken trypsinogen genes may have evolved in coincidental or concerted fashion. Images Figure 6 Figure 7 PMID:7733885

Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

PubMed

van Deenen, Nicole; Bachmann, Anne-Lena; Schmidt, Thomas; Schaller, Hubert; Sand, Jennifer; Prüfer, Dirk; Schulze Gronover, Christian

2012-04-01

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases.

PubMed

Kitabgi, Patrick

2006-10-01

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six amino acid neurotensin-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by endoproteases that belong to the recently identified family of pro-protein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Pro-NT/NN processing gives rise mainly to NT and NN in the brain, to NT and a large peptide ending with the NN sequence at its C-terminus (large NN) in the gut and to NT, large NN and a large peptide ending with the NT sequence (large NT) in the adrenals. Recent evidence indicates that PC1, PC2 and PC5-A are the pro-hormone convertases responsible for the processing patterns observed in the gut, brain and adrenals, respectively. As NT, NN, large NT and large NN are all endowed with biological activity, the evidence reviewed here supports the idea that post-translational processing of pro-NT/NN in tissues may generate biological diversity.

Mars Exploration 2003 to 2013 - An Integrated Perspective: Time Sequencing the Missions

NASA Technical Reports Server (NTRS)

Briggs, G.; McKay, C.

2000-01-01

The science goals for the Mars exploration program, together with the HEDS precursor environmental and technology needs, serve as a solid starting point for re-planning the program in an orderly way. Most recently, the community has recognized the significance of subsurface sampling as a key component in "following the water". Accessing samples from hundreds and even thousands of meters beneath the surface is a challenge that will call for technology development and for one or more demonstration missions. Recent mission failures and concerns about the complexity of the previously planned MSR missions indicate that, before we are ready to undertake sample return and deep sampling, the Mars exploration program needs to include: 1) technology development missions; and 2) basic landing site assessment missions. These precursor missions should demonstrate the capability for reliable & accurate soft landing and in situ propellant production. The precursor missions will need to carry out close-up site observations, ground-penetrating radar mapping from orbit and conduct seismic surveys. Clearly the programs should be planned as a single, continuous exploration effort. A prudent minimum list of missions, including surface rovers with ranges of more than 10 km, can be derived from the numerous goals and requirements; they can be sequenced in an orderly way to ensure that time is available to feed forward the results of the precursor missions. One such sequence of missions is proposed for the decade beginning in 2003.

Tuning the morphology, luminescence and magnetic properties of hexagonal-phase NaGdF4: Yb, Er nanocrystals via altering the addition sequence of the precursors

NASA Astrophysics Data System (ADS)

Zhao, Shuwen; Xia, Donglin; Zhao, Ruimin; Zhu, Hao; Zhu, Yiru; Xiong, Yuda; Wang, Youfa

2017-01-01

Hexagonal-phase NaGdF4: Yb, Er upconversion nanocrystals (UCNCs) with tunable morphology and properties were successfully prepared via a thermal decomposition method. The influences of the adding sequence of the precursors on the morphology, chemical composition, luminescence and magnetic properties were investigated by transmission electron microscopy (TEM), inductively coupled plasma-atomic emission spectrometry (ICP-AES), upconversion (UC) spectroscopy, and a vibrating sample magnetometer (VSM). It was found that the resulting nanocrystals, with different sizes ranging from 24 to 224 nm, are in the shape of spheres, hexagonal plates and flakes; moreover, the composition percentage of Yb3+-Er3+ and Gd3+ ions was found to vary in a regular pattern with the adding sequence. Furthermore, the intensity ratios of emission colors (f g/r, f g/p), and the magnetic mass susceptibility of hexagonal-phase NaGdF4: Yb, Er nanocrystals change along with the composition of the nanocrystals. A positive correlation between the susceptibility and f g/r of NaGdF4: Yb, Er was proposed. The decomposition processes of the precursors were investigated by a thermogravimetric (TG) analyzer. The result indicated that the decomposition of the resolved lanthanide trifluoroacetate is greatly different from lanthanide trifluoroacetate powder. It is of tremendous help to recognize the decomposition process of the precursors and to understand the related reaction mechanism.

Investigation of phase evolution of CaCu3Ti4O12 (CCTO) by in situ synchrotron high-temperature powder diffraction

NASA Astrophysics Data System (ADS)

Ouyang, Xin; Huang, Saifang; Zhang, Weijun; Cao, Peng; Huang, Zhaohui; Gao, Wei

2014-03-01

In situ synchrotron X-ray powder diffraction was used to study the high-temperature phase evolution of CaCu3Ti4O12 (CCTO) precursors prepared via solid-state and sol-gel methods. After the precursors are heated to 1225 °C, the CCTO phase is the main phase observed in the calcined powder, with the presence of some minor impurities. Comparing the two precursors, we found that the onset temperature for the CCTO phase formation is 800 °C in the sol-gel precursor, lower than that in the solid-state precursor (875 °C). Intermediate phases were only observed in the sol-gel precursor. Both precursors are able to be calcined to sub-micrometric sized powders. Based on the synchrotron data along with differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), the phase formation sequence and mechanism during calcination are proposed in this study.

Studies on Aspirin Crystals Generated by a Modified Vapor Diffusion Method.

PubMed

Mittal, Amit; Malhotra, Deepak; Jain, Preeti; Kalia, Anupama; Shunmugaperumal, Tamilvanan

2016-08-01

The objectives of the current investigation were (1) to study the influence of selected two different non-solvents (diethylether and dichloromethane) on the drug crystal formation of a model drug, aspirin (ASP-I) by the modified vapor diffusion method and (2) to characterize and compare the generated crystals (ASP-II and ASP-III) using different analytical techniques with that of unprocessed ASP-I. When compared to the classical vapor diffusion method which consumes about 15 days to generate drug crystals, the modified method needs only 12 h to get the same. Fourier transform-infrared spectroscopy (FT-IR) reveals that the internal structures of ASP-II and ASP-III crystals were identical when compared with ASP-I. Although the drug crystals showed a close similarity in X-ray diffraction patterns, the difference in the relative intensities of some of the diffraction peaks (especially at 2θ values of around 7.7 and 15.5) could be attributed to the crystal habit or crystal size modification. Similarly, the differential scanning calorimetry (DSC) study speculates that only the crystal habit modifications might occur but without involving any change in internal structure of the generated drug polymorphic form I. This is further substantiated from the scanning electron microscopy (SEM) pictures that indicated the formation of platy shape for the ASP-II crystals and needle shape for the ASP-III crystals. In addition, the observed slow dissolution of ASP crystals should indicate polymorph form I formation. Thus, the modified vapor diffusion method could routinely be used to screen and legally secure all possible forms of other drug entities too.

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora.

PubMed

González-Hernández, Ana; Borloo, Jimmy; Peelaers, Iris; Casaert, Stijn; Leclercq, Georges; Claerebout, Edwin; Geldhof, Peter

2018-01-01

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy

NASA Technical Reports Server (NTRS)

Maeda, A.; Sasaki, J.; Shichida, Y.; Yoshizawa, T.; Chang, M.; Ni, B.; Needleman, R.; Lanyi, J. K.

1992-01-01

The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.

GLU298ASP and 4G/5G Polymorphisms and the Risk of Ischemic Stroke in Young Individuals.

PubMed

Esparza-García, Juan Carlos; Santiago-Germán, David; Guadalupe Valades-Mejía, María; Hernández-Juárez, Jesus; Aguilar-Sosa, Eberth; Leaños-Miranda, Alfredo; Alvarado-Moreno, Antonio; Majluf-Cruz, Abraham; Isordia-Salas, Irma

2015-09-01

Polymorphisms in the endothelial nitric oxide synthase (eNOS) and in the plasminogen activator inhibitor -1 (PAI-1) genes have been implicated in stroke pathogenesis but results are still controversial. The aim of this study was to examine the possible contribution of Glu298Asp in the eNOS and 4G/5G in the PAI-1polymorphisms with ischemic stroke in a young Mexican population. In a case-control study, conducted between January 2006 and June 2010, 204 patients ≤45 years of age with ischemic stroke and 204 controls matched by age and gender, were recruited. The Glu298Asp and 4G/5G polymorphisms were determined in all participants by polymerase chain reaction-restriction fragment length polymorphism. There was a significant difference in the Glu298Asp genotype distribution (P=0.001) and allele frequency between the two groups (P=0.001). The 4G/5G genotype distribution (P=0.40) and the allele frequency was similar between groups; (P=0.13). There were independent factors for ischemic stroke: Asp carriage (GluAsp+AspAsp) (P=0.02); smoking (P=0.01); hypertension (P=0.03), and familial history of atherothrombotic disease (P=0.04). The Asp allele from the Gu298Asp gene represents an independent risk factor for ischemic stroke in a young Mexican population. In contrast, the 4G/5G was not associated with an increased risk for this disease in the same group of patients, as previously has been demonstrated in other populations.

Asn-tRNA in Lactobacillus bulgaricus is formed by asparaginylation of tRNA and not by transamidation of Asp-tRNA.

PubMed Central

Kim, S I; Nalaskowska, M; Germond, J E; Pridmore, D; Söll, D

1996-01-01

In many organisms (e.g., gram-positive eubacteria) Gin-tRNA is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln. We wondered whether a similar transamidation pathway also operates in the formation of Asn-tRNA in these organisms. Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferase of [14C]Asp-tRNA to [14C]Asn-tRNA. As no transamidation was observed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS). Two DNA fragments (from different locations of the L.bulgaricus chromosome) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene. The derived amino acid sequences of the two ORFs (432 amino acids) were the same and 41% identical with E.coli AsnRS. When one of the ORFs was expressed in E.coli, it complemented the temperature sensitivity of an E.coli asnS mutant. S-100 preparations of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine. Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties. This indicates that L.bulgaricus contains a functional AsnRS. Thus, the transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all gram-positive eubacteria. PMID:8758990

Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces.

PubMed

Rideout, D C; Lambert, M; Kendall, D A; Moe, G R; Osterman, D G; Tao, H P; Weinstein, I B; Kaiser, E T

1985-09-01

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.

Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

PubMed

Singh, Ram Sarup; Yadav, Mukesh

2012-07-01

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

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

Loew, G.H.; Axe, F.U.; Collins, J.R.

In this study, we have investigated the plausibility of a key postulated transformation of the proximal imidazole ligand (His 175) to an imidazolate by proton transfer to a nearby aspartate (Asp 235) absent in Mb. The proton relay system studied included not only models for the His 175 and Asp 235 residues but also for a nearby Trp 191 residue that could also interact with Asp 235 through hydrogen bonding and polarization. Two semiempirical quantum mechanical methods, Am1 and MNDO/H, with improved capabilities of describing H-bonded systems, were used to calculate the enthalpies of the three tautomeric forms of themore » proton relay system corresponding to the proton on the His, Asp, and Trp, respectively. These calculations were made for several models of the effect of the iron. Relative tautomeric enthalpies were calculated both with H-atom only optimization, keeping the heavy atoms fixed in their X-ray positions, and additional optimization that allowed the model Asp residue to relax. Transition-state enthalpies for the proton transfer from His to Asp were also calculated. The results of these studies suggest that the crucial postulated proton transfer from His to Asp is energetically favored, but only in the presence of the interaction of the iron with the imidazole ligand. Another stable form of the cluster, with competing proton transfer from the Trp to the Asp, was found only when the Asp position was allowed to optimize.« less

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu; Loukas, Alex; Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, QLD 4006

In order to clarify the structural basis of the pathogenesis-related-1 domain, Na-ASP-1, the first multi-domain ASP from the human hookworm parasite N. americanus, has been crystallized. 2.2 Å resolution data have been collected from a crystal belonging to the monoclinic space group P2{sub 1}. Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functionsmore » of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P2{sub 1} with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit.« less

Caspofungin exposure alters the core septin AspB interactome of Aspergillus fumigatus.

PubMed

Vargas-Muñiz, José M; Renshaw, Hilary; Waitt, Greg; Soderblom, Erik J; Moseley, M Arthur; Palmer, Jonathan M; Juvvadi, Praveen R; Keller, Nancy P; Steinbach, William J

2017-04-01

Aspergillus fumigatus, the main etiological agent of invasive aspergillosis, is a leading cause of death in immunocompromised patients. Septins, a conserved family of GTP-binding proteins, serve as scaffolding proteins to recruit enzymes and key regulators to different cellular compartments. Deletion of the A. fumigatus septin aspB increases susceptibility to the echinocandin antifungal caspofungin. However, how AspB mediates this response to caspofungin is unknown. Here, we characterized the AspB interactome under basal conditions and after exposure to a clinically relevant concentration of caspofungin. While A. fumigatus AspB interacted with 334 proteins, including kinases, cell cycle regulators, and cell wall synthesis-related proteins under basal growth conditions, caspofungin exposure altered AspB interactions. A total of 69 of the basal interactants did not interact with AspB after exposure to caspofungin, and 54 new interactants were identified following caspofungin exposure. We generated A. fumigatus deletion strains for 3 proteins (ArpB, Cyp4, and PpoA) that only interacted with AspB following exposure to caspofungin that were previously annotated as induced after exposure to antifungal agents, yet only PpoA was implicated in the response to caspofungin. Taken together, we defined how the septin AspB interactome is altered in the presence of a clinically relevant antifungal. Copyright © 2017 Elsevier Inc. All rights reserved.

Diversity and survivability of microbial community in ancient permafrost sediment of northeast Siberia

NASA Astrophysics Data System (ADS)

Liang, R.; Lau, M.; Vishnivetskaya, T. A.; Lloyd, K. G.; Pfiffner, S. M.; Rivkina, E.; Onstott, T. C.

2017-12-01

The prevalence of microorganisms in frozen permafrost has been well documented in ancient sediment up to several million years old. However, the long term survivability and metabolic activity of microbes over geological timespans remain underexplored. Siberian permafrost sediment was collected at various depths (1.4m, 11.8 m and 24.8m) to represent a wide range of geological time from thousands to millions of years. Extracellular (eDNA) and intracellular DNA (iDNA) was simultaneously recovered for sequencing to characterize the potentially extinct and extant microbial community. Additionally, aspartic acid racemization assay (D/L Asp) was used to infer the metabolic activity of microbes in ancient permafrost. As compared with the young sample (1.4m), DNA yield and content of aspartic acid dramatically decreased in old samples (11.8m and 24.8m). However, D/L Asp and eDNA/iDNA significantly increased with the geological age. Such findings suggested that ancient microbiomes might be subjected to racemization or even DNA/proteins degradation at subzero temperature over the wide geological time scale. Preliminary characterization of microbial community indicated that the majority of sequences in old samples were identified as bacteria and only a small fraction was identified as archaea from the iDNA pool. While the eDNA and iDNA fractions shared similar dominant taxa at phylum level, the relative abundance of Proteobacteria in eDNA library was much higher than iDNA. By contrast, the phylum affiliated with Firmicutes was more numerically abundant in the iDNA fraction. More dramatic differences were observed between eDNA and iDNA library at lower taxonomic levels. Particularly, the microbial lineages affiliated with the genera Methanoregula, Desulfosporosinus and Syntrophomonas were only detected in the iDNA library. Such taxonomic difference between the relic eDNA and iDNA suggested that numerous species become locally "extinct" whereas many other taxa might survive in ancient sediment. Ultimately, when coupling our current findings to the D/L Asp in cellular proteins and metaproteomics, a better understanding will be achieved about the microbial activity of the extant microbial community and their roles in biogeochemical cycling in ancient permafrost.

Structural and functional insights of β-glucosidases identified from the genome of Aspergillus fumigatus

NASA Astrophysics Data System (ADS)

Dodda, Subba Reddy; Aich, Aparajita; Sarkar, Nibedita; Jain, Piyush; Jain, Sneha; Mondal, Sudipa; Aikat, Kaustav; Mukhopadhyay, Sudit S.

2018-03-01

Thermostable glucose tolerant β-glucosidase from Aspergillus species has attracted worldwide interest for their potentiality in industrial applications and bioethanol production. A strain of Aspergillus fumigatus (AfNITDGPKA3) identified by our laboratory from straw retting ground showed higher cellulase activity, specifically the β-glucosidase activity, compared to other contemporary strains. Though A. fumigatus has been known for high cellulase activity, detailed identification and characterization of the cellulase genes from their genome is yet to be done. In this work we have been analyzed the cellulase genes from the genome sequence database of Aspergillus fumigatus (Af293). Genome analysis suggests two cellobiohydrolase, eleven endoglucanase and seventeen β-glucosidase genes present. β-Glucosidase genes belong to either Glycohydro1 (GH1 or Bgl1) or Glycohydro3 (GH3 or Bgl3) family. The sequence similarity suggests that Bgl1 and Bgl3 of A. fumagatus are phylogenetically close to those of A. fisheri and A. oryzae. The modelled structure of the Bgl1 predicts the (β/α)8 barrel type structure with deep and narrow active site, whereas, Bgl3 shows the (α/β)8 barrel and (α/β)6 sandwich structure with shallow and open active site. Docking results suggest that amino acids Glu544, Glu466, Trp408,Trp567,Tyr44,Tyr222,Tyr770,Asp844,Asp537,Asn212,Asn217 of Bgl3 and Asp224,Asn242,Glu440, Glu445, Tyr367, Tyr365,Thr994,Trp435,Trp446 of Bgl1 are involved in the hydrolysis. Binding affinity analyses suggest that Bgl3 and Bgl1 enzymes are more active on the substrates like 4-methylumbelliferyl glycoside (MUG) and p-nitrophenyl-β-D-1, 4-glucopyranoside (pNPG) than on cellobiose. Further docking with glucose suggests that Bgl1 is more glucose tolerant than Bgl3. Analysis of the Aspergillus fumigatus genome may help to identify a β-glucosidase enzyme with better property and the structural information may help to develop an engineered recombinant enzyme.

The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

PubMed

Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

2016-11-15

It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus

PubMed Central

Asojo, Oluwatoyin A.; Loukas, Alex; Inan, Mehmet; Barent, Rick; Huang, Jicai; Plantz, Brad; Swanson, Amber; Gouthro, Mark; Meagher, Michael M.; Hotez, Peter J.

2005-01-01

Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P21 with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit. PMID:16511050

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor*

PubMed Central

Kobayashi, Hidetomo; Utsunomiya, Hiroko; Yamanaka, Hiroyasu; Sei, Yoshihisa; Katunuma, Nobuhiko; Okamoto, Keinosuke; Tsuge, Hideaki

2009-01-01

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 Å resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs. PMID:19654332

Interface design and reinforced features of arrowroot (Maranta arundinacea) starch/polyester-based membranes: Preparation, antioxidant activity, and cytocompatibility.

PubMed

Wu, Chin-San; Liao, Hsin-Tzu

2017-01-01

The structural, mechanical, antioxidant, and cytocompatibility properties of membranes prepared from the polyhydroxyalkanoate (PHA) and arrowroot (Maranta arundinacea) starch powder (ASP) blend (PHA/ASP) were studied. The acrylic acid-grafted PHA (PHA-g-AA) and the coupling agent treated ASP (TASP) were used to enhance the desired characteristics of these membranes. The PHA-g-AA/TASP membranes had better mechanical properties than the PHA/ASP membrane. This effect was attributed to greater compatibility between the grafted PHA and TASP. The water resistance of the PHA-g-AA/TASP membranes was greater than that of the PHA/ASP membranes, and a cytocompatibility evaluation with human foreskin fibroblasts (FBs) indicated that both materials were nontoxic. Moreover, both ASP and TASP enhanced the polyphenol content and antioxidant properties of the membranes. PHA-g-AA/TASP and PHA/ASP membranes had better antioxidant activity than the control group. Copyright © 2016 Elsevier B.V. All rights reserved.

Biomass characterization of laboratory-scale thermophilic-mesophilic wastewater treatment processes.

PubMed

Suvilampi, J; Lehtomäki, A; Rintala, J

2006-01-01

Two thermophilic-mesophilic wastewater treatment processes, one as the combination of the thermophilic activated sludge process (ASP), followed by the mesophilic ASP and the other as thermophilic suspended carrier biofilm process (SCBP), followed by the mesophilic ASP, were used to study sludge characteristics and floc formation. Thermophilic bacteria in both ASP and SCBP were able to form flocs, which were <50 microm in size and had a weak structure and irregular shape. Flocs in both the mesophilic ASPs were larger in size (50-500 microm) and had more compact structures. Filamentous bacteria played an important role in both the thermophilic and mesophilic processes by forming bridges between small flocs. Both thermophilic processes showed a high density of dispersed particles, such as free bacteria. When hydraulic retention time (HRT) was decreased the biofilm was retained in the thermophilic SCBP better than the flocs in the thermophilic ASP. The mesophilic ASPs efficiently removed dispersed particles originating from the thermophilic processes.

A Gene Encoding a Hevein-Like Protein from Elderberry Fruits Is Homologous to PR-4 and Class V Chitinase Genes1

PubMed Central

Van Damme, Els J.M.; Charels, Diana; Roy, Soma; Tierens, Koenraad; Barre, Annick; Martins, José C.; Rougé, Pierre; Van Leuven, Fred; Does, Mirjam; Peumans, Willy J.

1999-01-01

We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene. PMID:10198114

Characterization of rat calcitonin mRNA.

PubMed Central

Amara, S G; David, D N; Rosenfeld, M G; Roos, B A; Evans, R M

1980-01-01

A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA. Images PMID:6933496

Multistress Regulation in Escherichia coli: Expression of osmB Involves Two Independent Promoters Responding either to σS or to the RcsCDB His-Asp Phosphorelay

PubMed Central

Boulanger, Alice; Francez-Charlot, Anne; Conter, Annie; Castanié-Cornet, Marie-Pierre; Cam, Kaymeuang; Gutierrez, Claude

2005-01-01

Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2. The downstream promoter, osmBp2, is induced after osmotic shock or upon entry into stationary phase in a σS-dependent manner. The upstream promoter, osmBp1, is independent of σS and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its −35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a σS-dependent promoter and a second one independent of σS but controlled by stress-specific transcription factors. PMID:15838058

Isolation and Functional Characterization of an Acidic Myotoxic Phospholipase A₂ from Colombian Bothrops asper Venom.

PubMed

Posada Arias, Silvia; Rey-Suárez, Paola; Pereáñez J, Andrés; Acosta, Cristian; Rojas, Mauricio; Delazari Dos Santos, Lucilene; Ferreira, Rui Seabra; Núñez, Vitelbina

2017-10-26

Myotoxic phospholipases A₂ (PLA₂) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA₂ (BaCol PLA₂) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA₂ had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA₂ showed structural homology with other acidic PLA₂ isolated from Bothrops venoms, including a non-myotoxic PLA₂ from Costa Rican B. asper . In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA₂ had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA₂ caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm.

Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family

PubMed Central

2010-01-01

Background A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor. Results Examination of candidate TOMM precursors has revealed a subclass with an uncharacteristically long leader sequence closely related to the enzyme nitrile hydratase. Members of this nitrile hydratase leader peptide (NHLP) family lack the metal-binding residues required for catalysis. Instead, NHLP sequences display the classic Gly-Gly cleavage motif and have C-terminal regions rich in heterocyclizable residues. The NHLP family exhibits a correlated species distribution and local clustering with an ABC transport system. This study also provides evidence that a separate family, annotated as Nif11 nitrogen-fixing proteins, can serve as natural product precursors (N11P), but not always of the TOMM variety. Indeed, a number of cyanobacterial genomes show extensive N11P paralogous expansion, such as Nostoc, Prochlorococcus and Cyanothece, which replace the TOMM cluster with lanthionine biosynthetic machinery. Conclusions This study has united numerous TOMM gene clusters with their cognate substrates. These results suggest that two large protein families, the nitrile hydratases and Nif11, have been retailored for secondary metabolism. Precursors for TOMMs and lanthionine-containing peptides derived from larger proteins to which other functions are attributed, may be widespread. The functions of these natural products have yet to be elucidated, but it is probable that some will display valuable industrial or medical activities. PMID:20500830

Peptide de novo sequencing of mixture tandem mass spectra

PubMed Central

Hotta, Stéphanie Yuki Kolbeck; Verano‐Braga, Thiago; Kjeldsen, Frank

2016-01-01

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co‐isolation and thus prone to false identifications. The deconvolution approach matched complementary b‐, y‐ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co‐isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20–35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications. PMID:27329701

The Global Epidemiology of Syphilis in the Past Century - A Systematic Review Based on Antenatal Syphilis Prevalence.

PubMed

Kenyon, Chris Richard; Osbak, Kara; Tsoumanis, Achilleas

2016-05-01

How can we explain the uneven decline of syphilis around the world following the introduction of penicillin? In this paper we use antenatal syphilis prevalence (ASP) to investigate how syphilis prevalence varied worldwide in the past century, and what risk factors correlate with this variance. 1) A systematic review using PubMed and Google Scholar was conducted to identify countries with published data relating to ASP estimates from before 1952 until the present. Eleven countries were identified (Canada, Denmark, Finland, India, Japan, Norway, Singapore, South Africa, United States of America (USA), United Kingdom (UK) and Zimbabwe). The ASP epidemic curve for each population was depicted graphically. In South Africa and the USA, results are reported separately for the black and white populations. 2) National antenatal syphilis prevalence estimates for 1990 to 1999 and 2008 were taken from an Institute for Health Metrics and Evaluation database on the prevalence of syphilis in low risk populations compiled for the Global Burden of Diseases study and from a recent review paper respectively. National ASPs were depicted graphically and regional median ASPs were calculated for both time periods. 3) Linear regression was used to test for an association between ASP in 1990-1999 and 2008 and four risk factors (efficacy of syphilis screening/treatment, health expenditure, GDP per capita and circumcision prevalence). WHO world regions were included as potential explanatory variables. In most populations, ASP dropped to under 1% before 1960. In Zimbabwe and black South Africans, ASP was high in the pre-penicillin period, dropped in the post-penicillin period, but then plateaued at around 6% until the end of the 20th century when ASP dropped to just above 1%. In black Americans, ASP declined in the post penicillin period, but plateaued at 3-5% thereafter. ASP was statistically significantly higher in sub-Saharan Africa in 1990-1999 and 2008 than in the other world regions (P < 0.001). On multivariate analysis in both time periods, ASP was only associated with residence in sub-Saharan Africa. Further research is necessary to elucidate the reasons for the higher prevalence of syphilis in sub-Saharan Africa.

Discovering ligands for a microRNA precursor with peptoid microarrays

PubMed Central

Chirayil, Sara; Chirayil, Rachel; Luebke, Kevin J.

2009-01-01

We have screened peptoid microarrays to identify specific ligands for the RNA hairpin precursor of miR-21, a microRNA involved in cancer and heart disease. Microarrays were printed by spotting a library of 7680 N-substituted oligoglycines (peptoids) onto glass slides. Two compounds on the array specifically bind RNA having the sequence and predicted secondary structure of the miR-21 precursor hairpin and have specific affinity for the target in solution. Their binding induces a conformational change around the hairpin loop, and the most specific compound recognizes the loop sequence and a bulged uridine in the proximal duplex. Functional groups contributing affinity and specificity were identified, and by varying a critical methylpyridine group, a compound with a dissociation constant of 1.9 μM for the miR-21 precursor hairpin and a 20-fold discrimination against a closely-related hairpin was created. This work describes a systematic approach to discovery of ligands for specific pre-defined novel RNA structures. It demonstrates discovery of new ligands for an RNA for which no specific lead compounds were previously known by screening a microarray of small molecules. PMID:19561197

Spectrum-based method to generate good decoy libraries for spectral library searching in peptide identifications.

PubMed

Cheng, Chia-Ying; Tsai, Chia-Feng; Chen, Yu-Ju; Sung, Ting-Yi; Hsu, Wen-Lian

2013-05-03

As spectral library searching has received increasing attention for peptide identification, constructing good decoy spectra from the target spectra is the key to correctly estimating the false discovery rate in searching against the concatenated target-decoy spectral library. Several methods have been proposed to construct decoy spectral libraries. Most of them construct decoy peptide sequences and then generate theoretical spectra accordingly. In this paper, we propose a method, called precursor-swap, which directly constructs decoy spectral libraries directly at the "spectrum level" without generating decoy peptide sequences by swapping the precursors of two spectra selected according to a very simple rule. Our spectrum-based method does not require additional efforts to deal with ion types (e.g., a, b or c ions), fragment mechanism (e.g., CID, or ETD), or unannotated peaks, but preserves many spectral properties. The precursor-swap method is evaluated on different spectral libraries and the results of obtained decoy ratios show that it is comparable to other methods. Notably, it is efficient in time and memory usage for constructing decoy libraries. A software tool called Precursor-Swap-Decoy-Generation (PSDG) is publicly available for download at http://ms.iis.sinica.edu.tw/PSDG/.

Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model

PubMed Central

BOUSSEROUEL, SOUAD; LE GRANDOIS, JULIE; GOSSÉ, FRANCINE; WERNER, DALAL; BARTH, STEPHAN W.; MARCHIONI, ERIC; MARESCAUX, JACQUES; RAUL, FRANCIS

2013-01-01

Shoots of white asparagus are a popular vegetable dish, known to be rich in many bioactive phytochemicals reported to possess antioxidant, and anti-inflammatory and antitumor activities. We evaluated the anticancer mechanisms of a methanolic extract of Asparagus officinalis L. shoots (Asp) on human colon carcinoma cells (SW480) and their derived metastatic cells (SW620), and Asp chemopreventive properties were also assessed in a model of colon carcinogenesis. SW480 and SW620 cell proliferation was inhibited by 80% after exposure to Asp (80 μg/ml). We demonstrated that Asp induced cell death through the activation of TRAIL DR4/DR5 death receptors leading to the activation of caspase-8 and caspase-3 and to cell apoptosis. By specific blocking agents of DR4/DR5 receptors we were able to prevent Asp-triggered cell death confirming the key role of DR4/DR5 receptors. We found also that Asp (80 μg/ml) was able to potentiate the effects of the cytokine TRAIL on cell death even in the TRAIL-resistant metastatic SW620 cells. Colon carcinogenesis was initiated in Wistar rats by intraperitoneal injections of azoxymethane (AOM), once a week for two weeks. One week after (post-initiation) rats received daily Asp (0.01%, 14 mg/kg body weight) in drinking water. After 7 weeks of Asp-treatment the colon of rats exhibited a 50% reduction of the number of preneoplastic lesions (aberrant crypt foci). In addition Asp induced inhibition of several pro-inflammatory mediators, in association with an increased expression of host-defense mediators. In the colonic mucosa of Asp-treated rats we also confirmed the pro-apoptotic effects observed in vitro including the activation of the TRAIL death-receptor signaling pathway. Taken together, our data highlight the chemopreventive effects of Asp on colon carcinogenesis and its ability to promote normal cellular homeostasis. PMID:23754197

Efficacy and safety of once-daily insulin degludec/insulin aspart compared with once-daily insulin glargine in participants with Type 2 diabetes: a randomized, treat-to-target study.

PubMed

Kumar, S; Jang, H C; Demirağ, N G; Skjøth, T V; Endahl, L; Bode, B

2017-02-01

To investigate, in a 26-week, open-label, randomized, treat-to-target trial, the efficacy and safety of insulin degludec/insulin aspart (IDegAsp) once daily vs insulin glargine (IGlar) once daily in adults with Type 2 diabetes, inadequately controlled on basal insulin. Participants were randomized (1:1) to IDegAsp once daily or IGlar once daily in combination with existing oral antidiabetic drugs. IDegAsp once daily was administered with the main evening meal or the largest meal of the day (agreed at baseline); dosing time was maintained throughout the trial. Participants titrated their insulin dose weekly to a mean pre-breakfast self-measured plasma glucose target [3.9-4.9 mmol/l (70-89 mg/dl)]. IDegAsp once daily was non-inferior to IGlar once daily in reducing HbA 1c after 26 weeks [mean estimated treatment difference IDegAsp once daily - IGlar once daily: -0.03% (95% CI -0.20, 0.14)]. The evening meal glucose increment was significantly lower with IDegAsp once daily vs IGlar once daily [estimated treatment difference IDegAsp once daily - IGlar once daily: -1.32 mmol/l (95% CI -1.93, -0.72); P < 0.05]. The overall confirmed hypoglycaemia rate was higher with IDegAsp once daily (estimated rate ratio 1.43; 95% CI 1.07, 1.92; P < 0.05). The rate of nocturnal hypoglycaemia did not significantly differ between the IDegAsp and IGlar groups [estimated rate ratio 0.80 (95% CI 0.49, 1.30); not significant]. In participants with Type 2 diabetes inadequately controlled on basal insulin, IDegAsp once daily improved glycaemic control and was non-inferior to IGlar once daily. IDegAsp led to higher rates of overall hypoglycaemia than IGlar, with no significant difference in rates of nocturnal hypoglycaemia. © 2016 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.

Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

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

Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang, E-mail: zhuanghu475000@sina.com

Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP responsemore » element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced by ASP.« less

Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers

PubMed Central

Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Kiwada, Tatsuto; Shiba, Kazuhiro; Odani, Akira

2013-01-01

68Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases. PMID:24391942

Antibiotic Stewardship Programs in U.S. Acute Care Hospitals: Findings From the 2014 National Healthcare Safety Network Annual Hospital Survey.

PubMed

Pollack, Lori A; van Santen, Katharina L; Weiner, Lindsey M; Dudeck, Margaret A; Edwards, Jonathan R; Srinivasan, Arjun

2016-08-15

The National Action Plan to Combat Antibiotic Resistant Bacteria calls for all US hospitals to improve antibiotic prescribing as a key prevention strategy for resistance and Clostridium difficile Antibiotic stewardship programs (ASPs) will be important in this effort but implementation is not well understood. We analyzed the 2014 National Healthcare Safety Network Annual Hospital Survey to describe ASPs in US acute care hospitals as defined by the Center for Disease Control and Prevention's (CDC) Core Elements for Hospital ASPs. Univariate analyses were used to assess stewardship infrastructure and practices by facility characteristics and a multivariate model determined factors associated with meeting all ASP core elements. Among 4184 US hospitals, 39% reported having an ASP that met all 7 core elements. Although hospitals with greater than 200 beds (59%) were more likely to have ASPs, 1 in 4 (25%) of hospitals with less than 50 beds reported achieving all 7 CDC-defined core elements of a comprehensive ASP. The percent of hospitals in each state that reported all seven elements ranged from 7% to 58%. In the multivariate model, written support (adjusted relative risk [RR] 7.2 [95% confidence interval [CI], 6.2-8.4]; P < .0001) or salary support (adjusted RR 1.5 [95% CI, 1.4-1.6]; P < .0001) were significantly associated with having a comprehensive ASP. Our findings show that ASP implementation varies across the United States and provide a baseline to monitor progress toward national goals. Comprehensive ASPs can be established in facilities of any size and hospital leadership support for antibiotic stewardship appears to drive the establishment of ASPs. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

ASP6537, a novel highly selective cyclooxygenase-1 inhibitor, exerts potent antithrombotic effect without "aspirin dilemma".

PubMed

Sakata, Chinatsu; Kawasaki, Tomihisa; Kato, Yasuko; Abe, Masaki; Suzuki, Ken-ichi; Ohmiya, Makoto; Funatsu, Toshiyuki; Morita, Yoshiaki; Okada, Masamichi

2013-07-01

Aspirin inhibits both the cyclooxygenase (COX)-1-dependent production of thromboxane A2 (TXA2) in platelets and COX-2-dependent production of anti-aggregatory prostaglandin I2 (PGI2) in vessel walls, resulting in "aspirin dilemma." Our objective is to investigate whether ASP6537 can overcome aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect. We evaluated the inhibitory effects of ASP6537 on recombinant human COX-1 (rhCOX-1) and rhCOX-2 activities using a COX-1/2 selectivity test. To determine whether ASP6537 induces aspirin dilemma, we examined the effects of ASP6537 on in vitro TXA2 and PGI2 metabolite production from platelets and isolated aorta of guinea pigs, and on plasma concentrations of TXA2 and PGI2 metabolites in aged rats. Finally, we evaluated the antithrombotic effects and ulcerogenic activity of ASP6537 using an electrically induced carotid arterial thrombosis model and a gastric ulcer model in guinea pigs. The IC50 ratios of rhCOX-2 to rhCOX-1 for ASP6537 and aspirin were >142,000 and 1.63 fold, respectively. ASP6537 inhibited TXA2 production more selectively than aspirin in in vitro and in vivo TXA2/PGI2 production studies. ASP6537 exerted a significant antithrombotic effect at ≥3 mg/kg, while aspirin tended to inhibit thrombosis at 300 mg/kg but it was not statistically significant. Further, ASP6537 did not induce ulcer formation at 100 mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of ≥100 mg/kg. ASP6537 functions as a highly selective COX-1 inhibitor with a superior ability to aspirin for normalizing TXA2/PGI2 balance, and exerts antithrombotic effect without ulcerogenic effect. Copyright © 2013 Elsevier Ltd. All rights reserved.

D-aspartic acid supplementation combined with 28 days of heavy resistance training has no effect on body composition, muscle strength, and serum hormones associated with the hypothalamo-pituitary-gonadal axis in resistance-trained men.

PubMed

Willoughby, Darryn S; Leutholtz, Brian

2013-10-01

It was hypothesized that D-aspartic acid (D-ASP) supplementation would not increase endogenous testosterone levels or improve muscular performance associated with resistance training. Therefore, body composition, muscle strength, and serum hormone levels associated with the hypothalamo-pituitary-gonadal axis were studied after 28 days of resistance training and D-ASP supplementation. Resistance-trained men resistance trained 4 times/wk for 28 days while orally ingesting either 3 g of placebo or 3 g of D-ASP. Data were analyzed with 2 × 2 analysis of variance (P < .05). Before and after resistance training and supplementation, body composition and muscle strength, serum gonadal hormones, and serum D-ASP and d-aspartate oxidase (DDO) were determined. Body composition and muscle strength were significantly increased in both groups in response to resistance training (P < .05) but not different from one another (P > .05). Total and free testosterone, luteinizing hormone, gonadotropin-releasing hormone, and estradiol were unchanged with resistance training and D-ASP supplementation (P > .05). For serum D-ASP and DDO, D-ASP resulted in a slight increase compared with baseline levels (P > .05). For the D-ASP group, the levels of serum DDO were significantly increased compared with placebo (P < .05). The gonadal hormones were unaffected by 28 days of D-ASP supplementation and not associated with the observed increases in muscle strength and mass. Therefore, at the dose provided, D-ASP supplementation is ineffective in up-regulating the activity of the hypothalamo-pituitary-gonadal axis and has no anabolic or ergogenic effects in skeletal muscle. © 2013 Elsevier Inc. All rights reserved.

Developing a financial framework for academic service partnerships: models of the United States and Europe.

PubMed

De Geest, Sabina; Sullivan Marx, Eileen M; Rich, Victoria; Spichiger, Elisabeth; Schwendimann, Rene; Spirig, Rebecca; Van Malderen, Greet

2010-09-01

Academic service partnerships (ASPs) are structured linkages between academe and service which have demonstrated higher levels of innovation. In the absence of descriptions in the literature on financial frameworks to support ASPs, the purpose of this paper is to present the supporting financial frameworks of a Swiss and a U.S. ASP. This paper used a case study approach. Two frameworks are presented. The U.S. model presented consists of a variety of ASPs, all linked to the School of Nursing of the University of Pennsylvania. The structural integration and governance system is elucidated. Each ASP has its own source of revenue or grant support with the goal to be fiscally in the black. Joint appointments are used as an instrument to realize these ASPs. The Swiss ASP entails a detailed description of the financial framework of one ASP between the Institute of Nursing Science at the University of Basel and the Inselspital Bern University Hospital. Balance in the partnership, in terms of both benefit and cost between both partners, was a main principle that guided the development of the financial framework and the translation of the ASP in budgetary terms. The model builds on a number of assumptions and provides the partnership management within a simple framework for monitoring and evaluation of the progress of the partnership. In operationalizing an ASP, careful budgetary planning should be an integral part of the preparation and evaluation of the collaboration. The proposed Swiss and U.S. financial frameworks allow doing so. Outcomes of care can be improved with strong nursing service and academic partnerships. Sustaining such partnerships requires attention to financial and contractual arrangements.

[Effects of Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly peptides on hormonal activity and thyroid morphology in hypophysectomized mature and old birds].

PubMed

Kuznik, B I; Pateiuk, A V; Rusaeva, N S; Baranchugova, L M; Obydenko, V I

2011-01-01

The aim of the paper was to investigate effects of Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly peptides which were designed and synthesized on the basis of amino acid study of the hypophyseal anterior and posterior lobe peptides on the thyroid morphology and hormonal activity in mature chicken and old birds. Hypophysectomy was established to produce atrophic changes in the thyroid gland and development of secondary hypothyrosis. Administration of Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly tetrapeptides significantly prevented these impairments by increasing the levels of the thyroid-stimulating hormone (TSH) as well as T3 and T4. Restoration of the thyroid functions and morphology was registered to be greater in one-year-old chicken as compared to five-year-old ones.

Efficacy and safety of native versus pegylated Escherichia coli asparaginase for treatment of adults with high-risk, Philadelphia chromosome-negative acute lymphoblastic leukemia.

PubMed

Ribera, Josep-Maria; Morgades, Mireia; Montesinos, Pau; Martino, Rodrigo; Barba, Pere; Soria, Beatriz; Bermúdez, Arancha; Moreno, María-José; González-Campos, José; Vives, Susana; Gil, Cristina; Abella, Eugenia; Guàrdia, Ramon; Martínez-Carballeira, Daniel; Martínez-Sánchez, Pilar; Amigo, María-Luz; Mercadal, Santiago; Serrano, Alfons; López-Martínez, Aurelio; Vall-Llovera, Ferran; Sánchez-Sánchez, María-José; Peñarrubia, María-Jesús; Calbacho, María; Méndez, Jose-Angel; Bergua, Juan; Cladera, Antonia; Tormo, Mar; García-Belmonte, Daniel; Feliu, Evarist; Ciudad, Juana; Orfao, Alberto

2017-11-22

Native or pegylated (PEG) asparaginase (ASP) are commonly used in treatment of acute lymphoblastic leukemia (ALL), but have been scarcely compared in the same trial in adult patients. Native vs. PEG-ASP administered according to availability in each center were prospectively evaluated in adults with high-risk ALL. Ninety-one patients received native ASP and 35 PEG-ASP in induction. No significant differences were observed in complete remission, minimal residual disease levels after induction and after consolidation, disease-free survival, and overall survival. No significant differences in grades 3-4 toxicity were observed in the induction period, although a trend for higher hepatic toxicity was observed in patients receiving PEG-ASP. In this trial the type of ASP did not influence patient response and outcome.

Anaphylactic reaction to polyethylene-glycol conjugated-asparaginase: premedication and desensitization may not be sufficient.

PubMed

Sahiner, Umit M; Yavuz, S Tolga; Gökce, Muge; Buyuktiryaki, Betul; Altan, Ilhan; Aytac, Selin; Tuncer, Murat; Tuncer, Ayfer; Sackesen, Cansin

2013-08-01

In hypersensitive reactions to native L-asparaginase, either premedication and desensitization or substitution with polyethylene glycol conjugated asparaginase (PEG-ASP) is preferred. Anaphylaxis with PEG-ASP is rare. An 8-year-old girl and a 2.5-year-old boy, both diagnosed as having acute lymphoblastic leukemia, presented with native L-asparaginase hypersensitivity and substitution with PEG-ASP was preferred. They received a premedication (methylprednisolone, hydroxyzine and ranitidine) followed by desensitization with PEG-ASP infusion. Both patients developed anaphylaxis with peg-asparaginase. These are the first reported cases of anaphylactic reaction to PEG-ASP, despite the application of both premedication and desensitization. Anaphylaxis with PEG-ASP is very rare and premedication and desensitization protocols may not prevent these hypersensitive reactions. © 2013 The Authors. Pediatrics International © 2013 Japan Pediatric Society.

Phosphate-Catalyzed Succinimide Formation from Asp Residues: A Computational Study of the Mechanism.

PubMed

Kirikoshi, Ryota; Manabe, Noriyoshi; Takahashi, Ohgi

2018-02-24

Aspartic acid (Asp) residues in proteins and peptides are prone to the non-enzymatic reactions that give biologically uncommon l-β-Asp, d-Asp, and d-β-Asp residues via the cyclic succinimide intermediate (aminosuccinyl residue, Suc). These abnormal Asp residues are known to have relevance to aging and pathologies. Despite being non-enzymatic, the Suc formation is thought to require a catalyst under physiological conditions. In this study, we computationally investigated the mechanism of the Suc formation from Asp residues that were catalyzed by the dihydrogen phosphate ion, H₂PO₄ - . We used Ac-l-Asp-NHMe (Ac = acetyl, NHMe = methylamino) as a model compound. The H₂PO₄ - ion (as a catalyst) and two explicit water molecules (as solvent molecules stabilizing the negative charge) were included in the calculations. All of the calculations were performed by density functional theory with the B3LYP functional. We revealed a phosphate-catalyzed two-step mechanism (cyclization-dehydration) of the Suc formation, where the first step is predicted to be rate-determining. In both steps, the reaction involved a proton relay mediated by the H₂PO₄ - ion. The calculated activation barrier for this mechanism (100.3 kJ mol -1 ) is in reasonable agreement with an experimental activation energy (107 kJ mol -1 ) for the Suc formation from an Asp-containing peptide in a phosphate buffer, supporting the catalytic mechanism of the H₂PO₄ - ion that is revealed in this study.

Drug release patterns and cytotoxicity of PEG-poly(aspartate) block copolymer micelles in cancer cells.

PubMed

Eckman, Allison M; Tsakalozou, Eleftheria; Kang, Nayon Y; Ponta, Andrei; Bae, Younsoo

2012-07-01

To test physicochemical and biological properties of PEG-poly(aspartate) [PEG-p(Asp)] block copolymer micelles entrapping doxorubicin hydrochloride (DOX) through ionic interaction. PEG-p(Asp) was synthesized from 5 kDa PEG and 20 Asp units. Carboxyl groups of p(Asp) were present as benzyl ester [PEG-p(Asp/Bz)], sodium salt [PEG-p(Asp/Na)] or free acid [PEG-p(Asp/H)]. Block copolymers and DOX were mixed at various ratios to prepare polymer micelles, which were subsequently characterized to determine particle size, drug loading and release patterns, and cytotoxicity against prostate (PC3 and DU145) and lung (A549) cancer cell lines. PEG-p(Asp/Bz), Na- and H-micelles entrapped 1.1, 56.8 and 40.6 wt.% of DOX, respectively. Na- and H-micelles (<100 nm) showed time-dependent DOX release at pH 7.4, which was accelerated at pH 5.0. Na-micelles were most stable at pH 7.4, retaining 31.8% of initial DOX for 48 h. Cytotoxicity of Na-micelles was 23.2% (A549), 28.5% (PC3) and 45.9% (DU145) more effective than free DOX. Ionic interaction appeared to entrap DOX efficiently in polymer micelles from PEG-p(Asp) block copolymers. Polymer micelles possessing counter ions (Na) of DOX in the core were the most stable, releasing drugs for prolonged time in a pH-dependent manner, and suppressing cancer cells effectively.

Identification, by molecular cloning, of a novel type of I2-superfamily conotoxin precursor and two novel I2-conotoxins from the worm-hunter snail Conus spurius from the Gulf of México.

PubMed

Zamora-Bustillos, Roberto; Aguilar, Manuel B; Falcón, Andrés

2010-03-01

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I(2)-conotoxins and three of I(2)-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I(2)) or 23- residue signal peptide (I(2)-like), a 31-residue "pro" region (I(2)-like), and a 32-residue mature toxin region (I(2) and I(2)-like). In addition, all the precursors have a 13-residue "post" region which contains a gamma-carboxylation recognition sequence that directs the gamma-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a "post" region has been found in precursors of I-conotoxins that also contain a "pro" region. The "post" peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that gamma-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I(2)-like-conotoxins of C. spurius are more related to I(2)-conotoxins than to I(1)- and I(3)-conotoxins from other species, and that they might represent a new subgroup of the I(2)-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes. (c) 2009 Elsevier Inc. All rights reserved.

Effect of tetrapeptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly on the structure and function of the thyroid gland in neonatally hypophysectomized chickens.

PubMed

Kuznik, B I; Pateyuk, A V; Rusaeva, N S

2008-01-01

Tetrapeptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly were synthesized on the basis of amino acid composition of pituitary cytomedins. Administration of these tetrapeptides to hypophysectomized chickens for 40 days was followed by an increase in the concentrations of thyrotropic hormone and thyroid hormones and recovery of thyroid gland structure.

Enhancing and Promoting Recovery In Attentionally Impaired People Diagnosed With Schizophrenia: Results From A Randomized Controlled Trial Of Attention Shaping In A Partial Hospital Program.

PubMed

Silverstein, Steven M; Roché, Matthew W; Khan, Zaynab; Carson, Sarah J; Malinovsky, Igor; Newbill, William A; Menditto, Anthony A; Wilkniss, Sandra M

2014-01-01

The attentional impairments associated with schizophrenia are well-documented and profound. Psychopharmacological and most psychosocial interventions have been shown to have limited effect in improving attentional capacity. That said, one form of psychosocial treatment, attention shaping procedures (ASP), has been repeatedly demonstrated to produce significant and meaningful change in various aspects of participant attentiveness behaviors. To date, studies of ASP have been limited in that they have been conducted primarily with inpatients, have not assessed the generalizability of ASP's effects, and have not explored whether reinforcement is required to be contingent on performance of attentive behaviors. To address these limitations we conducted the first randomized clinical trial of ASP with people diagnosed with schizophrenia who are being treated in a partial hospital program. Our results indicate that ASP is effective in improving attention in people with schizophrenia in these types of programs, the effects of ASP generalize outside of the immediate treatment context to both other treatment groups and real world functioning, and contingent reinforcement is a critical ingredient of ASP. This project provides further evidence for the benefits of use of ASP in the recovery-oriented treatment of people diagnosed with schizophrenia who have significant attentional impairments.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function*

PubMed Central

Cui, Guiying; Freeman, Cody S.; Knotts, Taylor; Prince, Chengyu Z.; Kuang, Christopher; McCarty, Nael A.

2013-01-01

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg352-Asp993 and Arg347-Asp924, that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg347 not only interacts with Asp924 but also interacts with Asp993. The tripartite interaction Arg347-Asp924-Asp993 mainly contributes to maintaining a stable s2 open subconductance state. The Arg352-Asp993 salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg352 and Asp993, channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle. PMID:23709221

Strategies for improving antibiotic use in Qatar: a survey of pharmacists' perceptions and experiences.

PubMed

Pawluk, Shane; Black, Emily; El-Awaisi, Alla

2015-02-01

The objectives of this study were to identify antimicrobial stewardship activities in Qatar, identify pharmacist involvement in activities and summarize perceived barriers for implementation of antimicrobial stewardship programs (ASPs). A cross-sectional survey was developed based on study objectives and completed by pharmacists in Qatar. Most hospital settings have implemented components of ASP. Lack of infectious disease specialists and training of healthcare providers was the most common barrier to implementation or expansion of ASP identified in the hospital and community settings respectively. Pharmacists report some components of ASP have been implemented; however, barriers must be overcome to further expand ASPs. © 2014 Royal Pharmaceutical Society.

Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus.

PubMed

Sebban, P; Maróti, P; Schiffer, M; Hanson, D K

1995-07-04

Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

PubMed

Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

2016-06-07

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket.

PubMed

Acosta-Maspons, Alexis; Sepúlveda-García, Edgar; Sánchez-Baldoquín, Laura; Marrero-Gutiérrez, Junier; Pons, Tirso; Rocha-Sosa, Mario; González, Lien

2014-01-01

Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

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

Takata, Takumi; Shimo-Oka, Tadashi; Kojima, Masami

Although proteins are generally composed of L-{alpha}-amino acids, D-{beta}-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several D-{beta}-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-{beta}-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel D-{beta}-Asp-containing proteins from rabbit lens. The results indicate that {beta}-crystallin A3, {beta}-crystallin A4, {beta}-crystallin B1, {beta}-crystallin B2, {beta}-crystallin B3,more » {gamma}-crystallin C, {gamma}-crystallin D, and {lambda}-crystallin in rabbit lens contain D-{beta}-Asp residues. Furthermore, the occurrence of D-{beta}-Asp residues increases with infrared ray (IR) irradiation. Additionally, some D-{beta}-Asp-containing proteins only appear after IR irradiation. One such protein is the {alpha}-enolase, which shows homology to {tau}-crystallin.« less

Elucidating the Role of Many-Body Forces in Liquid Water. I. Simulations of Water Clusters on the VRT (ASP-W) Potential Surfaces

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

Goldman, N; Saykally, R J

We test the new VRT(ASP-W)II and VRT(ASP-W)III potentials by employing Diffusion Quantum Monte Carlo simulations to calculate the vibrational ground-state properties of water clusters. These potentials are fits of the highly detailed ASP-W ab initio potential to (D{sub 2}O){sub 2} microwave and far-IR data, and along with the SAPT5s potentials, are the most accurate water dimer potential surfaces in the literature. The results from VRT(ASP-W)II and III are compare to those from the original ASP-W potential, the SAPT5s family of potentials, and several bulk water potentials. Only VRT(ASP-W)II and the spectroscopically ''tuned'' SAPT5st (with N-body induction included) accurately reproduce themore » vibrational ground-state structures of water clusters up to the hexamer. Finally, the importance of many-body induction and three-body dispension are examined, and it is shown that the latter can have significant effects on water cluster properties despite its small magnitude.« less

OXA-48 carbapenemase-producing Salmonella enterica serovar Kentucky isolate of sequence type 198 in a patient transferred from Libya to Switzerland.

PubMed

Seiffert, Salome N; Perreten, Vincent; Johannes, Sönke; Droz, Sara; Bodmer, Thomas; Endimiani, Andrea

2014-01-01

Here, we report a case of OXA-48-producing Salmonella enterica serovar Kentucky of sequence type 198 (ST198) from perianal screening cultures of a patient transferred from Libya to Switzerland. The blaOXA-48 gene was carried by Tn1999.2 and located on an ∼60-kb IncL/M plasmid. This Salmonella strain also possessed the blaVEB-8, aac(6)-Ib, tet(A), sul1, and mphA resistance genes and substitutions in GyrA (Ser83Phe and Asp87Asn) and ParC (Ser80Ile). This finding emphasizes that prompt screening strategies are essential to prevent the dissemination of carbapenemase producers imported from countries where they are endemic.

Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.

PubMed

Kita, Masaki; Black, David StC; Ohno, Osamu; Yamada, Kaoru; Kigoshi, Hideo; Uemura, Daisuke

2009-12-23

The duck-billed platypus (Ornithorhynchus anatinus) is one of the few venomous Australian mammals. We previously found that its crude venom potently induces Ca(2+) influx in human neuroblastoma IMR-32 cells. Guided by this bioassay, we identified 11 novel peptides, including the heptapeptide H-His-Asp-His-Pro-Asn-Pro-Arg-OH (1). Compounds 1-4 and 5-11 coincided with the 6-9 N-terminal residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP) and the 132-150 part of OvCNP precursor peptide, respectively. Heptapeptide 1, which is one of the primary components of the venom fluid (approximately 200 ng/microL), induced a significant increase in [Ca(2+)](i) in IMR-32 cells at 75 microM. To the best of our knowledge, this is the first example of the isolation of the N-terminal linear fragments of CNPs in any mammal.

De novo transcriptome sequencing reveals a considerable bias in the incidence of simple sequence repeats towards the downstream of 'Pre-miRNAs' of black pepper.

PubMed

Joy, Nisha; Asha, Srinivasan; Mallika, Vijayan; Soniya, Eppurathu Vasudevan

2013-01-01

Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of '43 pre-miRNA candidates bearing different types of SSR motifs'. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted 'pre-miRNA candidates bearing SSRs'. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted 'pre-miRNA candidates'. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of 'tandem repeats' in miRNAs.

De novo Transcriptome Sequencing Reveals a Considerable Bias in the Incidence of Simple Sequence Repeats towards the Downstream of ‘Pre-miRNAs’ of Black Pepper

PubMed Central

Joy, Nisha; Asha, Srinivasan; Mallika, Vijayan; Soniya, Eppurathu Vasudevan

2013-01-01

Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of ‘43 pre-miRNA candidates bearing different types of SSR motifs’. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted ‘pre-miRNA candidates bearing SSRs’. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted ‘pre-miRNA candidates’. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of ‘tandem repeats’ in miRNAs. PMID:23469176

Molecular cloning and expression of a gene for a factor which stabilizes formation of inhibitor-mitochondrial ATPase complex from Saccharomyces cerevisiae.

PubMed

Akashi, A; Yoshida, Y; Nakagoshi, H; Kuroki, K; Hashimoto, T; Tagawa, K; Imamoto, F

1988-10-01

Stabilizing factor, a 9 kDa protein, stabilizes and facilitates formation of the complex between mitochondrial ATP synthase and its intrinsic inhibitor protein. A clone containing the gene encoding the 9 kDa protein was selected from a yeast genomic library to determine the structure of its precursor protein. As deduced from the nucleotide sequence, the precursor of the yeast 9 kDa stabilizing factor contains 86 amino acid residues and has a molecular weight of 10,062. From the predicted sequence we infer that the stabilizing factor precursor contains a presequence of 23 amino acid residues at its amino terminus. We also used S1 mapping to determine the initiation site of transcription under glucose-repressed or derepressed conditions. These experiments suggest that transcription of this gene starts at three different sites and that only one of them is not affected by the presence of glucose.

Effects of Asp-179 mutations in TEMpUC19 beta-lactamase on susceptibility to beta-lactams.

PubMed Central

Vakulenko, S B; Tóth, M; Taibi, P; Mobashery, S; Lerner, S A

1995-01-01

To examine the effect of disruption of the salt bridge (between Arg-164 and Asp-179 [numbering of Ambler et al. (Biochem J. 267:269-272, 1991)]) that anchors the conserved omega-loop in class A beta-lactamases, we obtained mutant enzymes with each of the 19 other amino acid residues replacing Asp-179 in the TEM beta-lactamase encoded by pUC19 and studied the level of resistance to various beta-lactams conferred by each enzyme. All mutations of Asp-179 compromised the level of resistance to ampicillin, but most of them enhanced resistance to ceftazidime. In contrast, mutations of Asp-179 generally impaired the low levels of resistance to cefepime and aztreonam. One might expect to find clinical isolates with mutant TEM beta-lactamases with replacements of Asp-179 that express an expanded spectrum of resistance to beta-lactams including ceftazidime. PMID:7486939

Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana)

PubMed Central

Cajimat, Maria N. B.; Milazzo, Mary Louise; Borchert, Jeff N.; Abbott, Ken D.; Bradley, Robert D.; Fulhorst, Charles F.

2008-01-01

The results of analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that an arenavirus isolated from a Mexican woodrat (Neotoma mexicana) captured in Arizona is a strain of a novel species (proposed name Skinner Tank virus) and that arenaviruses isolated from Mexican woodrats captured in Colorado, New Mexico, and Utah are strains of Whitewater Arroyo virus or species phylogenetically closely related to Whitewater Arroyo virus. Pairwise comparisons of glycoprotein precursor sequences and nucleocapsid protein sequences revealed a high level of divergence among the viruses isolated from the Mexican woodrats captured in Colorado, New Mexico, and Utah and the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat (Neotoma albigula) captured in New Mexico. Conceptually, the viruses from Colorado, New Mexico, and Utah and strain AV 9310135 could be grouped together in a species complex in the family Arenaviridae, genus Arenavirus. PMID:18304671

Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

PubMed Central

Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

2006-01-01

RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

Platelet inhibition during ticagrelor monotherapy versus ticagrelor plus aspirin in patients with coronary artery disease (TEMPLATE study): study protocol for a randomised controlled trial.

PubMed

Baos, Sarah; Underwood, Wendy; Culliford, Lucy; Reeves, Barnaby C; Rogers, Chris A; Bowles, Ruth; Johnson, Tom; Baumbach, Andreas; Mumford, Andrew

2017-11-09

Dual antiplatelet therapy (DAPT) with aspirin (ASP) and a P2Y 12 blocker is currently standard care after percutaneous coronary intervention (PCI) with stent insertion, and aims to inhibit platelet function in order to prevent stent thrombosis. The P2Y 12 blocker ticagrelor (TIC) has greater antiplatelet effect than the previously used members of this class, such as clopidogrel. In healthy volunteers, TIC is sufficient to cause strong platelet inhibition, with little additional effect from ASP. Omission of ASP may improve the safety of antiplatelet regimes by reducing bleeding. However, the effect of single antiplatelet treatment with TIC, compared to DAPT with TIC + ASP, has not been studied in detail in patients with coronary artery disease. To compare TIC with TIC + ASP, we have initiated a single centre, open-label randomised controlled trial (TEMPLATE study) in adults receiving DAPT following PCI with a sample size of 110 patients. Patients are invited to join the study when, as part of standard care, they are due to switch from DAPT (ASP + any P2Y 12 blocker) to single antiplatelet treatment with ASP alone after 6-12 months. Patients are randomised to receive either TIC or TIC + ASP for 4 weeks. All patients then revert to standard care with ASP alone. Blood samples and clinical data are collected at three study visits: at baseline during treatment with ASP + any P2Y 12 blocker (visit 1); approximately 4 weeks after visit 1 during treatment with either TIC or TIC + ASP (visit 2); and approximately 8 weeks after visit 1 when treatment has reverted to ASP alone (visit 3). The primary outcome is the extent of platelet inhibition, measured by light transmission aggregation, flow cytometry, flow chamber and plasma biomarker tests. The primary analysis will compare the extent of platelet inhibition between the TIC and TIC + ASP groups at visit 2, adjusted for baseline platelet reactivity. Secondary analyses will compare the extent of platelet inhibition at visit 2 with that at visit 3. This is the first study to compare in detail the extent of platelet inhibition in patients who are receiving TIC compared with TIC + ASP. The study findings will complement larger-scale trials of the clinical efficacy and safety of TIC compared to TIC + ASP. ISRCTN registry, identifier ISRCTN84335288 . Registered on 23 June 2014.

Plant MicroRNA Prediction by Supervised Machine Learning Using C5.0 Decision Trees.

PubMed

Williams, Philip H; Eyles, Rod; Weiller, Georg

2012-01-01

MicroRNAs (miRNAs) are nonprotein coding RNAs between 20 and 22 nucleotides long that attenuate protein production. Different types of sequence data are being investigated for novel miRNAs, including genomic and transcriptomic sequences. A variety of machine learning methods have successfully predicted miRNA precursors, mature miRNAs, and other nonprotein coding sequences. MirTools, mirDeep2, and miRanalyzer require "read count" to be included with the input sequences, which restricts their use to deep-sequencing data. Our aim was to train a predictor using a cross-section of different species to accurately predict miRNAs outside the training set. We wanted a system that did not require read-count for prediction and could therefore be applied to short sequences extracted from genomic, EST, or RNA-seq sources. A miRNA-predictive decision-tree model has been developed by supervised machine learning. It only requires that the corresponding genome or transcriptome is available within a sequence window that includes the precursor candidate so that the required sequence features can be collected. Some of the most critical features for training the predictor are the miRNA:miRNA(∗) duplex energy and the number of mismatches in the duplex. We present a cross-species plant miRNA predictor with 84.08% sensitivity and 98.53% specificity based on rigorous testing by leave-one-out validation.

A Caenorhabditis elegans Mass Spectrometric Resource for Neuropeptidomics

NASA Astrophysics Data System (ADS)

Van Bael, Sven; Zels, Sven; Boonen, Kurt; Beets, Isabel; Schoofs, Liliane; Temmerman, Liesbet

2018-05-01

Neuropeptides are important signaling molecules used by nervous systems to mediate and fine-tune neuronal communication. They can function as neurotransmitters or neuromodulators in neural circuits, or they can be released as neurohormones to target distant cells and tissues. Neuropeptides are typically cleaved from larger precursor proteins by the action of proteases and can be the subject of post-translational modifications. The short, mature neuropeptide sequences often entail the only evolutionarily reasonably conserved regions in these precursor proteins. Therefore, it is particularly challenging to predict all putative bioactive peptides through in silico mining of neuropeptide precursor sequences. Peptidomics is an approach that allows de novo characterization of peptides extracted from body fluids, cells, tissues, organs, or whole-body preparations. Mass spectrometry, often combined with on-line liquid chromatography, is a hallmark technique used in peptidomics research. Here, we used an acidified methanol extraction procedure and a quadrupole-Orbitrap LC-MS/MS pipeline to analyze the neuropeptidome of Caenorhabditis elegans. We identified an unprecedented number of 203 mature neuropeptides from C. elegans whole-body extracts, including 35 peptides from known, hypothetical, as well as from completely novel neuropeptide precursor proteins that have not been predicted in silico. This set of biochemically verified peptide sequences provides the most elaborate C. elegans reference neurpeptidome so far. To exploit this resource to the fullest, we make our in-house database of known and predicted neuropeptides available to the community as a valuable resource. We are providing these collective data to help the community progress, amongst others, by supporting future differential and/or functional studies. [Figure not available: see fulltext.

The evolution of neuropeptide signalling: insights from echinoderms.

PubMed

Semmens, Dean C; Elphick, Maurice R

2017-09-01

Neuropeptides are evolutionarily ancient mediators of neuronal signalling that regulate a wide range of physiological processes and behaviours in animals. Neuropeptide signalling has been investigated extensively in vertebrates and protostomian invertebrates, which include the ecdysozoans Drosophila melanogaster (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda). However, until recently, an understanding of evolutionary relationships between neuropeptide signalling systems in vertebrates and protostomes has been impaired by a lack of genome/transcriptome sequence data from non-ecdysozoan invertebrates. The echinoderms-a deuterostomian phylum that includes sea urchins, sea cucumbers and starfish-have been particularly important in providing new insights into neuropeptide evolution. Sequencing of the genome of the sea urchin Strongylocentrotus purpuratus (Class Echinoidea) enabled discovery of (i) the first invertebrate thyrotropin-releasing hormone-type precursor, (ii) the first deuterostomian pedal peptide/orcokinin-type precursors and (iii) NG peptides-the 'missing link' between neuropeptide S in tetrapod vertebrates and crustacean cardioactive peptide in protostomes. More recently, sequencing of the neural transcriptome of the starfish Asterias rubens (Class Asteroidea) enabled identification of 40 neuropeptide precursors, including the first kisspeptin and melanin-concentrating hormone-type precursors to be identified outside of the chordates. Furthermore, the characterization of a corazonin-type neuropeptide signalling system in A. rubens has provided important new insights into the evolution of gonadotropin-releasing hormone-related neuropeptides. Looking forward, the discovery of multiple neuropeptide signalling systems in echinoderms provides opportunities to investigate how these systems are used to regulate physiological and behavioural processes in the unique context of a decentralized, pentaradial bauplan. © The Author 2017. Published by Oxford University Press.

A Caenorhabditis elegans Mass Spectrometric Resource for Neuropeptidomics

NASA Astrophysics Data System (ADS)

Van Bael, Sven; Zels, Sven; Boonen, Kurt; Beets, Isabel; Schoofs, Liliane; Temmerman, Liesbet

2018-01-01

Neuropeptides are important signaling molecules used by nervous systems to mediate and fine-tune neuronal communication. They can function as neurotransmitters or neuromodulators in neural circuits, or they can be released as neurohormones to target distant cells and tissues. Neuropeptides are typically cleaved from larger precursor proteins by the action of proteases and can be the subject of post-translational modifications. The short, mature neuropeptide sequences often entail the only evolutionarily reasonably conserved regions in these precursor proteins. Therefore, it is particularly challenging to predict all putative bioactive peptides through in silico mining of neuropeptide precursor sequences. Peptidomics is an approach that allows de novo characterization of peptides extracted from body fluids, cells, tissues, organs, or whole-body preparations. Mass spectrometry, often combined with on-line liquid chromatography, is a hallmark technique used in peptidomics research. Here, we used an acidified methanol extraction procedure and a quadrupole-Orbitrap LC-MS/MS pipeline to analyze the neuropeptidome of Caenorhabditis elegans. We identified an unprecedented number of 203 mature neuropeptides from C. elegans whole-body extracts, including 35 peptides from known, hypothetical, as well as from completely novel neuropeptide precursor proteins that have not been predicted in silico. This set of biochemically verified peptide sequences provides the most elaborate C. elegans reference neurpeptidome so far. To exploit this resource to the fullest, we make our in-house database of known and predicted neuropeptides available to the community as a valuable resource. We are providing these collective data to help the community progress, amongst others, by supporting future differential and/or functional studies.

A Caenorhabditis elegans Mass Spectrometric Resource for Neuropeptidomics.

PubMed

Van Bael, Sven; Zels, Sven; Boonen, Kurt; Beets, Isabel; Schoofs, Liliane; Temmerman, Liesbet

2018-05-01

Neuropeptides are important signaling molecules used by nervous systems to mediate and fine-tune neuronal communication. They can function as neurotransmitters or neuromodulators in neural circuits, or they can be released as neurohormones to target distant cells and tissues. Neuropeptides are typically cleaved from larger precursor proteins by the action of proteases and can be the subject of post-translational modifications. The short, mature neuropeptide sequences often entail the only evolutionarily reasonably conserved regions in these precursor proteins. Therefore, it is particularly challenging to predict all putative bioactive peptides through in silico mining of neuropeptide precursor sequences. Peptidomics is an approach that allows de novo characterization of peptides extracted from body fluids, cells, tissues, organs, or whole-body preparations. Mass spectrometry, often combined with on-line liquid chromatography, is a hallmark technique used in peptidomics research. Here, we used an acidified methanol extraction procedure and a quadrupole-Orbitrap LC-MS/MS pipeline to analyze the neuropeptidome of Caenorhabditis elegans. We identified an unprecedented number of 203 mature neuropeptides from C. elegans whole-body extracts, including 35 peptides from known, hypothetical, as well as from completely novel neuropeptide precursor proteins that have not been predicted in silico. This set of biochemically verified peptide sequences provides the most elaborate C. elegans reference neurpeptidome so far. To exploit this resource to the fullest, we make our in-house database of known and predicted neuropeptides available to the community as a valuable resource. We are providing these collective data to help the community progress, amongst others, by supporting future differential and/or functional studies. Graphical Abstract ᅟ.

Neutropenia associated with vincristine and L-asparaginase induction chemotherapy for canine lymphoma.

PubMed

Northrup, Nicole C; Rassnick, Kenneth M; Snyder, Laura A; Stone, Michael S; Kristal, Orna; Cotter, Susan M; Moore, Antony S

2002-01-01

Vincristine (VCR) and L-asparaginase (L-ASP) are commonly used to treat canine lymphoma. As single agents, these drugs are not myelosuppressive. However, in combination, VCR and L-ASP cause severe neutropenia in some dogs. It has been recommended that L-ASP be administered 12-24 hours after VCR to minimize toxicity. The purpose of this retrospective study was to determine the prevalence of neutropenia after VCR/L-ASP induction therapy for canine lymphoma and to evaluate risk factors for myelosuppression, especially the interval between VCR and L-ASP administration. Medical records of 147 dogs were reviewed. L-ASP was given 0 (n = 50), 6 (n = 23), 18 (n = 20), or 24 (n = 54) hours after VCR. Forty percent of the dogs were neutropenic 7 days after VCR/L-ASP, and 18% had neutrophil counts of <1,000 cells/microL. The median neutrophil count was 3,712 cells/microL (range 0-30,968 cells/microL). No correlation was found between administration interval and day 7 neutrophil count (P = .84) or development of gastrointestinal signs, including vomiting (P = .80), diarrhea (P = .52), and decreased appetite (P = .30). No significant predictors of neutropenia were identified. Higher clinical stage and substage b were associated with decreased appetite after treatment (P = .04 and .01, respectively). Sixteen percent of the dogs were hospitalized. This study demonstrates that VCR/L-ASP induction for canine lymphoma may result in neutropenia but that separation of VCR and L-ASP administration may not be necessary to avoid toxicity.

Different profile of thrombin generation in children with acute lymphoblastic leukaemia treated with native or pegylated asparaginase: A cohort study.

PubMed

Rozen, Laurence; Noubouossie, Denis; Dedeken, Laurence; Huybrechts, Sophie; Lê, Phu Quoc; Ferster, Alina; Demulder, Anne

2017-02-01

Asparaginase (Asp) and corticosteroid (CS) treatment in patients with acute lymphoblastic leukaemia (ALL) is associated with an increased risk of thrombotic events. Characterization of global haemostatic phenotypes of patients with ALL during Asp therapy. Thrombin generation (TG) was monitored in platelet-poor plasma of 56 children treated for a B lineage ALL (36 with native, 20 with PEG Asp) using 1 pM tissue factor and 4 μM phospholipids, with and without thrombomodulin. Protein C activity (PC), free protein S (PS), antithrombin (AT) and fibrinogen levels were also measured. Elevated endogenous thrombin potential (ETP) and peak of TG were noted at diagnosis, throughout the Induction phase and Late Intensification but was significantly less for PEG than for native Asp (P < 0.001), while age, sex, type of corticosteroid during Induction and molecular response had no significant effect. The reduction of ETP after addition of thrombomodulin was significantly lower in ALL children compared with that in controls, suggesting impairment in PS/PC pathway. Three patients experienced thrombosis: two treated with native and one with PEG Asp. The two patients with native Asp had, at the time of thrombosis, a prothrombotic profile. Treatment with Asp, in combination with CS, enhances TG in children with ALL, more significantly with native than PEG Asp, which is present early at diagnosis, persists during Induction and reappears during Late Intensification. This is consistent with the high incidence of thrombotic events described during these phases of therapy. The less pronounced effect of PEG Asp remains to be elucidated. © 2016 Wiley Periodicals, Inc.

Low acylation stimulating protein levels are associated with cardiometabolic disorders–secondary to autoimmune activation?

PubMed Central

Onat, Altan; Altay, Servet; Yüksel, Murat; Karadeniz, Yusuf; Can, Günay; Yüksel, Hüsniye; Ademoğlu, Evin

2017-01-01

Objective: We investigated the possible association of serum acylation stimulating protein (ASP) with cardiometabolic disorders and the evidence of autoimmune activation. Methods: Population-based randomly selected 1024 participants were cross-sectionally and prospectively analyzed. ASP concentrations were measured with a validated ELISA kit. Correlations were sought separately in subjects with no cardiometabolic disorders (n=427) designated as “healthy.” Results: ASP was positively correlated with total testosterone and inversely correlated with platelet activating factor (PAF), PAF-acetylhydrolase (AH), in each gender, and positively correlated in “healthy” men with lipoprotein [Lp](a) and apolipoprotein B. Correlations of ASP with PAF values ≥22 nmol/L were abolished, contrasted to a strongly inverse one in subjects with PAF <22 nmol/L. In linear regression analyses in the whole sample, ASP was inversely associated independently with PAF and PAF-AH and, in men, positively with Lp(a) and sex hormone-binding globulin. Prevalent and (at 2.0 years’ follow-up) incident metabolic syndrome (MetS, n=393), diabetes (n=154), and coronary heart disease (CHD, n=171) were analyzed by sex-, age-, and Lp(a)-adjusted logistic regression, using tertiles of ASP and PAF. The lower two (<42 nmol/L) ASP tertiles were a risk factor in combined sexes for MetS and diabetes. In women, incident CHD was predicted by either reduced or elevated ASP tertiles. Conclusion: Findings can be explained by the notion of operation of immune responses against both ASP and oxidized PAF-like lipids of Lp(a) to yield for “reduced” values and increased likelihood of cardiometabolic disorders. PMID:27599666

Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis.

PubMed

Williamson, Heather R; Sehanobish, Esha; Shiller, Alan M; Sanchez-Amat, Antonio; Davidson, Victor L

2017-02-21

The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

The Global Epidemiology of Syphilis in the Past Century – A Systematic Review Based on Antenatal Syphilis Prevalence

PubMed Central

Kenyon, Chris Richard; Osbak, Kara; Tsoumanis, Achilleas

2016-01-01

Background How can we explain the uneven decline of syphilis around the world following the introduction of penicillin? In this paper we use antenatal syphilis prevalence (ASP) to investigate how syphilis prevalence varied worldwide in the past century, and what risk factors correlate with this variance. Methods 1) A systematic review using PubMed and Google Scholar was conducted to identify countries with published data relating to ASP estimates from before 1952 until the present. Eleven countries were identified (Canada, Denmark, Finland, India, Japan, Norway, Singapore, South Africa, United States of America (USA), United Kingdom (UK) and Zimbabwe). The ASP epidemic curve for each population was depicted graphically. In South Africa and the USA, results are reported separately for the black and white populations. 2) National antenatal syphilis prevalence estimates for 1990 to 1999 and 2008 were taken from an Institute for Health Metrics and Evaluation database on the prevalence of syphilis in low risk populations compiled for the Global Burden of Diseases study and from a recent review paper respectively. National ASPs were depicted graphically and regional median ASPs were calculated for both time periods. 3) Linear regression was used to test for an association between ASP in 1990–1999 and 2008 and four risk factors (efficacy of syphilis screening/treatment, health expenditure, GDP per capita and circumcision prevalence). WHO world regions were included as potential explanatory variables. Results In most populations, ASP dropped to under 1% before 1960. In Zimbabwe and black South Africans, ASP was high in the pre-penicillin period, dropped in the post-penicillin period, but then plateaued at around 6% until the end of the 20th century when ASP dropped to just above 1%. In black Americans, ASP declined in the post penicillin period, but plateaued at 3–5% thereafter. ASP was statistically significantly higher in sub-Saharan Africa in 1990–1999 and 2008 than in the other world regions (P < 0.001). On multivariate analysis in both time periods, ASP was only associated with residence in sub-Saharan Africa. Conclusions Further research is necessary to elucidate the reasons for the higher prevalence of syphilis in sub-Saharan Africa. PMID:27167068

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

PubMed

Huang, Yang-Bin; Li, Jiang; Gao, Xin; Sun, Jiu-Ru; Lu, Yi; Feng, Tao; Fei, Jian; Cui, Da-Fu; Xia, Qi-Chang; Ren, Jun; Zhang, You-Shang

2006-08-01

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Array Simulations Platform (ASP) predicts NASA Data Link Module (NDLM) performance

NASA Technical Reports Server (NTRS)

Snook, Allen David

1993-01-01

Through a variety of imbedded theoretical and actual antenna patterns, the array simulation platform (ASP) enhanced analysis of the array antenna pattern effects for the KTx (Ku-Band Transmit) service of the NDLM (NASA Data Link Module). The ASP utilizes internally stored models of the NDLM antennas and can develop the overall pattern of antenna arrays through common array calculation techniques. ASP expertly assisted in the diagnosing of element phase shifter errors during KTx testing and was able to accurately predict the overall array pattern from combinations of the four internally held element patterns. This paper provides an overview of the use of the ASP software in the solving of array mis-phasing problems.

Effect of short peptides on expression of signaling molecules in organotypic pineal cell culture.

PubMed

Khavinson, V Kh; Linkova, N S; Chalisova, N I; Dudkov, A V; Koncevaya, E A

2011-11-01

We demonstrated the influence of short peptides on the expression of signaling molecules in organotypic culture of the pineal gland from 3-month-old rats. Peptides Ala-Glu-Asp-Gly and Lys-Glu-Asp stimulate the expression of proliferative protein Ki-67 in pineal gland culture. These peptides as well as Glu-Asp-Arg and Lys-Glu do not affect the expression of apoptosis marker AIF. The synthesis of transcription factor CGRP by pinealocytes was stimulated only by Ala-Glu-Asp-Gly. Thus, peptide Ala-Glu-Asp-Gly tissue-specifically stimulates proliferative and secretory activities of pinealocytes, which can be used for recovery of pineal gland functions at the molecular level.

Athlete support personnel and anti-doping: Knowledge, attitudes, and ethical stance.

PubMed

Mazanov, J; Backhouse, S; Connor, J; Hemphill, D; Quirk, F

2014-10-01

Athlete support personnel (ASP) failing to meet responsibilities under the World Anti-Doping Code risk sanction. It is unclear whether the poor knowledge of responsibilities seen in sports physicians and coaches applies to other ASP (e.g., administrators, chiropractors, family, nutritionists, physiotherapists, psychologists, and trainers). A purposive sample of Australian ASP (n = 292) responded to a survey on knowledge of anti-doping rules (35 true/false questions), ethical beliefs and practice, and attitudes toward performance enhancement. Some ASP declined to participate, claiming doping was irrelevant to their practice. Physicians were most knowledgeable (30.8/35), with family and trainers the least (26.0/35). ASP reported that improvements were needed to support anti-doping education (e.g., basis for anti-doping) and practice (e.g., rules). ASP also had a slightly negative attitude toward performance enhancement. Linear regression showed that being a sports physician, providing support at the elite level, and 15 years of experience influenced knowledge. The results confirm gaps in knowledge, suggesting that stronger engagement with ASP anti-doping education and practice is needed. Applying the principles of andragogy could help foster active engagement through emphasis on active inquiry, rather than passive reception of content. Future work on the context within which ASP experience anti-doping is needed, exploring acquisition and translation of knowledge into practice. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Self-assembly of proglycinin and hybrid proglycinin synthesized in vitro from cDNA

PubMed Central

Dickinson, Craig D.; Floener, Liliane A.; Lilley, Glenn G.; Nielsen, Niels C.

1987-01-01

An in vitro system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized in vitro from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The in vitro assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein. Images PMID:16593868

Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

PubMed Central

Prabhu, Yogikala; Burgos, Patricia V.; Schindler, Christina; Farías, Ginny G.; Magadár, Javier G.; Bonifacino, Juan S.

2012-01-01

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway. PMID:22553349

A Review of Quality Measures for Assessing the Impact of Antimicrobial Stewardship Programs in Hospitals

PubMed Central

Akpan, Mary Richard; Ahmad, Raheelah; Shebl, Nada Atef; Ashiru-Oredope, Diane

2016-01-01

The growing problem of antimicrobial resistance (AMR) has led to calls for antimicrobial stewardship programs (ASP) to control antibiotic use in healthcare settings. Key strategies include prospective audit with feedback and intervention, and formulary restriction and preauthorization. Education, guidelines, clinical pathways, de-escalation, and intravenous to oral conversion are also part of some programs. Impact and quality of ASP can be assessed using process or outcome measures. Outcome measures are categorized as microbiological, patient or financial outcomes. The objective of this review was to provide an overview of quality measures for assessing ASP and the reported impact of ASP in peer-reviewed studies, focusing particularly on patient outcomes. A literature search of papers published in English between 1990 and June 2015 was conducted in five databases using a combination of search terms. Primary studies of any design were included. A total of 63 studies were included in this review. Four studies defined quality metrics for evaluating ASP. Twenty-one studies assessed the impact of ASP on antimicrobial utilization and cost, 25 studies evaluated impact on resistance patterns and/or rate of Clostridium difficile infection (CDI). Thirteen studies assessed impact on patient outcomes including mortality, length of stay (LOS) and readmission rates. Six of these 13 studies reported non-significant difference in mortality between pre- and post-ASP intervention, and five reported reductions in mortality rate. On LOS, six studies reported shorter LOS post intervention; a significant reduction was reported in one of these studies. Of note, this latter study reported significantly (p < 0.001) higher unplanned readmissions related to infections post-ASP. Patient outcomes need to be a key component of ASP evaluation. The choice of metrics is influenced by data and resource availability. Controlling for confounders must be considered in the design of evaluation studies to adequately capture the impact of ASP and it is important for unintended consequences to be considered. This review provides a starting point toward compiling standard outcome metrics for assessing ASP. PMID:27025520

Incremental Value of Live/Real Time Three-Dimensional over Two-Dimensional Transesophageal Echocardiography in the Assessment of Atrial Septal Pouch.

PubMed

Elsayed, Mahmoud; Hsiung, Ming C; Meggo-Quiroz, L David; Elguindy, Mostafa; Uygur, Begum; Tandon, Rohit; Guvenc, Tolga; Keser, Nurgul; Vural, Mustafa G; Bulur, Serkan; Chahwala, Jugal R; Abtahi, Firoozeh; Nanda, Navin C

2015-12-01

An atrial septal pouch (ASP) results from partial fusion of the septum primum and the septum secundum, and depending on the site of fusion, the pouch can be left-sided (LASP) or right-sided (RASP). LASPs have been described in association with thrombi found in patients admitted with acute strokes, raising awareness of its potential cardioembolic role, especially in those with no other clearly identifiable embolic source. We retrospectively studied 39 patients in whom the presence of an ASP had been identified by three-dimensional transesophageal echocardiography (3DTEE) and who had a two-dimensional transesophageal echocardiogram (2DTEE) performed during the same clinical encounter. The incremental value provided by 3DTEE over 2DTEE included the detection of six ASPs not found by 2DTEE; the detection of two ASPs in the same subject (in four patients) not identified by 2DTEE; larger ASP measurements of length and height in over 80% of the cases; and measurement of the ASP width (elevational axis) for the calculation of the area of the ASP opening, because of its unique capability to view the pouch en face. In addition, the volume of ASP and of the echogenic masses contained in the ASP (four of 39 patients) could be calculated by 3DTEE, which is a superior parameter of size characterization when compared to individual dimensions. One of these patients who presented with ischemic stroke diagnosed by magnetic resonance imaging had a large (>2 cm) mass in a LASP, with echolucencies similar to those seen in thrombi and associated with clot lysis and resolution. This mass completely disappeared on anticoagulant therapy lending credence that it was most likely a thrombus. There was no history of stroke or any other type of embolic event in the other three patients with masses in ASP. In conclusion, this retrospective study highlights the incremental value of 3DTEE over 2DTEE in the comprehensive assessment and characterization of ASPs, which can aid in the clarification of their role in cryptogenic stroke patients. © 2015, Wiley Periodicals, Inc.

Active Play in After-school Programmes: development of an intervention and description of a matched-pair cluster-randomised trial assessing physical activity play in after-school programmes.

PubMed

Riiser, Kirsti; Helseth, Sølvi; Ellingsen, Hanna; Fallang, Bjørg; Løndal, Knut

2017-08-04

Interventions delivered in after-school programmes (ASPs) have the potential to become a means of ensuring adequate physical activity among schoolchildren. This requires a motivational climate, allowing for self-determined play. If trained, ASP staff may represent a valuable resource for supporting such play. Increasing knowledge and supportive skills among ASP staff may also potentially increase their motivation for work. The purpose of this article is to describe the development of the 'Active Play in ASP' intervention, which aims to promote physical activity among first graders attending ASP, and to present a protocol for a matched-pair cluster-randomised trial to evaluate the intervention. Informed by experiences from practice, evidence-based knowledge and theory, the intervention was developed in a stepwise process including focus group meetings and a small-scale pilot test. The intervention contains a course programme for ASP staff to increase their skills in how to support physical activity through play. In a cluster randomised controlled trial, the ASPs will be matched and randomly allocated to receive the 7-month intervention or to a control group. Outcomes will be assessed at baseline, after 7 and 19 months. First graders attending the ASPs included are eligible. The primary outcome will be accelerometer-determined minutes in moderate to vigorous physical activity in the ASP. The study uses a mixed methods approach including observations and interviews to provide rich descriptions of the concept of children's physical activity in ASP. Moreover, the trial will assess whether the ASP staff benefits from participation in the intervention in terms of increased work motivation. Lastly, process evaluations of programme fidelity, satisfaction and suggestions on improvement will be performed. The study is approved by the Data Protection Official for Research (reference no 46008). Results will be presented in conferences and peer-reviewed journals. Clinical Trials (NCT02954614), pre-results. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Insulin Degludec/Insulin Aspart Administered Once Daily at Any Meal, With Insulin Aspart at Other Meals Versus a Standard Basal-Bolus Regimen in Patients With Type 1 Diabetes

PubMed Central

Hirsch, Irl B.; Bode, Bruce; Courreges, Jean-Pierre; Dykiel, Patrik; Franek, Edward; Hermansen, Kjeld; King, Allen; Mersebach, Henriette; Davies, Melanie

2012-01-01

OBJECTIVE To evaluate efficacy and tolerability of a co-formulation of insulin degludec and insulin aspart (IDegAsp) with insulin aspart (IAsp) at other meals compared with basal-bolus therapy using insulin detemir (IDet) and IAsp. RESEARCH DESIGN AND METHODS Adults (n = 548) with type 1 diabetes (A1C 7.0–10.0%; BMI ≤35.0 kg/m2) were randomized 2:1 in a 26-week, multinational, parallel-group, treat-to-target trial to IDegAsp or IDet. IDegAsp was given with a meal, and IDet was given in the evening, with a second (breakfast) dose added if needed. RESULTS Non-inferiority for IDegAsp versus IDet was confirmed; A1C improved by 0.75% with IDegAsp and 0.70% with IDet to 7.6% in both groups (estimated treatment difference IDegAsp − IDet: –0.05% [95% CI –0.18 to 0.08]). There was no statistically significant difference between IDegAsp and IDet in the rates of severe hypoglycemia (0.33 and 0.42 episodes/patient-year, respectively) or overall confirmed (plasma glucose <3.1 mmol/L) hypoglycemia (39.17 and 44.34 episodes/patient-year, respectively). Nocturnal confirmed hypoglycemia rate was 37% lower with IDegAsp than IDet (3.71 vs. 5.72 episodes/patient-year, P < 0.05). Weight gain was 2.3 and 1.3 kg with IDegAsp and IDet, respectively (P < 0.05). Total insulin dose was 13% lower in the IDegAsp group (P < 0.0001). No treatment differences were detected in Health-Related Quality of Life, laboratory measurements, physical examination, vital signs, electrocardiograms, fundoscopy, or adverse events. CONCLUSIONS IDegAsp in basal-bolus therapy with IAsp at additional mealtimes improves overall glycemic control and was non-inferior to IDet, with a reduced risk of nocturnal hypoglycemia and fewer injections in comparison with IDet + IAsp basal-bolus therapy. PMID:22933438

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

PubMed

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Effects of aspirin on immobile behavior and endocrine and immune changes in the forced swimming test: comparison to fluoxetine and imipramine.

PubMed

Guan, Xi-ting; Shao, Feng; Xie, Xi; Chen, Lin; Wang, Weiwen

2014-09-01

Aspirin (ASP) is the most commonly used non-steroidal anti-inflammatory drug in the world. Recent clinical and preclinical evidence suggests that ASP may also exert psychoactive effects. It remains unclear whether ASP has antidepressant-like activity, and any molecular mechanisms underlying such activity have yet to be elucidated. Using the forced swimming test (FST), a well-established animal model of depression widely used to screen potential antidepressants in rodents, we investigated the effects of subacute treatment with ASP (0, 6, 12, 25, and 50mg/kg, i.p.) on immobility in the FST, and on FST-induced changes in endocrine and immune parameters in rats, in comparison to the clinical antidepressants imipramine (IMI) and fluoxetine (FLU). Serum levels of corticosterone, pro-inflammatory cytokine interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay. ASP dose-dependently decreased immobility in the FST, without altering the locomotor activity in the open-field test. The inhibitory effects of higher doses (25 and 50mg/kg) of ASP on immobility were similar to that of FLU and IMI at a dose of 10mg/kg. In addition, the levels of corticosterone, IL-6, and TNF-α in peripheral blood were significantly increased after the FST exposure. IMI, but not FLU and ASP at any dose tested, significantly attenuated corticosterone responses in the FST. Both FLU and IMI treatment reduced the increase of IL-6 and TNF-α levels following the FST exposure. ASP dose-dependently decreased FST-induced increase of cytokine levels, as manifested by significantly stronger effects on IL-6 and TNF-α levels at higher doses (25 and 50mg/kg) than the lowest dose of ASP (6 mg/kg). In all, these results indicate that ASP treatment dose-dependently decreased the immobility time and the release of pro-inflammatory cytokines in the FST, suggesting that the anti-inflammatory effects of ASP might be involved in the antidepressant-like effect. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family.

PubMed

Amiche, M; Seon, A A; Wroblewski, H; Nicolas, P

2000-07-01

A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four-step protocol. Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria) and Gram-positive eubacteria, and also, though to a lesser extent, towards Gram-negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Observation of bacterial cells using reflected light fluorescence microscopy after DNA-staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion-conducting channels through the plasma membrane. CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic alpha-helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms. PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain. Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino-acid substitutions in the preproregion of the brain precursor. The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N-terminal preproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, and to the D-amino-acid-containing opioid heptapeptides dermorphins and deltorphins. Because the structures and cidal mechanisms of dermatoxin, dermaseptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C-terminal domains of precursors of the dermaseptins family.

Characterizing the structural ensemble of γ-secretase using a multiscale molecular dynamics approach† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc00980a Click here for additional data file.

PubMed Central

Aguayo-Ortiz, Rodrigo; Chávez-García, Cecilia; Straub, John E.

2017-01-01

γ-Secretase is an intramembrane-cleaving aspartyl protease that plays an essential role in the processing of a variety of integral membrane proteins. Its role in the ultimate cleavage step in the processing of amyloid precursor protein to form amyloid-β (Aβ) peptide makes it an important therapeutic target in Alzheimer's disease research. Significant recent advances have been made in structural studies of this critical membrane protein complex. However, details of the mechanism of activation of the enzyme complex remain unclear. Using a multiscale computational modeling approach, combining multiple coarse-grained microsecond dynamic trajectories with all-atom models, the structure and two conformational states of the γ-secretase complex were evaluated. The transition between enzymatic state 1 and state 2 is shown to critically depend on the protonation states of the key catalytic residues Asp257 and Asp385 in the active site domain. The active site formation, related to our γ-secretase state 2, is observed to involve a concerted movement of four transmembrane helices from the catalytic subunit, resulting in the required localization of the catalytic residues. Global analysis of the structural ensemble of the enzyme complex was used to identify collective fluctuations important to the mechanism of substrate recognition and demonstrate that the corresponding fluctuations observed were uncorrelated with structural changes associated with enzyme activation. Overall, this computational study provides essential insight into the role of structure and dynamics in the activation and function of γ-secretase. PMID:28970936

The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin).

PubMed

Cutfield, J F; Cutfield, S M; Carne, A; Emdin, S O; Falkmer, S

1986-07-01

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse-phase high-performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N-terminal sequence for pancreatic polypeptide was determined. The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N-terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not readily form crystals containing zinc-insulin hexamers, despite the presence of the coordinating B10 His.

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations.

PubMed

Thompson, Damien; Lazennec, Christine; Plateau, Pierre; Simonson, Thomas

2008-05-15

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand. 2007 Wiley-Liss, Inc.

Aspergillus fumigatus proteases, Asp f 5 and Asp f 13, are essential for airway inflammation and remodelling in a murine inhalation model.

PubMed

Namvar, S; Warn, P; Farnell, E; Bromley, M; Fraczek, M; Bowyer, P; Herrick, S

2015-05-01

In susceptible individuals, exposure to Aspergillus fumigatus can lead to the development of atopic lung diseases such as allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitization (SAFS). Protease allergens including Asp f 5 and Asp f 13 from Aspergillus fumigatus are thought to be important for initiation and progression of allergic asthma. To assess the importance of secreted protease allergens Asp f 5 (matrix metalloprotease) and Asp f 13 (serine protease) in Aspergillus fumigatus-induced inflammation, airway hyperactivity, atopy and airway wall remodelling in a murine model following chronic exposure to secreted allergens. BALB/c mice were repeatedly intranasally dosed over the course of 5 weeks with culture filtrate from wild-type (WT), Asp f 5 null (∆5) or Asp f 13 null (∆13) strains of Aspergillus fumigatus. Airway hyper-reactivity was measured by non-invasive whole-body plethysmography, Th2 response and airway inflammation by ELISA and cell counts, whilst airway remodelling was assessed by histological analysis. Parent WT and ∆5 culture filtrates showed high protease activity, whilst protease activity in ∆13 culture filtrate was low. Chronic intranasal exposure to the three different filtrates led to comparable airway hyper-reactivity and Th2 response. However, protease allergen deleted strains, in particular ∆13 culture filtrate, induced significantly less airway inflammation and remodelling compared to WT culture filtrate. Aspergillus fumigatus-secreted allergen proteases, Asp f 5 and Asp f 13, are important for recruitment of inflammatory cells and remodelling of the airways in this murine model. However, deletion of a single allergen protease fails to alleviate airway hyper-reactivity and allergic immune response. Targeting protease activity of Aspergillus fumigatus in conditions such as SAFS or ABPA may have beneficial effects in preventing key aspects of airway pathology. © 2014 John Wiley & Sons Ltd.

Impact of antimicrobial stewardship programme on hospitalized patients at the intensive care unit: a prospective audit and feedback study.

PubMed

Khdour, Maher R; Hallak, Hussein O; Aldeyab, Mamoon A; Nasif, Mowaffaq A; Khalili, Aliaa M; Dallashi, Ahamad A; Khofash, Mohammad B; Scott, Michael G

2018-04-01

Inappropriate use of antibiotics is one of the most important factors contributing to the emergence of drug resistant pathogens. The purpose of this study was to measure the clinical impact of antimicrobial stewardship programme (ASP) interventions on hospitalized patients at the Intensive care unit at Palestinian Medical Complex. A prospective audit with intervention and feedback by ASP team within 48-72 h of antibiotic administration began in September 2015. Four months of pre-ASP data were compared with 4 months of post-ASP data. Data collected included clinical and demographic data; use of antimicrobials measured by defined daily doses, duration of therapy, length of stay, readmission and all-cause mortality. Overall, 176 interventions were made the ASP team with an average acceptance rate of 78.4%. The most accepted interventions were dose optimization (87.0%) followed by de-escalation based on culture results with an acceptance rate of 84.4%. ASP interventions significantly reduces antimicrobial use by 24.3% (87.3 defined daily doses/100 beds vs. 66.1 defined daily doses/100 beds P < 0.001). The median (interquartile range) of length of stay was significantly reduced post ASP [11 (3-21) vs. 7 (4-19) days; P < 0.01]. Also, the median (interquartile range) of duration of therapy was significantly reduced post-ASP [8 (5-12) days vs. 5 (3-9); P = 0.01]. There was no significant difference in overall 30-day mortality or readmission between the pre-ASP and post-ASP groups (26.9% vs. 23.9%; P = 0.1) and (26.1% vs. 24.6%; P = 0.54) respectively. Our prospective audit and feedback programme was associated with positive impact on antimicrobial use, duration of therapy and length of stay. © 2017 The British Pharmacological Society.

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

PubMed

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P < 0.05). However, RafCAAX mutant can enhance pcDI-[12Asp]K-ras4B cell growth (P < 0.05). (1) [12Asp]K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

Elucidating the role of many-body forces in liquid water. I. Simulations of water clusters on the VRT(ASP-W) potential surfaces.

PubMed

Goldman, Nir; Saykally, R J

2004-03-08

We test two new potentials for water, fit to vibration-rotation tunneling (VRT) data by employing diffusion quantum Monte Carlo simulations to calculate the vibrational ground-state properties of water clusters. These potentials, VRT(ASP-W)II and VRT(ASP-W)III, are fits of the highly detailed ASP-W (anisotropic site potential with Woermer dispersion) ab initio potential to (D(2)O)(2) microwave and far-infrared data, and along with the SAPT5s (five-site symmetry adapted perturbation theory) potentials, are the most accurate water dimer potential surfaces in the literature. The results from VRT(ASP-W)II and III are compared to those from the original ASP-W potential, the SAPT5s family of potentials, and several bulk water potentials. Only VRT(ASP-W)III and the spectroscopically "tuned" SAPT5st (with N-body induction included) accurately reproduce the vibrational ground-state structures of water clusters up to the hexamer. Finally, the importance of many-body induction and three-body dispersion are examined, and it is shown that the latter can have significant effects on water cluster properties despite its small magnitude.

Elucidating the role of many-body forces in liquid water. I. Simulations of water clusters on the VRT(ASP-W) potential surfaces

NASA Astrophysics Data System (ADS)

Goldman, Nir; Saykally, R. J.

2004-03-01

We test two new potentials for water, fit to vibration-rotation tunneling (VRT) data by employing diffusion quantum Monte Carlo simulations to calculate the vibrational ground-state properties of water clusters. These potentials, VRT(ASP-W)II and VRT(ASP-W)III, are fits of the highly detailed ASP-W (anisotropic site potential with Woermer dispersion) ab initio potential to (D2O)2 microwave and far-infrared data, and along with the SAPT5s (five-site symmetry adapted perturbation theory) potentials, are the most accurate water dimer potential surfaces in the literature. The results from VRT(ASP-W)II and III are compared to those from the original ASP-W potential, the SAPT5s family of potentials, and several bulk water potentials. Only VRT(ASP-W)III and the spectroscopically "tuned" SAPT5st (with N-body induction included) accurately reproduce the vibrational ground-state structures of water clusters up to the hexamer. Finally, the importance of many-body induction and three-body dispersion are examined, and it is shown that the latter can have significant effects on water cluster properties despite its small magnitude.

Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

PubMed

Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M; Yue, Junming

2017-01-01

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. Results Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. Conclusions This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes. PMID:22394504

Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells

PubMed Central

Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M.; Yue, Junming

2017-01-01

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells. PMID:28123598

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.

PubMed

Fukasawa, Kayoko M; Hirose, Junzo; Hata, Toshiyuki; Ono, Yukio

2006-09-26

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.

Peptide de novo sequencing of mixture tandem mass spectra.

PubMed

Gorshkov, Vladimir; Hotta, Stéphanie Yuki Kolbeck; Verano-Braga, Thiago; Kjeldsen, Frank

2016-09-01

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20-35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Steiner, B.; Cousot, D.; Trzeciak, A.

The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single bindingmore » site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.« less

Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae

PubMed Central

2013-01-01

Background Secondary metabolite production, a hallmark of filamentous fungi, is an expanding area of research for the Aspergilli. These compounds are potent chemicals, ranging from deadly toxins to therapeutic antibiotics to potential anti-cancer drugs. The genome sequences for multiple Aspergilli have been determined, and provide a wealth of predictive information about secondary metabolite production. Sequence analysis and gene overexpression strategies have enabled the discovery of novel secondary metabolites and the genes involved in their biosynthesis. The Aspergillus Genome Database (AspGD) provides a central repository for gene annotation and protein information for Aspergillus species. These annotations include Gene Ontology (GO) terms, phenotype data, gene names and descriptions and they are crucial for interpreting both small- and large-scale data and for aiding in the design of new experiments that further Aspergillus research. Results We have manually curated Biological Process GO annotations for all genes in AspGD with recorded functions in secondary metabolite production, adding new GO terms that specifically describe each secondary metabolite. We then leveraged these new annotations to predict roles in secondary metabolism for genes lacking experimental characterization. As a starting point for manually annotating Aspergillus secondary metabolite gene clusters, we used antiSMASH (antibiotics and Secondary Metabolite Analysis SHell) and SMURF (Secondary Metabolite Unknown Regions Finder) algorithms to identify potential clusters in A. nidulans, A. fumigatus, A. niger and A. oryzae, which we subsequently refined through manual curation. Conclusions This set of 266 manually curated secondary metabolite gene clusters will facilitate the investigation of novel Aspergillus secondary metabolites. PMID:23617571

Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68.

PubMed

Pons, Tirso; Naumoff, Daniil G; Martínez-Fleites, Carlos; Hernández, Lázaro

2004-02-15

Multiple-sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes. A detailed analysis of the site-directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis. These residues are close in space in the 5-bladed beta-propeller fold determined for Cellvibrio japonicus alpha-L-arabinanase Arb43A [Nurizzo et al., Nat Struct Biol 2002;9:665-668] and Bacillus subtilis endo-1,5-alpha-L-arabinanase. A sequence-structure compatibility search using 3D-PSSM, mGenTHREADER, INBGU, and SAM-T02 programs predicted indistinctly the 5-bladed beta-propeller fold of Arb43A and the 6-bladed beta-propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68. We conclude that the identified acidic residues are located at the active site of a beta-propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration. Also, we propose that the beta-propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction. Copyright 2003 Wiley-Liss, Inc.

Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients.

PubMed

Poyau, A; Buchet, K; Bouzidi, M F; Zabot, M T; Echenne, B; Yao, J; Shoubridge, E A; Godinot, C

2000-02-01

We have studied the fibroblasts of three patients suffering from Leigh syndrome associated with cytochrome c oxidase deficiency (LS-COX-). Their mitochondrial DNA was functional and all nuclear COX subunits had a normal sequence. The expression of transcripts encoding mitochondrial and nuclear COX subunits was normal or slightly increased. Similarly, the OXA1 transcript coding for a protein involved in COX assembly was increased. However, several COX-protein subunits were severely depressed, indicating deficient COX assembly. Surf1, a factor involved in COX biogenesis, was recently reported as mutated in LS-COX- patients, all mutations predicting a truncated protein. Sequence analysis of SURF1 gene in our three patients revealed seven heterozygous mutations, six of which were new : an insertion, a nonsense mutation, a splicing mutation of intron 7 in addition to three missense mutations. The mutation G385 A (Gly124-->Glu) changes a Gly that is strictly conserved in Surfl homologs of 12 species. The substitution G618 C (Asp202-->His), changing an Asp that is conserved only in mammals, appears to be a polymorphism. The mutation T751 C changes Ile246 to Thr, a position at which a hydrophobic amino acid is conserved in all eukaryotic and some bacterial species. Replacing Ile246 by Thr disrupts a predicted beta sheet structure present in all higher eukaryotes. COX activity could be restored in fibroblasts of the three patients by complementation with a retroviral vector containing normal SURF1 cDNA. These mutations identify domains essential to Surf1 protein structure and/or function.

Proton conduction within the reaction centers of Rhodobacter capsulatus: the electrostatic role of the protein.

PubMed

Maróti, P; Hanson, D K; Baciou, L; Schiffer, M; Sebban, P

1994-06-07

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor QB. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213-->Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from QB (class 3 strain, Ala-Ala + Arg-M231-->Leu; class 4 strain, Ala-Ala + Asn-M43-->Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to QB compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain > 2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (> or = 60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.

Proteolytic events in the post-translational processing of somatostatin precursors from rat brain cortex and anglerfish pancreatic islets.

PubMed

Cohen, P; Morel, A; Gluschankof, P; Gomez, S; Nicolas, P

1985-01-01

An Arg-Lys esteropeptidase which converts somatostatin-28 (S-28) into somatostatin-14 (S-14) was detected in rat brain cortical extracts using a synthetic undecapeptide substrate mimicking the octacosapeptide sequence at the restriction site. This enzyme system was unable to release either the octacosapeptide or S-14 from the 15,000 mol wt (15K) rat hypothalamic precursor. This argues in favor of sequential degradation of the precursor into S-14 via S-28 as an obligatory intermediate. Another in vivo processing system was analyzed in the anglerfish pancreatic Brockmann organs. Here, cloning of two cDNA corresponding to two mRNA species predicts two distinct somatostatins precursors, called prosomatostatins I and II (Hobart et al., Nature 288:137, 1980). While a single S-14 can be detected in extracts made from this pancreatic tissue, indistinguishable from the mammalian species, two S-28 species could be separated by HPLC. Immunochemical and biochemical evidence indicates that the second species should correspond to anglerfish S-28 (AF S-28), the product of prosomatostatin-II processing in vivo. Amino acid analysis, together with the determined complete amino acid sequence of this peptide, demonstrates that this is indeed the case and that AF S-28 contains in its C-terminal half the [Tyr7, Gly10] derivative of S-14. These observations give an example of a AF S-28 being a terminal active product of prosomatostatin processing. They suggest that this octacosapeptide, which is potent on the inhibition of growth hormone release by anterior pituitary cells, may play such a role in the gastrointestinal tract of the anglerfish. These results, while not excluding alternative routes, give support to a sequential processing of the 15 K precursor----S-28----S-14.

Ground state destabilization from a positioned general base in the ketosteroid isomerase active site.

PubMed

Ruben, Eliza A; Schwans, Jason P; Sonnett, Matthew; Natarajan, Aditya; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

2013-02-12

We compared the binding affinities of ground state analogues for bacterial ketosteroid isomerase (KSI) with a wild-type anionic Asp general base and with uncharged Asn and Ala in the general base position to provide a measure of potential ground state destabilization that could arise from the close juxtaposition of the anionic Asp and hydrophobic steroid in the reaction's Michaelis complex. The analogue binding affinity increased ~1 order of magnitude for the Asp38Asn mutation and ~2 orders of magnitude for the Asp38Ala mutation, relative to the affinity with Asp38, for KSI from two sources. The increased level of binding suggests that the abutment of a charged general base and a hydrophobic steroid is modestly destabilizing, relative to a standard state in water, and that this destabilization is relieved in the transition state and intermediate in which the charge on the general base has been neutralized because of proton abstraction. Stronger binding also arose from mutation of Pro39, the residue adjacent to the Asp general base, consistent with an ability of the Asp general base to now reorient to avoid the destabilizing interaction. Consistent with this model, the Pro mutants reduced or eliminated the increased level of binding upon replacement of Asp38 with Asn or Ala. These results, supported by additional structural observations, suggest that ground state destabilization from the negatively charged Asp38 general base provides a modest contribution to KSI catalysis. They also provide a clear illustration of the well-recognized concept that enzymes evolve for catalytic function and not, in general, to maximize ground state binding. This ground state destabilization mechanism may be common to the many enzymes with anionic side chains that deprotonate carbon acids.

Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.

PubMed

Zhang, Jun-Hui; Jiang, Yu-Yan; Lin, Ying; Sun, Yu-Fei; Zheng, Sui-Ping; Han, Shuang-Yan

2013-01-01

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.

Probing the Catalytic Mechanism of Vibrio harveyi GH20 β-N-Acetylglucosaminidase by Chemical Rescue

PubMed Central

Meekrathok, Piyanat; Suginta, Wipa

2016-01-01

Background Vibrio harveyi GH20 β-N-acetylglucosaminidase (VhGlcNAcase) is a chitinolytic enzyme responsible for the successive degradation of chitin fragments to GlcNAc monomers, activating the onset of the chitin catabolic cascade in marine Vibrios. Methods Two invariant acidic pairs (Asp303-Asp304 and Asp437-Glu438) of VhGlcNAcase were mutated using a site-directed mutagenesis strategy. The effects of these mutations were examined and the catalytic roles of these active-site residues were elucidated using a chemical rescue approach. Enhancement of the enzymic activity of the VhGlcNAcase mutants was evaluated by a colorimetric assay using pNP-GlcNAc as substrate. Results Substitution of Asp303, Asp304, Asp437 or Glu438 with Ala/Asn/Gln produced a dramatic loss of the GlcNAcase activity. However, the activity of the inactive D437A mutant was recovered in the presence of sodium formate. Our kinetic data suggest that formate ion plays a nucleophilic role by mimicking the β-COO-side chain of Asp437, thereby stabilizing the reaction intermediate during both the glycosylation and the deglycosylation steps. Conclusions Chemical rescue of the inactive D437A mutant of VhGlcNAcase by an added nucleophile helped to identify Asp437 as the catalytic nucleophile/base, and hence its acidic partner Glu438 as the catalytic proton donor/acceptor. General Significance Identification of the catalytic nucleophile of VhGlcNAcases supports the proposal of a substrate-assisted mechanism of GH20 GlcNAcases, requiring the catalytic pair Asp437-Glu438 for catalysis. The results suggest the mechanistic basis of the participation of β-N-acetylglucosaminidase in the chitin catabolic pathway of marine Vibrios. PMID:26870945

D-Aspartic acid and nitric oxide as regulators of androgen production in boar testis.

PubMed

Lamanna, Claudia; Assisi, Loredana; Vittoria, Alfredo; Botte, Virgilio; Di Fiore, Maria Maddalena

2007-01-15

D-Aspartic acid (D-Asp) and nitric oxide (NO) are two biologically active molecules playing important functions as neurotransmitters and neuromodulators of nerve impulse and as regulators of hormone production by endocrine organs. We studied the occurrence of D-Asp and NO as well as their effects on testosterone synthesis in the testis of boar. This model was chosen for our investigations because it contains more Leydig cells than other mammals. Indirect immunofluorescence applied to cryostat sections was used to evaluate the co-localization of D-Asp and of the enzyme nitric oxide synthase (NOS) in the same Leydig cells. D-Asp and NOS often co-existed in the same Leydig cells and were found, separately, in many other testicular cytotypes. D-Asp level was dosed by an enzymatic method performed on boar testis extracts and was 40+/-3.6 nmol/g of fresh tissue. NO measurement was carried out using a biochemical method by NOS activity determination and expressed as quantity of nitrites produced: it was 155.25+/-21.9 nmol/mg of tissue. The effects of the two molecules on steroid hormone production were evaluated by incubating testis homogenates, respectively with or without D-Asp and/or the NO-donor L-arginine (L-Arg). After incubation, the testosterone presence was measured by immunoenzymatic assay (EIA). These in vitro experiments showed that the addition of D-Asp to incubated testicular homogenates significantly increased testosterone concentration, whereas the addition of L-Arg decreased the hormone production. Moreover, the inclusion of L-Arg to an incubation medium of testicular homogenates with added D-Asp, completely inhibited the stimulating effects of this enantiomer. Our results suggest an autocrine action of both D-Asp and NO on the steroidogenetic activity of the Leydig cell.

Aspartic acid functions as carbonyl trapper to inhibit the formation of advanced glycation end products by chemical chaperone activity.

PubMed

Prasanna, Govindarajan; Saraswathi, N T

2016-05-01

Advanced glycation end products (AGEs) were implicated in pathology of numerous diseases. In this study, we present the bioactivity of aspartic acid (Asp) to inhibit the AGEs. Hemoglobin and bovine serum albumin (BSA) were glycated with glucose, fructose, and ribose in the presence and absence of Asp (100-200 μM). HbA1c inhibition was investigated using human blood and characterized by micro-column ion exchange chromatography. The effect of methyl glyoxal (MG) on hemoglobin and BSA was evaluated by fluorescence spectroscopy and gel electrophoresis. The effect of MG on red blood cells morphology was characterized by scanning electron micrographs. Molecular docking was performed on BSA with Asp. Asp is capable of inhibiting the formation of fluorescent AGEs by reacting with the reducing sugars. The presence of Asp as supplement in whole blood reduced the HbA1c% from 8.8 to 6.1. The presence of MG showed an increase in fluorescence and the presence of Asp inhibited the glycation thereby the fluorescence was quenched. MG also affected the electrophoretic mobility of hemoglobin and BSA by forming high molecular weight aggregates. Normal RBCs showed typical biconcave shape. MG modified RBCs showed twisted and elongated shape whereas the presence of ASP tends to protect RBC from twisting. Asp interacted with arginine residues of bovine serum albumin particularly ARG 194, ARG 198, and ARG 217 thereby stabilized the protein complex. We conclude that Asp has dual functions as a chemical chaperone to stabilize protein and as a dicarbonyl trapper, and thereby it can prevent the complications caused by glycation.

Neural network versus activity-specific prediction equations for energy expenditure estimation in children.

PubMed

Ruch, Nicole; Joss, Franziska; Jimmy, Gerda; Melzer, Katarina; Hänggi, Johanna; Mäder, Urs

2013-11-01

The aim of this study was to compare the energy expenditure (EE) estimations of activity-specific prediction equations (ASPE) and of an artificial neural network (ANNEE) based on accelerometry with measured EE. Forty-three children (age: 9.8 ± 2.4 yr) performed eight different activities. They were equipped with one tri-axial accelerometer that collected data in 1-s epochs and a portable gas analyzer. The ASPE and the ANNEE were trained to estimate the EE by including accelerometry, age, gender, and weight of the participants. To provide the activity-specific information, a decision tree was trained to recognize the type of activity through accelerometer data. The ASPE were applied to the activity-type-specific data recognized by the tree (Tree-ASPE). The Tree-ASPE precisely estimated the EE of all activities except cycling [bias: -1.13 ± 1.33 metabolic equivalent (MET)] and walking (bias: 0.29 ± 0.64 MET; P < 0.05). The ANNEE overestimated the EE of stationary activities (bias: 0.31 ± 0.47 MET) and walking (bias: 0.61 ± 0.72 MET) and underestimated the EE of cycling (bias: -0.90 ± 1.18 MET; P < 0.05). Biases of EE in stationary activities (ANNEE: 0.31 ± 0.47 MET, Tree-ASPE: 0.08 ± 0.21 MET) and walking (ANNEE 0.61 ± 0.72 MET, Tree-ASPE: 0.29 ± 0.64 MET) were significantly smaller in the Tree-ASPE than in the ANNEE (P < 0.05). The Tree-ASPE was more precise in estimating the EE than the ANNEE. The use of activity-type-specific information for subsequent EE prediction equations might be a promising approach for future studies.

Exploiting excess sharing: a more powerful test of linkage for affected sib pairs than the transmission/disequilibrium test.

PubMed Central

Wicks, J

2000-01-01

The transmission/disequilibrium test (TDT) is a popular, simple, and powerful test of linkage, which can be used to analyze data consisting of transmissions to the affected members of families with any kind pedigree structure, including affected sib pairs (ASPs). Although it is based on the preferential transmission of a particular marker allele across families, it is not a valid test of association for ASPs. Martin et al. devised a similar statistic for ASPs, Tsp, which is also based on preferential transmission of a marker allele but which is a valid test of both linkage and association for ASPs. It is, however, less powerful than the TDT as a test of linkage for ASPs. What I show is that the differences between the TDT and Tsp are due to the fact that, although both statistics are based on preferential transmission of a marker allele, the TDT also exploits excess sharing in identity-by-descent transmissions to ASPs. Furthermore, I show that both of these statistics are members of a family of "TDT-like" statistics for ASPs. The statistics in this family are based on preferential transmission but also, to varying extents, exploit excess sharing. From this family of statistics, we see that, although the TDT exploits excess sharing to some extent, it is possible to do so to a greater extent-and thus produce a more powerful test of linkage, for ASPs, than is provided by the TDT. Power simulations conducted under a number of disease models are used to verify that the most powerful member of this family of TDT-like statistics is more powerful than the TDT for ASPs. PMID:10788332

Exploiting excess sharing: a more powerful test of linkage for affected sib pairs than the transmission/disequilibrium test.

PubMed

Wicks, J

2000-06-01

The transmission/disequilibrium test (TDT) is a popular, simple, and powerful test of linkage, which can be used to analyze data consisting of transmissions to the affected members of families with any kind pedigree structure, including affected sib pairs (ASPs). Although it is based on the preferential transmission of a particular marker allele across families, it is not a valid test of association for ASPs. Martin et al. devised a similar statistic for ASPs, Tsp, which is also based on preferential transmission of a marker allele but which is a valid test of both linkage and association for ASPs. It is, however, less powerful than the TDT as a test of linkage for ASPs. What I show is that the differences between the TDT and Tsp are due to the fact that, although both statistics are based on preferential transmission of a marker allele, the TDT also exploits excess sharing in identity-by-descent transmissions to ASPs. Furthermore, I show that both of these statistics are members of a family of "TDT-like" statistics for ASPs. The statistics in this family are based on preferential transmission but also, to varying extents, exploit excess sharing. From this family of statistics, we see that, although the TDT exploits excess sharing to some extent, it is possible to do so to a greater extent-and thus produce a more powerful test of linkage, for ASPs, than is provided by the TDT. Power simulations conducted under a number of disease models are used to verify that the most powerful member of this family of TDT-like statistics is more powerful than the TDT for ASPs.

Phylogenetic appearance of Neuropeptide S precursor proteins in tetrapods

PubMed Central

Reinscheid, Rainer K.

2007-01-01

Sleep and emotional behavior are two hallmarks of vertebrate animal behavior, implying that specialized neuronal circuits and dedicated neurochemical messengers may have been developed during evolution to regulate such complex behaviors. Neuropeptide S (NPS) is a newly identified peptide transmitter that activates a typical G protein-coupled receptor. Central administration of NPS produces profound arousal, enhances wakefulness and suppresses all stages of sleep. In addition, NPS can alleviate behavioral responses to stress by producing anxiolytic-like effects. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates with the exception of fish. A high level of sequence conservation, especially of aminoterminal structures was detected, indicating stringent requirements for agonist-induced receptor activation. Duplication of the NPS precursor gene was only found in one out of two marsupial species with sufficient genome coverage (Monodelphis domestica; opossum), indicating that the duplicated opossum NPS sequence might have arisen as an isolated event. Pharmacological analysis of both Monodelphis NPS peptides revealed that only the closely related NPS peptide retained agonistic activity at NPS receptors. The duplicated precursor might be either a pseudogene or could have evolved different receptor selectivity. Together, these data show that NPS is a relatively recent gene in vertebrate evolution whose appearance might coincide with its specialized physiological functions in terrestrial vertebrates. PMID:17293003

R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

PubMed

Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

2016-01-01

The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.

A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance.

PubMed

Ali, Akhtar; Raddatz, Natalia; Aman, Rashid; Kim, Songmi; Park, Hyeong Cheol; Jan, Masood; Baek, Dongwon; Khan, Irfan Ullah; Oh, Dong-Ha; Lee, Sang Yeol; Bressan, Ray A; Lee, Keun Woo; Maggio, Albino; Pardo, Jose M; Bohnert, Hans J; Yun, Dae-Jin

2016-07-01

A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K(+) TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na(+) from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K(+) transporter in the presence of Na(+) in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1(N-D)) complemented K(+)-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1(N) (-) (D) and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1 Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na(+) and K(+) based on the n/d variance in the pore region. This change also dictated inward-rectification for Na(+) transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats. © 2016 American Society of Plant Biologists. All Rights Reserved.

The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences.

PubMed

Bossak, Karolina; Drew, Simon C; Stefaniak, Ewelina; Płonka, Dawid; Bonna, Arkadiusz; Bal, Wojciech

2018-05-01

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 10 13  M -1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 10 14  M -1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function. Copyright © 2018 Elsevier Inc. All rights reserved.

Distribution of Diego blood group alleles and identification of four novel mutations on exon 19 of SLC4A1 gene in the Chinese Han population by polymerase chain reaction sequence-based typing.

PubMed

Xu, X G; He, J; He, Y M; Tao, S D; Ying, Y L; Zhu, F M; Lv, H J; Yan, L X

2011-04-01

The Diego blood group system plays an important role in transfusion medicine. Genotyping of DI1 and DI2 alleles is helpful for the investigation into haemolytic disease of the newborn (HDN) and for the development of rare blood group databases. Here, we set up a polymerase chain reaction sequence-based typing (PCR-SBT) method for genotyping of Diego blood group alleles. Specific primers for exon 19 of the solute carrier family 4, anion exchanger, member1 (SLC4A1) gene were designed, and our PCR-SBT method was established and optimized for Diego genotyping. A total of 1053 samples from the Chinese Han population and the family members of a rare proband with DI1/DI1 genotype were investigated by the PCR-SBT method. An allele-specific primer PCR (PCR-ASP) was used to verify the reliability of the PCR-SBT method. The frequencies of DI1 and DI2 alleles in the Chinese Han population were 0.0247 and 0.9753, respectively. Six new single nucleotide polymorphisms (SNPs) were found in the sequenced regions of the SLC4A1 gene, and four novel SNPs located in the exon 19, in which one SNP could cause an amino acid alteration of Ala858Ser on erythrocyte anion exchanger protein 1. The genotypes for Diego blood group were identical among 41 selected samples with PCR-ASP and PCR-SBT. The PCR-SBT method can be used in Diego genotyping as a substitute of serological technique when the antisera is lacking and was suitable for screening large numbers of donors in rare blood group databases. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

PubMed Central

Batista, F R; Hernández, L; Fernández, J R; Arrieta, J; Menéndez, C; Gómez, R; Támbara, Y; Pons, T

1999-01-01

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases. PMID:9895294

Multinational Consensus: Insulin Initiation with Insulin Degludec/Aspart (IDegAsp).

PubMed

Kalra, Sanjay; Atkin, Stephen; Cervera, Antonio; Das, Ashok Kumar; Demir, Ozgur; Demir, Tevfik; Fariduddin, Md; Vo, Khoa Tuan; Ku, Bon Jeong; Kumar, Ajay; Latif, Zafar A; Malek, Rachid; Matawaran, Bien J; Mehta, Roopa; Tran, Nam Quang; Panelo, Araceli; Ruder, Sundeep; Saldana, Joel Rodriquez; Shaikh, Khalid A; Shakya, Amit; Shrestha, Dina; Unnikrishnan, A G

2018-05-23

Insulin degludec/aspart (IDegAsp) is the first soluble insulin co-formulation, combining a long-acting insulin degludec (IDeg) and rapid-acting insulin aspart (IAsp). In type 2 diabetes patients with oral antidiabetes agent (OAD) inadequacy, insulin initiation with IDegAsp once daily provides superior long-term glycemic control compared to insulin glargine, with similar fasting plasma glucose (FPG) and insulin doses, and numerically lower rates of overall and nocturnal hypoglycemia. Furthermore, in patients with uncontrolled type 2 diabetes previously treated with insulins, IDegAsp twice daily effectively improves glycated hemoglobin and FPG, with fewer hypoglycemic episodes versus premix insulins and basal bolus therapy. In patients with type 1 diabetes mellitus, IDegAsp once daily with two doses of IAsp is a convenient, yet effective, regimen as compared to the conventional 4-5 injection-based basal bolus therapy. IDegAsp is an appropriate and reasonable option for initiation of insulin therapy in both type 1 and type 2 diabetes.

Face-selective crystal growth behavior of L-aspartic acid in the presence of L-asparagine

NASA Astrophysics Data System (ADS)

Sato, Hiroyasu; Doki, Norihito; Yoshida, Saki; Yokota, Masaaki; Shimizu, Kenji

2016-02-01

The kinetic mechanism of L-asparagine (L-Asn) action on L-aspartic acid (L-Asp) crystal growth, namely the face-selective effect of L-Asn on the L-Asp crystal growth rate in each direction, was examined. In the a-axis direction, the effect of L-Asn on the L-Asp crystal growth rate was small. Enhancement and inhibition of L-Asp crystal growth, and interestingly the dissolution of the L-Asp crystal face, were observed in the b-axis direction, depending on the amount of L-Asn added. In the c-axis direction, the L-Asp crystal growth rate decreased with the increase in the amount of L-Asn added, and the experimental results were well fitted with a Langmuir adsorption isotherm. The study showed that there were crystal growth conditions where enhancement and inhibition, as well as inhibition and dissolution, coexisted in the presence of an additive with a structure similar to the growing crystal.

Lack of association between the APEX1 Asp148Glu polymorphism and sporadic amyotrophic lateral sclerosis.

PubMed

Coppedè, Fabio; Lo Gerfo, Annalisa; Carlesi, Cecilia; Piazza, Selina; Mancuso, Michelangelo; Pasquali, Livia; Murri, Luigi; Migliore, Lucia; Siciliano, Gabriele

2010-02-01

Impairments in DNA repair enzymes have been observed in amyotrophic lateral sclerosis (ALS) tissues, particularly in the activity of the apurinic/apyrimidinic endonuclease 1 (APEX1). Moreover, it was suggested that the common APEX1 Asp148Glu polymorphism might be associated with ALS risk. To further address this question we performed the present study aimed at evaluating the contribution of the APEX1 Asp148Glu polymorphism in sporadic ALS (sALS) risk and clinical presentation, including age and site of onset and disease progression. We screened 134 sALS Italian patients and 129 matched controls for the presence of the APEX1 Asp148Glu polymorphism. No difference in APEX1 Asp148Glu allele and genotype frequencies was found between the groups, nor was the polymorphism associated with age and site of onset or disease progression. Present results do not support a role for the APEX1 Asp148Glu polymorphism in sALS pathogenesis in the Italian population.

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

PubMed Central

Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

2014-01-01

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

Hydrogeochemical changes before and during the 2016 Amatrice-Norcia seismic sequence (central Italy).

PubMed

Barberio, Marino Domenico; Barbieri, Maurizio; Billi, Andrea; Doglioni, Carlo; Petitta, Marco

2017-09-15

Seismic precursors are an as yet unattained frontier in earthquake studies. With the aim of making a step towards this frontier, we present a hydrogeochemical dataset associated with the 2016 Amatrice-Norcia seismic sequence (central Apennines, Italy), developed from August 24 th , with an M w 6.0 event, and culminating on October 30 th , with an M w 6.5 mainshock. The seismic sequence occurred during a seasonal depletion of hydrostructures, and the four strongest earthquakes (M w  ≥ 5.5) generated an abrupt uplift of the water level, recorded up to 100 km away from the mainshock area. Monitoring a set of selected springs in the central Apennines, a few hydrogeochemical anomalies were observed months before the onset of the seismic swarm, including a variation of pH values and an increase of As, V, and Fe concentrations. Cr concentrations increased immediately after the onset of the seismic sequence. On November 2016, these elements recovered to their usual low concentrations. We interpret these geochemical anomalies as reliable seismic precursors for a dilational tectonic setting.

Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus

PubMed Central

Oroszlan, Stephen; Henderson, Louis E.; Stephenson, John R.; Copeland, Terry D.; Long, Cedric W.; Ihle, James N.; Gilden, Raymond V.

1978-01-01

The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the gag gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH2 terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous gag gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the gag gene is proposed. PMID:206897

The antimicrobial stewardship program in Gulf Cooperation Council (GCC) states: insights from a regional survey.

PubMed

Enani, Mushira A

2016-01-01

The purpose of the current study is to describe the prevalence and characteristics of antimicrobial stewardship programs (ASP) in Gulf Cooperation Council (GCC) states to explore opportunities and overcome barriers to effective ASP implementation. A cross-sectional questionnaire survey was used to evaluate the current status of ASP: major stewardship components, barriers of implementation and program impact in acute care hospitals of GCC states. Forty-seven healthcare professionals responded from four GCC states, the majority from Saudi Arabia (81%). Twenty-nine (62%) participating hospitals had ASP in place. Of these established programs, 35 (75%) reported lack of funding and personnel as major barriers to program implementation. The top three objectives cited for the hospital ASP were to reduce resistance (72.3%), improve clinical outcomes (70.2%) and reduce costs (44.7%). The reported impact of existing ASP was reduction of inappropriate prescribing (68%), reduction of broad spectrum antibiotic use (63.8%), reduction of healthcare-associated infections (61.7%), reduction of length of stay or mortality metrics (59.6%), reduction in direct antibiotic cost (57.4%) and reduction of reported antibiotic resistance (55.3%). Survey participants from GCC states who have implemented ASP report significant impacts in the reduction of broad spectrum antibiotic use, hospital-acquired infection, inappropriate prescribing and antimicrobial resistance. These findings suggest a promising opportunity to enhance existing ASP through sharing of best practices and support the development of regional guidelines across GCC states.

The antimicrobial stewardship program in Gulf Cooperation Council (GCC) states: insights from a regional survey

PubMed Central

2015-01-01

Objectives: The purpose of the current study is to describe the prevalence and characteristics of antimicrobial stewardship programs (ASP) in Gulf Cooperation Council (GCC) states to explore opportunities and overcome barriers to effective ASP implementation. Methods: A cross-sectional questionnaire survey was used to evaluate the current status of ASP: major stewardship components, barriers of implementation and program impact in acute care hospitals of GCC states. Results: Forty-seven healthcare professionals responded from four GCC states, the majority from Saudi Arabia (81%). Twenty-nine (62%) participating hospitals had ASP in place. Of these established programs, 35 (75%) reported lack of funding and personnel as major barriers to program implementation. The top three objectives cited for the hospital ASP were to reduce resistance (72.3%), improve clinical outcomes (70.2%) and reduce costs (44.7%). The reported impact of existing ASP was reduction of inappropriate prescribing (68%), reduction of broad spectrum antibiotic use (63.8%), reduction of healthcare-associated infections (61.7%), reduction of length of stay or mortality metrics (59.6%), reduction in direct antibiotic cost (57.4%) and reduction of reported antibiotic resistance (55.3%). Conclusion: Survey participants from GCC states who have implemented ASP report significant impacts in the reduction of broad spectrum antibiotic use, hospital-acquired infection, inappropriate prescribing and antimicrobial resistance. These findings suggest a promising opportunity to enhance existing ASP through sharing of best practices and support the development of regional guidelines across GCC states. PMID:28989448

The volitional inhibition of anticipatory ocular pursuit using a stop signal.

PubMed

Jarrett, Christian Beresford; Barnes, Graham R

2003-10-01

Unlike limb movements, smooth pursuit eye movements cannot normally be performed in the absence of a target. However, when subjects have a high expectancy of an imminent target appearance, the situation changes, and anticipatory smooth pursuit (ASP) tends to precede target onset by several hundred milliseconds. The velocity of this ASP is scaled predictively according to expected target velocity. And when an upcoming target is unexpectedly altered, or fails to appear, ASP continues regardless for approximately 150-200 ms before modification by visual feedback begins [J. Neurophysiol., 84 (2000) 2340]. These and other observations led to the earlier suggestion that ASP might be ballistic, being pre-programmed from start to finish. Two experiments with different timing parameters were therefore performed to test this hypothesis using a version of Logan's [Psychol. Rev., 91 (1984) 295] stop signal task. The aim was to test whether ASP could be stopped at will, and if so, whether the time taken to stop varied as a function of the time since ASP onset. Results showed that in response to a stop signal, ASP can be inhibited at any point in its trajectory, and for the majority of subjects in experiment 1, and all the subjects in experiment 2, with a latency that does not change significantly with target speed or time since ASP onset. These results provide the first demonstration that anticipatory movements can be stopped volitionally in response to a stop signal. Possible cognitive and neurophysiological mechanisms underlying this process are discussed.

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Ameliorative effect of Pimpinella anisum oil on immunohistochemical and ultrastuctural changes of cerebellum of albino rats induced by aspartame.

PubMed

Abdul-Hamid, Manal; Gallaly, Sanaa Rida

2014-05-01

The study aims to investigate the protective effect of Pimpinella anisum oil on aspartame (ASP) which resulted in cerebellar changes. The rats were divided into four equal groups: Group 1: (control group): served as control animals. Group 2: control P. anisum oil received .5 mL/kg/d/b wt. once daily. Group 3 (ASP group): received daily 250 mg/kg/b wt. of ASP dissolved in distilled water and given orally to the animals by intra-gastric tube for 2 months. Group 4: received .5 mL/kg/b wt. of prophylactic P. anisum oil once daily, followed by ASP after 2 h for 2 months. The histopathological approach revealed marked changes in the Purkinje cells, myleinated nerve fibers and granular cells of ASP-treated animals. Some of these cells appeared with deeply stained cytoplasm. Ultrastructural examination showed Purkinje cells with dilated rough endoplasmic reticulum and condensed mitochondria. Granular cells appeared with less c nuclei and surrounded by dissolution of most Mossy rosettes structures. Most myelinated nerve fibers showed thickening of myelinated sheath and others showed splitting of their myelin sheath. The histopathological, immunohistochemical and ultrastructural alterations were much less observed in concomitant use of P. anisum oil with ASP. Cerebellar cortex is considered target areas of ASP neurotoxicity, while P. anisum oil, when used in combination with ASP displays a protective action against neurotoxicity.

ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide.

PubMed

Dubreuil, P; Fulcrand, P; Rodriguez, M; Laur, J; Bali, J P; Martinez, J

1990-06-19

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.

An Approach for Peptide Identification by De Novo Sequencing of Mixture Spectra.

PubMed

Liu, Yi; Ma, Bin; Zhang, Kaizhong; Lajoie, Gilles

2017-01-01

Mixture spectra occur quite frequently in a typical wet-lab mass spectrometry experiment, which result from the concurrent fragmentation of multiple precursors. The ability to efficiently and confidently identify mixture spectra is essential to alleviate the existent bottleneck of low mass spectra identification rate. However, most of the traditional computational methods are not suitable for interpreting mixture spectra, because they still take the assumption that the acquired spectra come from the fragmentation of a single precursor. In this manuscript, we formulate the mixture spectra de novo sequencing problem mathematically, and propose a dynamic programming algorithm for the problem. Additionally, we use both simulated and real mixture spectra data sets to verify the merits of the proposed algorithm.

Embedding Positive Behavior Intervention and Supports in Afterschool Programs

ERIC Educational Resources Information Center

Farrell, Anne F.; Collier-Meek, Melissa A.; Pons, Shelby R.

2013-01-01

There is growing recognition that after-school programs (ASPs) provide opportunities for positive youth development. Many ASPs focus on behavior and socio-emotional challenges, provide evidence-based interventions to improve homework completion and academic skills, and offer physical activities and nutritious foods. Generally speaking, ASPs offer…

Improved ASP I/O performance. [For 370/195

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

Engert, D.E.

1975-11-01

An improvement to the I/0 routines of ASP is described, which can save 25% of the EXCPs issued by ASP. Unused buffers in the buffer pool are used to retain parts of the single track table, and only two modules are changed. 4 figures (auth)

A novel COL11A1 missense mutation in siblings with non-ocular Stickler syndrome.

PubMed

Kohmoto, Tomohiro; Tsuji, Atsumi; Morita, Kei-Ichi; Naruto, Takuya; Masuda, Kiyoshi; Kashimada, Kenichi; Enomoto, Keisuke; Morio, Tomohiro; Harada, Hiroyuki; Imoto, Issei

2016-01-01

Stickler syndrome (STL) is an autosomal, dominantly inherited, clinically variable and genetically heterogeneous connective tissue disorder characterized by ocular, auditory, orofacial and skeletal abnormalities. We conducted targeted resequencing using a next-generation sequencer for molecular diagnosis of a 2-year-old girl who was clinically suspected of having STL with Pierre Robin sequence. We detected a novel heterozygous missense mutation, NM_001854.3:n.4838G>A [NM_001854.3 (COL11A1_v001):c.4520G>A], in COL11A1, resulting in a Gly to Asp substitution at position 1507 [NM_001854.3(COL11A1_i001)] within one of the collagen-like domains of the triple helical region. The same mutation was detected in her 4-year-old brother with cleft palate and high-frequency sensorineural hearing loss.

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

NASA Technical Reports Server (NTRS)

Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

Answer Set Programming and Other Computing Paradigms

ERIC Educational Resources Information Center

Meng, Yunsong

2013-01-01

Answer Set Programming (ASP) is one of the most prominent and successful knowledge representation paradigms. The success of ASP is due to its expressive non-monotonic modeling language and its efficient computational methods originating from building propositional satisfiability solvers. The wide adoption of ASP has motivated several extensions to…

Canadian Ranger Rifle: Human Factors Requirements Validation

DTIC Science & Technology

2010-08-01

index-eng.asp retrieved 9 February 2010 2 http://www.armee.forces.gc.ca/land-terre/cr-rc/history- histoire -eng.asp retrieved 9 February 2010 3 http... histoire -eng.asp Department of National Defence. (2010). Canadian Ranger Patrol (CRPG). Retrieved June 3, 2010, from http://www.army.forces.gc.ca

Cardioprotection activity and mechanism of Astragalus polysaccharide in vivo and in vitro.

PubMed

Liu, Debin; Chen, Lei; Zhao, Jianye; Cui, Kang

2018-05-01

Astragalus polysaccharides (ASP) is extracted from Astragalus, and is the main active ingredient of Astragalus membranaceus. The purpose of this study was to investigate the protective effect of ASP on rat cardiomyocytes damage induced by myocardial ischemia and reperfusion injury (MVRI) and isoprenaline(ISO) in vivo and in vitro. The model of cardiomyocytes damage was induced using MVRI in a rat in vivo and also using ISO in cell. After ASP intervention, the protective effect of ASP on cardiomyocytes was evaluated by animal experimental and cell experimental. The results show that ASP can relieve the increase of cell volume in myocardium, reduce the apoptosis of cell in myocardial tissue caused by MVRI in vivo. At the cellular level, ASP can reverse the decrease of cell activity induced by ISO, inhibit the apoptosis, and decrease the levels of intracellular reactive oxygen species. Mechanistically at the molecular level, these effects are elicited via down-regulation of the protein levels of caspase-3 and bax and up-regulation of the protein levels of bcl-2 in both in vivo and in vitro. These results demonstrate that ASP has a protective efficacy in MVRI/ISO-treated cardiomyocytes by inhibiting the apoptosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Selectivity Mechanism of the Voltage-gated Proton Channel, HV1

NASA Astrophysics Data System (ADS)

Dudev, Todor; Musset, Boris; Morgan, Deri; Cherny, Vladimir V.; Smith, Susan M. E.; Mazmanian, Karine; Decoursey, Thomas E.; Lim, Carmay

2015-05-01

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores, and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp-Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O+ protonated the SF, breaking the Asp-Arg linkage and opening the conduction pathway, whereas Na+ or Cl- was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp-Lys SF behaved like the Asp-Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH0-H2O0-Arg+ interactions with hydronium but unfavorable Asp--X-/X+-Arg+ interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis, and signaling.

Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

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

Chou, Bin; Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582; Hiromatsu, Kenji, E-mail: khiromatsu@fukuoka-u.ac.jp

2010-02-12

Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +}more » T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.« less

Proteolytic cleavage of polymeric tau protein by caspase-3: implications for Alzheimer disease.

PubMed

Jarero-Basulto, Jose J; Luna-Muñoz, Jose; Mena, Raul; Kristofikova, Zdena; Ripova, Daniela; Perry, George; Binder, Lester I; Garcia-Sierra, Francisco

2013-12-01

Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.

Chronic administration of Angelica sinensis polysaccharide effectively improves fatty liver and glucose homeostasis in high-fat diet-fed mice.

PubMed

Wang, Kaiping; Cao, Peng; Wang, Hanxiang; Tang, Zhuohong; Wang, Na; Wang, Jinglin; Zhang, Yu

2016-05-18

This study aimed to investigate the therapeutic effects of Angelica sinensis polysaccharide (ASP), an active component derived from a water extract of Angelica sinensis, in high-fat diet (HFD)-fed BALB/c mice. The potential mechanisms underlying the activity of this compound were also considered. Specifically, serum and hepatic biochemical parameters were evaluated, and key proteins involved in the lipid/glucose metabolism were analyzed. Long-term feeding with a HFD induced severe fatty liver and hyperglycemia. Histological examination clearly showed that ASP reduced lipid accumulation in the liver and attenuated hepatic steatosis in HFD-fed mice. In addition, ASP markedly alleviated serum and liver lipid disorders and fatty liver via the upregulation of PPARγ expression and the activation of adiponectin-SIRT1-AMPK signaling. Furthermore, ASP also significantly relieved severe oxidative stress, demonstrating that ASP might attenuate nonalcoholic fatty liver disease via a "two-hit" mechanism. In addition, ASP reduced blood glucose levels and ameliorated insulin resistance via the regulation of related metabolic enzymes and by activating the PI3K/Akt pathway in HFD-fed mice. Our findings revealed that ASP might be used as an alternative dietary supplement or health care product to ameliorate metabolic syndrome in populations that consistently consume HFDs.

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

PubMed

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.