Adaptation of tick-borne encephalitis virus from human brain to different cell cultures induces multiple genomic substitutions.

PubMed

Ponomareva, Eugenia P; Ternovoi, Vladimir A; Mikryukova, Tamara P; Protopopova, Elena V; Gladysheva, Anastasia V; Shvalov, Alexander N; Konovalova, Svetlana N; Chausov, Eugene V; Loktev, Valery B

2017-10-01

The C11-13 strain from the Siberian subtype of tick-borne encephalitis virus (TBEV) was isolated from human brain using pig embryo kidney (PEK), 293, and Neuro-2a cells. Analysis of the complete viral genome of the C11-13 variants during six passages in these cells revealed that the cell-adapted C11-13 variants had multiple amino acid substitutions as compared to TBEV from human brain. Seven out of eight amino acids substitutions in the high-replicating C11-13(PEK) variant mapped to non-structural proteins; 13 out of 14 substitutions in the well-replicating C11-13(293) variant, and all four substitutions in the low-replicating C11-13(Neuro-2a) variant were also localized in non-structural proteins, predominantly in the NS2a (2), NS3 (6) and NS5 (3) proteins. The substitutions NS2a 1067 (Asn → Asp), NS2a 1168 (Leu → Val) in the N-terminus of NS2a and NS3 1745 (His → Gln) in the helicase domain of NS3 were found in all selected variants. We postulate that multiple substitutions in the NS2a, NS3 and NS5 genes play a key role in adaptation of TBEV to different cells.

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

PubMed

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-06-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

PubMed Central

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-01-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses. PMID:15890917

Detection and a possible link between parvovirus B19 and thyroid cancer.

PubMed

Etemadi, Ashkan; Mostafaei, Shayan; Yari, Kheirollah; Ghasemi, Amir; Minaei Chenar, Hamzeh; Moghoofei, Mohsen

2017-06-01

Human parvovirus B19 (B19) is a small, non-enveloped virus and belongs to Parvoviridae family. B19 persists in many tissues such as thyroid tissue and even thyroid cancer. The main aim of this study was to determine the presence of B19, its association with increased inflammation in thyroid tissue, and thus its possible role in thyroid cancer progression. Studies have shown that virus replication in non-permissive tissue leads to overexpression of non-structural protein and results in upregulation of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha. A total of 36 paraffin-embedded thyroid specimens and serum were collected from patients and 12 samples were used as control. Various methods were employed, including polymerase chain reaction, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. The results have shown the presence of B19 DNA in 31 of 36 samples (86.11%). Almost in all samples, the levels of non-structural protein 1, nuclear factor kappa B, tumor necrosis factor alpha, and interleukin 6 were simultaneously high. The presence of parvovirus B19 has a significant positive correlation with nuclear factor kappa B, tumor necrosis factor alpha, and interleukin 6 levels. This study suggests that B19 infection may play an important role in tumorigenesis and thyroid cancer development via the inflammatory mechanisms.

Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

PubMed

Szkola, A; Linares, E M; Worbs, S; Dorner, B G; Dietrich, R; Märtlbauer, E; Niessner, R; Seidel, M

2014-11-21

Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 μg L(-1) for ricin, 0.1 ± 0.1 μg L(-1) for SEB and 2.3 ± 1.7 μg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies.

PubMed Central

Gong, K; Wen, D Y; Ouyang, T; Rao, A T; Herzberg, M C

1995-01-01

Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s. PMID:7642300

Radio-metabolite analysis of carbon-11 biochemical partitioning to non-structural carbohydrates for integrated metabolism and transport studies.

PubMed

Babst, Benjamin A; Karve, Abhijit A; Judt, Tatjana

2013-06-01

Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 ((11)C) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive (11)C-based methodologies for the study of phloem transport. In this report, we present methods using (11)C-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of (11)C partitioning to starch, which was previously not possible. Because of the high specific activity of (11)C and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other (11)C assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.

Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

PubMed Central

Nüesch, Jürg P. F.; Corbau, Romuald; Tattersall, Peter; Rommelaere, Jean

1998-01-01

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C. PMID:9733839

Growth hormone isoforms release in response to physiological and pharmacological stimuli.

PubMed

Pagani, S; Cappa, M; Meazza, C; Ubertini, G; Travaglino, P; Bozzola, E; Bozzola, M

2008-06-01

Ten healthy subjects used to performing regular physical activity and eight subjects affected by idiopathic isolated GH deficiency (GHD) were enrolled; 22- and 20-kDa GH secretion and its biological activity were evaluated in response to pharmacological stimuli such as arginine, L-dopa or glucagon in GHD children, while the hormonal response to exercise was studied according to Bruce protocol in healthy subjects. We found a significant increase in 22- and 20-kDa GH level in healthy subjects after monitored physical exercise (MPE; basal 0.28+/-0.12 vs 7.37+/-2.08 ng/ml and basal 0.076+/-0.04 vs 0.18+/-0.05 ng/ml, respectively). Furthermore, the 22-kDa/20-kDa ratio significantly increased in children who had undergone MPE and the GH bioactivity basal mean value also increased significantly after exercise (basal 2.86+/-0.76 vs 7.64+/-1.9 ng/ml). The mean value of 22-kDa GH in GHD patients increased significantly following GH pharmacological stimulation (2.78+/-0.63 ng/ml) when compared with mean basal (0.20+/-0.11 ng/ml) value. In the GHD group the basal concentration of 20-kDa GH significantly increased following GH pharmacological stimulation (0.34+/-0.11 vs 0.72+/-0.2 ng/ml); the 22-kDa/20-kDa ratio significantly increased too. Likewise, GH bioactivity in children with GHD increased significantly after pharmacological stimulation test (basal 2.53+/-0.56 vs 7.33+/-1.26 ng/ml). Both GH isoform concentrations and their biological activity are significantly increased in healthy subjects after submaximal exercise protocol and in GHD children after pharmacological stimuli.

Transport of dissolved organic matter in Boom Clay: Size effects

NASA Astrophysics Data System (ADS)

Durce, D.; Aertsens, M.; Jacques, D.; Maes, N.; Van Gompel, M.

2018-01-01

A coupled experimental-modelling approach was developed to evaluate the effects of molecular weight (MW) of dissolved organic matter (DOM) on its transport through intact Boom Clay (BC) samples. Natural DOM was sampled in-situ in the BC layer. Transport was investigated with percolation experiments on 1.5 cm BC samples by measuring the outflow MW distribution (MWD) by size exclusion chromatography (SEC). A one-dimensional reactive transport model was developed to account for retardation, diffusion and entrapment (attachment and/or straining) of DOM. These parameters were determined along the MWD by implementing a discretisation of DOM into several MW points and modelling the breakthrough of each point. The pore throat diameter of BC was determined as 6.6-7.6 nm. Below this critical size, transport of DOM is MW dependent and two major types of transport were identified. Below MW of 2 kDa, DOM was neither strongly trapped nor strongly retarded. This fraction had an averaged capacity factor of 1.19 ± 0.24 and an apparent dispersion coefficient ranging from 7.5 × 10- 11 to 1.7 × 10- 11 m2/s with increasing MW. DOM with MW > 2 kDa was affected by both retardation and straining that increased significantly with increasing MW while apparent dispersion coefficients decreased. Values ranging from 1.36 to 19.6 were determined for the capacity factor and 3.2 × 10- 11 to 1.0 × 10- 11 m2/s for the apparent dispersion coefficient for species with 2.2 kDa < MW < 9.3 kDa. Straining resulted in an immobilisation of in average 49 ± 6% of the injected 9.3 kDa species. Our findings show that an accurate description of DOM transport requires the consideration of the size effects.

Transcriptional analysis of Penaeus stylirostris densovirus genes

USDA-ARS?s Scientific Manuscript database

Penaeus stylirostris densovirus (PstDNV) genome contains three open reading frames (ORFs), left, middle, and right, which encode a non-structural (NS) protein, an unknown protein, and a capsid protein (CP), respectively. Transcription mapping revealed that P2, P11 and P61 promoters transcribe the le...

Metabolic activation of sodium nitroprusside to nitric oxide in vascular smooth muscle.

PubMed

Kowaluk, E A; Seth, P; Fung, H L

1992-09-01

Sodium nitroprusside (SNP) is thought to exert its vasodilating activity, at least in part, by vascular activation to nitric oxide (NO), but the activation mechanism has not been delineated. This study has examined the potential for vascular metabolism of SNP to NO in bovine coronary arterial smooth muscle subcellular fractions using a sensitive and specific redox-chemiluminescence assay for NO. SNP was readily metabolized to NO in subcellular fractions, and the dominant site of metabolism appeared to be located in the membrane fractions. NO-generating activity was significantly enhanced by, but did not absolutely require, the addition of a NADPH-regenerating system, NADPH per se, NADH or cysteine. A correlation analysis of NO-generating activity (in the presence of a NADPH-regenerating system) with marker enzyme activities indicated that the SNP-directed NO-generating activity was primarily membrane-associated. Radiation inactivation target-size analysis revealed that the microsomal SNP-directed NO-generating activity was relatively insensitive to inactivation by radiation exposure, suggesting that the functioning catalytic unit might be quite small. A molecular weight of 5 to 11 kDa was estimated. NO-generating activity could be solubilized from the crude microsomes with 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and the solubilized extract was subjected to gel filtration chromatography. NO-generating activity was eluted in two peaks: one peak corresponding to an approximate molecular weight of 4 kDa, thus confirming the existence of a small molecular weight NO-generating activity, and a second activity peak corresponding to a molecular weight of 112 to 169 kDa, the functional significance of which is unclear at present.(ABSTRACT TRUNCATED AT 250 WORDS)

The small phytoplasma virulence effector SAP11 contains distinct domains required for nuclear targeting and CIN-TCP binding and destabilization.

PubMed

Sugio, Akiko; MacLean, Allyson M; Hogenhout, Saskia A

2014-05-01

Phytoplasmas are insect-transmitted bacterial phytopathogens that secrete virulence effectors and induce changes in the architecture and defense response of their plant hosts. We previously demonstrated that the small (± 10 kDa) virulence effector SAP11 of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) binds and destabilizes Arabidopsis CIN (CINCINNATA) TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 AND 2) transcription factors, resulting in dramatic changes in leaf morphogenesis and increased susceptibility to phytoplasma insect vectors. SAP11 contains a bipartite nuclear localization signal (NLS) that targets this effector to plant cell nuclei. To further understand how SAP11 functions, we assessed the involvement of SAP11 regions in TCP binding and destabilization using a series of mutants. SAP11 mutants lacking the entire N-terminal domain, including the NLS, interacted with TCPs but did not destabilize them. SAP11 mutants lacking the C-terminal domain were impaired in both binding and destabilization of TCPs. These SAP11 mutants did not alter leaf morphogenesis. A SAP11 mutant that did not accumulate in plant nuclei (SAP11ΔNLS-NES) was able to bind and destabilize TCP transcription factors, but instigated weaker changes in leaf morphogenesis than wild-type SAP11. Overall the results suggest that phytoplasma effector SAP11 has a modular organization in which at least three domains are required for efficient CIN-TCP destabilization in plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

DOE PAGES

Rames, Matthew; Yu, Yadong; Ren, Gang

2014-08-15

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electronmore » microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.« less

Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas

PubMed Central

Kang, Seongjoon; Odom, Obed W.; Thangamani, Saravanan; Herrin, David L.

2016-01-01

We are developing Chlamydomonas strains that can be used for safe and sustainable control of mosquitoes, because they produce proteins from Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast. Chlamydomonas has a number of advantages for this approach, including genetic controls that are not generally available with industrial algae. The Bti toxin has been used for mosquito control for > 30 years and does not engender resistance; it contains three Cry proteins, Cry4Aa (135 kDa), Cry4Ba (128 kDa) and Cry11Aa (72 kDa), and Cyt1Aa (25 kDa). To express the Cry proteins in the chloroplast, the three genes were resynthesized and cry4Aa was truncated to the first 700 amino acids (cry4Aa700); also, since they can be toxic to host cells, the inducible Cyc6:Nac2-psbD expression system was used. Western blots of total protein from the chloroplast transformants showed accumulation of the intact polypeptides, and the relative expression level was Cry11Aa > Cry4Aa700 > Cry4Ba. Quantitative western blots with purified Cry11Aa as a standard showed that Cry11Aa accumulated to 0.35% of total cell protein. Live cell bioassays in dH20 demonstrated toxicity of the cry4Aa700 and cry11Aa transformants to larvae of Aedes aegypti and Culex quinquefasciatus. These results demonstrate that the Cry proteins that are most toxic to Aedes and Culex mosquitoes, Cry4Aa and Cry11Aa, can be successfully expressed in the chloroplast of Chlamydomonas. PMID:28713202

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

NASA Technical Reports Server (NTRS)

Roswit, W. T.; McCourt, D. W.; Partridge, N. C.; Jeffrey, J. J.

1992-01-01

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

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

Chen, J.; Varner, J.E.

1985-07-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 asmore » a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.« less

The KnowRISK project: Tools and strategies to reduce non-structural damage

NASA Astrophysics Data System (ADS)

Sousa Oliveira, Carlos; Lopes, Mário; Mota de Sá, Francisco; Amaral Ferreia, Mónica; Candeias, Paulo; Campos Costa, Alfredo; Rupakhety, Rajesh; Meroni, Fabrizio; Azzaro, Raffaele; D'Amico, Salvatore; Langer, Horst; Musacchio, Gemma; Sousa Silva, Delta; Falsaperla, Susanna; Scarfì, Luciano; Tusa, Giuseppina; Tuvé, Tiziana

2016-04-01

The project KnowRISK (Know your city, Reduce seISmic risK through non-structural elements) is financed by the European Commission to develop prevention measures that may reduce non-structural damage in urban areas. Pilot areas of the project are within the three European participating countries, namely Portugal, Iceland and Italy. Non-structural components of a building include all those components that are not part of the structural system, more specifically the architectural, mechanical, electrical, and plumbing systems, as well as furniture, fixtures, equipment, and contents. Windows, partitions, granite veneer, piping, ceilings, air conditioning ducts and equipment, elevators, computer and hospital equipment, file cabinets, and retail merchandise are all examples of non-structural components that are vulnerable to earthquake damage. We will use the experience gained during past earthquakes, which struck in particular Iceland, Italy and Portugal (Azores). Securing the non-structural elements improves the safety during an earthquake and saves lives. This paper aims at identifying non-structural seismic protection measures in the pilot areas and to develop a portfolio of good practices for the most common and serious non-structural vulnerabilities. This systematic identification and the portfolio will be achieved through a "cross-knowledge" strategy based on previous researches, evidence of non-structural damage in past earthquakes. Shake table tests of a group of non-structural elements will be performed. These tests will be filmed and, jointly with portfolio, will serve as didactic supporting tools to be used in workshops with building construction stakeholders and in risk communication activities. A Practical Guide for non-structural risk reduction will be specifically prepared for citizens on the basis of the outputs of the project, taking into account the local culture and needs of each participating country.

Effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans

PubMed Central

Kim, Won-Seok; Jez, Joseph M.; Krishnan, Hari B.

2014-01-01

Expression of heterologous methionine-rich proteins to increase the overall sulfur amino acid content of soybean seeds has been only marginally successful, presumably due to low accumulation of transgenes in soybeans or due to gene silencing. Proteome rebalancing of seed proteins has been shown to promote the accumulation of foreign proteins. In this study, we have utilized RNAi technology to suppress the expression of the β-conglycinin, the abundant 7S seed storage proteins of soybean. Western blot and 2D-gel analysis revealed that β-conglycinin knockdown line (SAM) failed to accumulate the α′, α, and β-subunits of β-conglycinin. The proteome rebalanced SAM retained the overall protein and oil content similar to that of wild-type soybean. We also generated transgenic soybean lines expressing methionine-rich 11 kDa δ-zein under the control of either the glycinin or β-conglycinin promoter. The introgression of the 11 kDa δ-zein into β-conglycinin knockdown line did not enhance the accumulation of the 11 kDa δ-zein. However, when the same plants were grown in sulfur-rich medium, we observed 3- to 16-fold increased accumulation of the 11 kDa δ-zein. Transmission electron microscopy observation revealed that seeds grown in sulfur-rich medium contained numerous endoplasmic reticulum derived protein bodies. Our findings suggest that sulfur availability, not proteome rebalancing, is needed for high-level accumulation of heterologous methionine-rich proteins in soybean seeds. PMID:25426134

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

PubMed

Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

2012-01-01

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

MALDI-TOF mass spectrometry analysis of small molecular weight compounds (under 10 KDa) as biomarkers of rat hearts undergoing arecoline challenge.

PubMed

Chen, Tung-Sheng; Chang, Mu-Hsin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Day, Cecilia Hsuan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

2013-04-01

Statistical and clinical reports indicate that betel nut chewing is strongly associated with progression of oral cancer because some ingredients in betel nuts are potential cancer promoters, especially arecoline. Early diagnosis for cancer biomarkers is the best strategy for prevention of cancer progression. Several methods are suggested for investigating cancer biomarkers. Among these methods, gel-based proteomics approach is the most powerful and recommended tool for investigating biomarkers due to its high-throughput. However, this proteomics approach is not suitable for screening biomarkers with molecular weight under 10 KDa because of the characteristics of gel electrophoresis. This study investigated biomarkers with molecular weight under 10 KDa in rats with arecoline challenge. The centrifuging vials with membrane (10 KDa molecular weight cut-off) played a crucial role in this study. After centrifuging, the filtrate (containing compounds with molecular weight under 10 KDa) was collected and spotted on a sample plate for MALDI-TOF mass spectrometry analysis. Compared to control, three extra peaks (m/z values were 1553.1611, 1668.2097 and 1740.1832, respectively) were found in sera and two extra peaks were found in heart tissue samples (408.9719 and 524.9961, respectively). These small compounds should play important roles and may be potential biomarker candidates in rats with arecoline. This study successfully reports a mass-based method for investigating biomarker candidates with small molecular weight in different types of sample (including serum and tissue). In addition, this reported method is more time-efficient (1 working day) than gel-based proteomics approach (5~7 working days).

Characterization of a novel wheat endosperm protein belonging to the prolamin superfamily

USDA-ARS?s Scientific Manuscript database

Starch granule surface-associated proteins were separated by HPLC and identified by direct protein sequencing. Among the proteins identified was one that consisted of two polypeptide chains of 11 kDa and 19 kDa linked by disulfide bonds. Sequencing of tryptic peptides from each of the polypeptide ch...

Biochemistry of plant class IV chitinases and fungal chitinase-modifying proteins

USDA-ARS?s Scientific Manuscript database

Plant class IV chitinases have 2 domains, a small (3 kDa) amino-terminal domain with homology to carbohydrate binding peptides, and a larger (25 kDa) catalytic domain. The biological function of these chitinases is not known. But it is known that some pathogenic fungi secrete chitinase modifying pro...

SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra

NASA Astrophysics Data System (ADS)

Khaliq, Binish; Falke, Sven; Negm, Amr; Buck, Friedrich; Munawar, Aisha; Saqib, Maria; Mahmood, Seema; Ahmad, Malik Shoaib; Betzel, Christian; Akrem, Ahmed

2017-06-01

Oilseeds of the plant family Brassicaceae are important for providing both lipid and protein contents to human nutrition. Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (∼190 kDa) cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30% saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with cruciferin from Arabidopsis thaliana. The 50-55 kDa monomeric cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28-32 kDa and β-polypeptides of 17-20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7 and 8.0 (α-polypeptides) and 5.5-8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8 Å). Small-angle x-ray scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. SAXS showed that the radius of gyration is 4.24 ± 0.25 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra cruciferin was compared with 11S globulins.

Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3)*

PubMed Central

Dutta, Dipanjan; Chattopadhyay, Shiladitya; Bagchi, Parikshit; Halder, Umesh Chandra; Nandi, Satabdi; Mukherjee, Anupam; Kobayashi, Nobumichi; Taniguchi, Koki; Chawla-Sarkar, Mamta

2011-01-01

Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225–258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225–258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225–258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225–258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells. PMID:21489987

Role of one N-linked oligosaccharide chain on canine herpesvirus gD in its biological activity.

PubMed

Maeda, K; Yokoyama, N; Fujita, K; Xuan, X; Mikami, T

1997-12-01

The YP11mu strain of a plaque-selected canine herpesvirus (CHV) encoded a smaller molecular weight (MW) of gD than those of other strains including YP2 strain (Xuan et al., 1990). When nucleotide sequence of the mutated gD of YP11mu strain (gD(YP11mu)) was compared with that of gDs of other CHV strains, gD(YP11mu) lacked 12 nucleotides encoding 4 amino acids, NKTI, including one predicted potential N-linked glycosylation site and no other change was found in other regions. When the gD(YP11mu) and gD of YP2 strain (gD(YP2)) expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells were compared each other, both gDs reacted with a panel of monoclonal antibodies (MAbs) against CHV gD by indirect immunofluorescence analysis and the gD(YP11mu) possessed an MW of approximately 47-51 and 39-44 kDa in COS-7 and Sf9 cells, respectively, which were smaller than the expressed gD(YP2) (approximately 51-55 and 41-46 kDa, respectively) by immunoblot analysis. After treatment with tunicamycin, the MW of both gDs in Sf9 cells became approximately 37 kDa. When hemagglutination (HA) test using canine red blood cells (RBC) were carried out, lysates of Sf9 cells expressing CHV gDs agglutinated canine RBC. Serum from mice inoculated with lysates of Sf9 cells expressing the gDs possessed a high titer of virus-neutralizing (VN) activities against CHV. These results indicated that the deletion of 4 amino acids possessing approximately 4 kDa of glyco-chain from gD of CHV in mammalian cells does not affect HA activity and VN antibody-inducing activity and that this deletion of gD(YP11mu) might be a good selective marker for development of recombinant viruses as a live vaccine.

Performance-based seismic design of nonstructural building components: The next frontier of earthquake engineering

NASA Astrophysics Data System (ADS)

Filiatrault, Andre; Sullivan, Timothy

2014-08-01

With the development and implementation of performance-based earthquake engineering, harmonization of performance levels between structural and nonstructural components becomes vital. Even if the structural components of a building achieve a continuous or immediate occupancy performance level after a seismic event, failure of architectural, mechanical or electrical components can lower the performance level of the entire building system. This reduction in performance caused by the vulnerability of nonstructural components has been observed during recent earthquakes worldwide. Moreover, nonstructural damage has limited the functionality of critical facilities, such as hospitals, following major seismic events. The investment in nonstructural components and building contents is far greater than that of structural components and framing. Therefore, it is not surprising that in many past earthquakes, losses from damage to nonstructural components have exceeded losses from structural damage. Furthermore, the failure of nonstructural components can become a safety hazard or can hamper the safe movement of occupants evacuating buildings, or of rescue workers entering buildings. In comparison to structural components and systems, there is relatively limited information on the seismic design of nonstructural components. Basic research work in this area has been sparse, and the available codes and guidelines are usually, for the most part, based on past experiences, engineering judgment and intuition, rather than on objective experimental and analytical results. Often, design engineers are forced to start almost from square one after each earthquake event: to observe what went wrong and to try to prevent repetitions. This is a consequence of the empirical nature of current seismic regulations and guidelines for nonstructural components. This review paper summarizes current knowledge on the seismic design and analysis of nonstructural building components, identifying major knowledge gaps that will need to be filled by future research. Furthermore, considering recent trends in earthquake engineering, the paper explores how performance-based seismic design might be conceived for nonstructural components, drawing on recent developments made in the field of seismic design and hinting at the specific considerations required for nonstructural components.

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Venom Proteomics of Indonesian King Cobra, Ophiophagus hannah: Integrating Top-Down and Bottom-Up Approaches.

PubMed

Petras, Daniel; Heiss, Paul; Süssmuth, Roderich D; Calvete, Juan J

2015-06-05

We report on the first application of top-down mass spectrometry in snake venomics. De novo sequence tags generated by, and ProSight Lite supported analysis of, combined collisional based dissotiations (CID and HCD) recorded in a hybrid LTQ Orbitrap instrument in data-dependent mode identified a number of proteins from different toxin families, namely, 11 three-finger toxins (7-7.9 kDa), a Kunitz-type inhibitor (6.3 kDa), ohanin (11.9 kDa), a novel phospholipase A2 molecule (13.8 kDa), and the cysteine-rich secretory protein (CRISP) ophanin (25 kDa) from Indonesian king cobra venom. Complementary bottom-up MS/MS analyses contributed to the completion of a locus-resolved venom phenotypic map for Ophiophagus hannah, the world's longest venomous snake and a species of medical concern across its wide distribution range in forests from India to Southeast Asia. Its venom composition, comprising 32-35 proteins/peptides from 10 protein families, is dominated by α-neurotoxins and convincingly explains the main neurotoxic effects of human envenoming caused by king cobra bite. The integration of efficient chromatographic separation of the venom's components and locus-resolved toxin identification through top-down and bottom-up MS/MS-based species-specific database searching and de novo sequencing holds promise that the future will be bright for the field of venom research.

Regulation of Dab2 expression in intestinal and renal epithelia by development.

PubMed

Vázquez-Carretero, María D; García-Miranda, Pablo; Calonge, María L; Peral, María J; Ilundáin, Anunciación A

2011-01-01

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.

Pannus overgrowth after mitral valve replacement with a Carpentier-Edwards pericardial bioprosthesis.

PubMed

Oda, Takeshi; Kato, Seiya; Tayama, Eiki; Fukunaga, Shuji; Akashi, Hidetoshi; Aoyagi, Shigeaki

2009-01-01

A Carpentier-Edwards pericardial (CEP) bioprosthesis was explanted from an 81-year-old woman due to nonstructural dysfunction 9 years after mitral valve replacement. The nonstructural dysfunction produced severe regurgitation in the mitral position. During the surgery, excessive pannus overgrowth was seen on the left ventricular side of the CEP bioprosthesis. Pannus overgrowth was prominent on one leaflet. That leaflet was stiff and shortened due to the excessive overgrowth of pannus. In this patient, the distortion of one leaflet was the main reason for transvalvular leakage of the CEP bioprosthesis in the mitral position. A new CEP bioprosthesis was implanted in the mitral position. Pathological analysis revealed fibrotic pannus with a small amount of cellular material over the leaflets of the resected CEP valve. This change was marked on the distorted leaflet.

Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

PubMed

Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

2017-09-01

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

Molecular-specific urokinase antibodies

NASA Technical Reports Server (NTRS)

Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

2009-01-01

Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

CHANGES IN FERRITIN H- AND L-CHAINS IN CANINE LENSES WITH AGE-RELATED NUCLEAR CATARACT

PubMed Central

Goralska, Małgorzata; Nagar, Steven; Colitz, Carmen M.H.; Fleisher, Lloyd N.; McGahan, M. Christine

2014-01-01

PURPOSE To determine potential differences in the characteristics of the iron storage protein, ferritin and its heavy (H) and light (L) subunits in fiber cells from cataractous and normal lenses of older dogs. METHODS Lens fiber cell homogenates were analyzed by SDS-PAGE and ferritin chains were immunodetected with ferritin chain-specific antibodies. Ferritin concentration was measured by ELISA. Immunohistochemistry was used to localize ferritin chains in lens sections. RESULTS The concentration of assembled ferritin was comparable in normal and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was about 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver. The H-chain identified in cataractous fiber cells (29 kDa) differed from 21 kDa standard canine H-chain and from 12 kDa modified H-chain present in fiber cells of normal lenses. Histologic analysis revealed that the H-chain was distributed differently throughout cataractous lenses when compared to normal lenses. There was also a difference in subunit makeup of assembled ferritin between the two lens types. Ferritin from cataractous lenses contained more H-chain and bound 11-fold more iron than ferritin from normal lenses. CONCLUSIONS There are significant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses as compared to normal lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition the accumulation of H-chain in deeper fiber layers of the lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage. PMID:18708625

The KnowRISK project - Know your city, Reduce seISmic risK through non-structural elements

NASA Astrophysics Data System (ADS)

Sousa Oliveria, Carlos; Amaral Ferreira, Mónica; Lopez, Mário; Sousa Silva, Delta; Musacchio, Gemma; Rupakhety, Rajesh; Falsaperla, Susanna; Meroni, Fabrizio; Langer, Horst

2016-04-01

Historically, there is a tendency to focus on seismic structural performance of buildings, neglecting the potential for damage of non-structural elements. In particular, non-structural elements of buildings are their architectural parts (i.e. partitions, ceilings, cladding), electrical and mechanical components (i.e., distribution panels, piping, plumbing), and contents (e.g., furniture, bookcases, computers and desktop equipment). Damage of these elements often contributes significantly to earthquake impacts. In the 1999 Izmit Earthquake, Turkey, 50% of the injuries and 3% of human losses were caused by non-structural failures. In the 2010-2011 Christchurch Earthquakes (New Zealand), 40% of building damage was induced by non-structural malfunctions. Around 70%-85% of construction cost goes into these elements, and their damage can strongly influence the ability of communities to cope with and recover from earthquakes. The project Know your city, Reduce seISmic risK through non-structural elements (KnowRISK) aims at facilitating local communities' access to expert knowledge on non-structural seismic protection solutions. The project will study seismic scenarios critical for non-structural damage, produce a portfolio of non-structural protection measures and investigate the level of awareness in specific communities. We will implement risk communication strategies that will take into account the social and cultural background and a participatory approach to raise awareness in local communities. The paradox between the progress of scientific knowledge and the ongoing increase of losses from natural disasters worldwide is a well-identified gap in the UN Hyogo Framework for Action 2005-2015, in which one of the main priorities is the investment on "knowledge use, innovation and education to build a culture of safety and resilience". The KnowRISK is well aligned with these priorities and will contribute to participatory action aimed at: i) transferring expert knowledge on seismic risk and non-structural protective solutions into practical knowledge and ii) communication tools designed to engage communities in disaster risk reduction

Genome sequences of a mouse-avirulent and a mouse-virulent strain of Ross River virus.

PubMed

Faragher, S G; Meek, A D; Rice, C M; Dalgarno, L

1988-04-01

The nucleotide sequence of the genomic RNA of a mouse-avirulent strain of Ross River virus, RRV NB5092 (isolated in 1969), has been determined and the corresponding sequence for the prototype mouse-virulent strain, RRV T48 (isolated in 1959), has been completed. The RRV NB5092 genome is approximately 11,674 nucleotides in length, compared with 11,853 nucleotides for RRV T48. RRV NB5092 and RRV T48 have the same genome organization. For both viruses an untranslated region of 80 nucleotides at the 5' end of the genome is followed by a 7440-nucleotide open reading frame which is interrupted after 5586 nucleotides by a single opal termination codon. By homology with other alphaviruses, the 5586-nucleotide open reading frame encodes the nonstructural proteins nsP1, nsP2, and nsP3; a fourth nonstructural protein, nsP4, is produced by read-through of the opal codon. The RRV nonstructural proteins show strong homology with the corresponding proteins of Sindbis virus and Semliki Forest virus in terms of size, net charge, and hydropathy characteristics. However, homology is not uniform between or within the proteins; nsP1, nsP2, and nsP4 contain extended domains which are highly conserved between alphaviruses, while the C-terminal region of nsP3 shows little conservation in sequence or length between alphaviruses. An untranslated "junction" region of 44 nucleotides (for RRV NB5092) or 47 nucleotides (for RRV T48) separates the nonstructural and structural protein coding regions. The structural proteins (capsid-E3-E2-6K-E1) are translated from an open reading frame of 3762 nucleotides which is followed by a 3'-untranslated region of approximately 348 nucleotides (for RRV NB5092) or 524 nucleotides (for RRV T48). Excluding deletions and insertions, the genomes of RRV NB5092 and RRV T48 differ at 284 nucleotides, representing a sequence divergence of 2.38%. Sequence deletions or insertions were found only in the noncoding regions and include a 173-nucleotide deletion in the 3'-untranslated region of RRV NB5092, compared with RRV T48. In the coding regions, most of the nucleotide differences are silent; there are 36 amino acid differences in the nonstructural proteins and 12 in the structural proteins. The distribution of amino acid differences between the two RRV strains correlates with the location of domains which are poorly conserved in sequence between alphaviruses. The possible role of amino acid differences in envelope glycoproteins E1 and E2 in determining the different antigenic and biological properties of RRV NB5092 and RRV T48 is discussed.

Enhancement of Gag-specific but reduction of Env- and Pol-specific CD8+ T cell responses by simian immunodeficiency virus nonstructural proteins in mice.

PubMed

Zhang, Yinfeng; Sun, Caijun; Feng, Liqiang; Xiao, Lijun; Chen, Ling

2012-04-01

Accessory and regulatory proteins (nonstructural proteins) have received increasing attention as components in novel HIV/SIV vaccine design. However, the complicated interactions between nonstructural proteins and structural proteins remain poorly understood, especially their effects on immunogenicity. In this study, the immunogenicity of structural proteins in the presence and absence of nonstructural proteins was compared. First, a series of recombinant plasmids and adenoviral vectors carrying various SIVmac239 nonstructural and structural genes was constructed. Then mice were primed with DNA plasmids and boosted with corresponding Ad5 vectors of different combinations, and the resulting immune responses were measured. Our results demonstrated that when the individual Gag, Pol, or Env gene products were coimmunized with the whole repertoire of nonstructural proteins, the Gag-specific CD8(+) T response was greatly enhanced, while the Env- and Pol-specific CD8(+) T responses were significantly reduced. The same pattern was not observed in CD4(+) T cell responses. Antibody responses against both the Gag and Env proteins were elicited more effectively when these structural antigens were immunized together with nonstructural antigens. These findings may provide helpful insights into the development of novel HIV/SIV vaccines.

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

PubMed Central

Oblong, J E; Lamppa, G K

1992-01-01

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet. Images PMID:1385116

Complete genome sequence of a Watermelon silver mottle virus isolate from China.

PubMed

Rao, Xueqin; Wu, Zhuyan; Li, Yuan

2013-06-01

The complete genome of a Watermelon silver mottle virus (WSMoV) (genus Tospovirus, family Bunyaviridae) isolate (WSMoV-GZ) from Guangdong province, China was sequenced. The genomes of WSMoV-GZ contained 3,603, 4,909, and 8,914 nt of small (S), medium (M), and large (L) RNA segments, respectively, and had a genomic organization characteristic of members of the genus Tospovirus. The amino acid sequence of the nucleocapsid (N) protein, S RNA-encoded nonstructural (NSs) protein, M RNA-encoded nonstructural (NSm) protein, Gn/Gc glycoprotein precursor, and RNA-dependent RNA polymerase (RdRp) protein showed 94.3-97.5 % identity with those of other WSMoV isolates. Phylogenetic analysis showed that the N protein of WSMoV-GZ was clustered together with those of the WSMoV isolates. The full sequence of WSMoV-GZ provides a reference genome for comparison with other tospoviruses.

Aspergillus oryzae GB-107 fermentation improves nutritional quality of food soybeans and feed soybean meals.

PubMed

Hong, Kee-Jong; Lee, Chan-Ho; Kim, Sung Woo

2004-01-01

This study evaluated the effect of fermentation on the nutritional quality of food-grade soybeans and feed-grade soybean meals. Soybeans and soybean meals were fermented by Aspergillus oryzae GB-107 in a bed-packed solid fermentor for 48 hours. After fermentation, their nutrient contents as well as trypsin inhibitor were measured and compared with those of raw soybeans and soybean meals. Proteins were extracted from fermented and non-fermented soybeans and soybean meals, and the peptide characteristics were evaluated after electrophoresis. Fermented soybeans and fermented soybean meals contained 10% more (P < .05) crude protein than raw soybeans and soybean meals. The essential amino acid profile was unchanged after fermentation. Fermentation eliminated (P < .05) most of the trypsin inhibitor from both soybeans and soybean meals. Fermentation increased the amount of small-size peptides (<20 kDa) (P < .05) compared with raw soybeans, while significantly decreasing large-size peptides (>60 kDa) (P < .05). Fermented soybean meal contained more (P < .01) small-size peptides (<20 kDa) than soybean meal. Fermented soybean meal did not contain large-size peptides (>60 kDa), whereas 22.1% of peptides in soybean meal were large-size (>60 kDa). Collectively, fermentation increased protein content, eliminated trypsin inhibitors, and reduced peptide size in soybeans and soybean meals. These effects of fermentation might make soy foods more useful in human diets as a functional food and benefit livestock as a novel feed ingredient.

Effect of gamma irradiation on the expressed proteins in the foodborne pathogen Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Trudeau, Karine; Dang Vu, Khanh; Shareck, François; Lacroix, Monique

2012-08-01

A capillary electrophoresis method with UV detection was developed to analyze protein composition of the foodborne pathogen Staphylococcus aureus. Bacterial samples containing 109 CFU/ml, obtained after two cycles of incubations of 24 h, were gamma irradiated at different doses of 1.2, 3.5 and 2.9 kGy to respectively create damage cells, to kill cells and to provoke viable but non cultivable cells (VBNC). It was observed that an irradiation at a sensitive dose of 1.2 kGy caused a significantly increase in the protein with molecular weight (MW) of 17.7 kDa (from 0.61% to 1.2%). This treatment also caused decreases in the expressed proteins with the MWs of 16.3 kDa (from 6.2% to 5.3%) and of 23.4 kDa (from 4.0% to 2.30%). Irradiation at a VBCN dose of 2.9 kGy caused increases in expressed proteins with the MWs of 17.7 kDa (from 0.61% to 3.43%), 18.7 kDa (from 1.04% to 4.30%), 19.5 kDa (from 0.71% to 2.30%), 21.1 kDa (from 1.20% to 3.80%). Moreover, this treatment (2.9 kGy) also caused significantly decreases (P≤0.05) in the expressed proteins with the MW of 30.7 kDa (from 8.6% to 5.15%), 36.3 kDa (from 3.1% to 2.7%) and 40.5 kDa (from 11.3% to 8.5%). Finally, for the irradiation at a lethal dose of 3.5 kGy, it can be found that the expressed proteins with the MW of 17.7 kDa, 18.7 kDa and 19.5 kDa were increased less than that of expressed proteins at the VCNC dose (2.9 kGy) and these might be the very important proteins which are responsible for the survival of the S. aureus. Further, there were also the decreases in expressed proteins with the MW of 30.7 kDa, 36.3 kDa and 75.1 kDa at this dose of treatment (3.5 kGy) which can be expected that these proteins are seriously affected at high dose of γ-irradiation treatment.

Nonstructural urban stormwater quality measures: building a knowledge base to improve their use.

PubMed

Taylor, André C; Fletcher, Tim D

2007-05-01

This article summarizes a research project that investigated the use, performance, cost, and evaluation of nonstructural measures to improve urban stormwater quality. A survey of urban stormwater managers from Australia, New Zealand, and the United States revealed a widespread trend of increasing use of nonstructural measures among leading stormwater management agencies, with at least 76% of 41 types of nonstructural measures being found to be increasing in use. Data gathered from the survey, an international literature review, and a multicriteria analysis highlighted four nonstructural measures of greatest potential value: mandatory town planning controls that promote the adoption of low-impact development principles and techniques; development of strategic urban stormwater management plans for a city, shire, or catchment; stormwater management measures and programs for construction/building sites; and stormwater management activities related to municipal maintenance operations such as maintenance of the stormwater drainage network and manual litter collections. Knowledge gained on the use and performance of nonstructural measures from the survey, literature review, and three trial evaluation projects was used to develop tailored monitoring and evaluation guidelines for these types of measure. These guidelines incorporate a new evaluation framework based on seven alternative styles of evaluation that range from simply monitoring whether a nonstructural measure has been fully implemented to monitoring its impact on waterway health. This research helps to build the stormwater management industry's knowledge base concerning nonstructural measures and provides a practical tool to address common impediments associated with monitoring and evaluating the performance and cost of these measures.

The human mitochondrial NADH: Ubiquinone oxidoreductase 51-kDa subunit oxidoreductase 51-kDa subunit maps adjacent to the glutathione S-transferase P1-1 gene on chromosome 11q13

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

Spencer, S.R.; Taylor, J.B.; Cowell, I.G.

The soluble glutathione transferases (GSTs) are a family of dimeric isoenymes catalyzing the conjugation of glutathione to hydrophobic electropiles. Their subunits can be grouped into four families, alpha, mu, pi, and theta, on the basis of their primary structures. In man, the pi class is represented by a single gene, GSTP1-1 (GST[pi]) localized to human chromosome 11, band q13. The oncogenes INT2, HSTF1, and PRAD1 are also localized at 11q13, and together with the GSTP1 locus and other gene loci mapped to 11q13, i.e., BCL1 and EMS1, they form a unit of DNA approximately 2000-2500 kb, known as the 11q13more » amplicon, which is often amplified in a range of solid tumors. Any gene locus at 11q13 is of interest because it may influence tumorigenesis. 14 refs., 1 fig.« less

Seismic performance of non-structural components and contents in buildings: an overview of NZ research

NASA Astrophysics Data System (ADS)

Dhakal, Rajesh P.; Pourali, Atefeh; Tasligedik, Ali Sahin; Yeow, Trevor; Baird, Andrew; MacRae, Gregory; Pampanin, Stefano; Palermo, Alessandro

2016-03-01

This paper summarizes the research on non-structural elements and building contents being conducted at University of Canterbury in New Zealand. Since the 2010-2011 series of Canterbury earthquakes, in which damage to non-structural components and contents contributed heavily to downtime and overall financial loss, attention to seismic performance and design of non-structural components and contents in buildings has increased exponentially in NZ. This has resulted in an increased allocation of resources to research leading to development of more resilient non-structural systems in buildings that would incur substantially less damage and cause little downtime during earthquakes. In the last few years, NZ researchers have made important developments in understanding and improving the seismic performance of secondary building elements such as partitions, facades, ceilings and contents.

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

Paterson, Carolyn P.; Ayalew, Lisanework E.; Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acidmore » 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.« less

Isolation and initial structural characterization of a 27 kDa protein from Zingiber officinale

NASA Astrophysics Data System (ADS)

Rasheed, Saima; Malik, Shoaib Ahmad; Falke, Sven; Arslan, Ali; Fazel, Ramin; Schlüter, Hartmut; Betzel, Christian; Choudhary, M. Iqbal

2018-03-01

Zingiber officinale Roscoe (Ginger) is a widely used traditional medicinal plant (for different ailments such as arthritis, constipation, and hypertension). This article describes the isolation and characterization of a so far unknown protein from ginger rhizomes applying ion exchange, affinity, size-exclusion chromatography, small angle X-ray scattering (SAXS), and mass spectrometry techniques. One-dimensional Coomassie-stained SDS-PAGE was performed under non-reducing conditions, showing one band corresponding to approx. 27 kDa. Dynamic light scattering (DLS) analysis of the protein solution revealed monodispersity and a monomeric state of the purified protein. Circular dichroism (CD) spectroscopy strongly indicated a β-sheet-rich protein, and disordered regions. MALDI-TOF-MS, and LC-MS/MS analysis resulted in the identification of 27.29 kDa protein, having 32.13% and 25.34% sequence coverage with Zingipain-1 and 2, respectively. The monomeric state and molecular weight were verified by small angle X-ray scattering (SAXS) studies. An elongated ab-initio model was calculated based on the scattering intensity distribution.

Predicting water-soluble carbohydrates and ethanol-soluble carbohydrates in cool-season grasses with near-infrared reflectance spectroscopy

USDA-ARS?s Scientific Manuscript database

Grazing animals may require a high or low total nonstructural carbohydrate diet for optimal health and production. Understanding how nonstructural carbohydrates fluctuate in Kentucky pastures and being able to quantify and monitor nonstructural carbohydrates in a timely manner will greatly aid in m...

Seasonal dynamics and age of stemwood nonstructural carbohydrates in temperate forest trees

Treesearch

Andrew D. Richardson; Mariah S. Carbone; Trevor F. Keenan; Claudia I. Czimczik; David Y. Hollinger; Paula Murakami; Paul G. Schaberg; Xiaomei Xu

2013-01-01

Nonstructural carbohydrate reserves support tree metabolism and growth when current photosynthates are insufficient, offering resilience in times of stress. We monitored stemwood nonstructural carbohydrate (starch and sugars) concentrations of the dominant tree species at three sites in the northeastern United States. We estimated the mean age of the starch and sugars...

Drought stress, growth, and nonstructural carbohydrate dynamics of pine trees in a semi-arid forest

NASA Astrophysics Data System (ADS)

Klein, Tamir; Yakir, Dan; Hoch, Günter

2014-05-01

• In trees under prolonged drought, both carbon uptake (C source) and growth (C sink) typically decrease. This correlation raises two important questions: (1) to what degree is tree growth limited by C availability; and (2) Is growth limited by concurrent C storage (e.g. as nonstructural carbohydrates, NSC). • To test the relationships between drought, growth, and C reserves, we monitored the changes in NSC levels and constructed stem growth chronologies of Pinus halepensis trees of three drought stress levels growing in Yatir forest, Israel, at the dry limit of forest existence. • Moderately stressed and stressed trees showed 37% and 21% of the stem growth of healthy trees in 2012; 71% and 31% of the sap flux density; and 79% and 66% of the final needle length. In spite of these large reductions, both starch and soluble sugars concentrations in branches of these trees were similar in all trees throughout the dry season (2-4% dry mass). At the same time the root starch concentrations of moderately stressed and stressed trees were 47% and 58% of that of healthy trees, but never below 2% d.m. • Our results suggest that the drought-induced growth reduction is associated with a general C shortage, rather than competition with concurrent C storage. The relatively small effect of drought stress level on NSC dynamics, the maintenance of a 2% d.m. starch, and the continued sap flow indicate that a whole-tree C starvation is not likely to occur in these trees growing at the edge of the desert. Special request: If the abstract is not accepted for presentation in this session, please consider for presentation in session BG2.11 Plant traits and biogeochemical cycles. Thank you.

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

PubMed Central

Hwang, Byung Joon; Toering, Stephanie; Francke, Uta; Chu, Gilbert

1998-01-01

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin. PMID:9632823

Characterization of yam bean (Pachyrhizus erosus) proteins.

PubMed

Morales-Arellano, G Y; Chagolla-López, A; Paredes-López, O; Barba de la Rosa, A P

2001-03-01

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.

Nutritive evaluation of a non-conventional leafy vegetable (Pereskia aculeata Miller).

PubMed

Takeiti, Cristina Y; Antonio, Graziella C; Motta, Eliana M P; Collares-Queiroz, Fernanda P; Park, Kil J

2009-01-01

Pereskia aculeata Miller is a native cactus that can be found in Brazil and is called 'ora-pro-nobis' (OPN). Many people from poor communities consume the dark green leaves of OPN as a vegetable. The objective of the present work was to evaluate the nutritional components in terms of proximate composition, minerals, vitamins, protein content and their in vitro protein digestibility. OPN leaves showed remarkable levels of total dietary fiber (39.1% dry basis), minerals (calcium, magnesium, manganese and zinc) and vitamins (vitamin A, vitamin C and folic acid). Among amino acids, tryptophan was the most abundant (20.5% of the total amino acids) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed small peptides, inferior to 6.5 kDa, and four major bands (61 kDa, 53 kDa, 33 kDa, and 15 kDa). The protein digestibility corrected amino acid score showed the lowest value of sulfur-amino acids (Met+Cys). OPN leaves could be considered a good source of minerals, vitamins and amino acids, and may serve as a potential functional ingredient.

77 FR 28533 - Special Conditions: Boeing, Model 737-800; Large Non-Structural Glass in the Passenger Compartment

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-15

...-0499; Notice No. 25-12-01-SC] Special Conditions: Boeing, Model 737-800; Large Non-Structural Glass in... associated with the installation of large non-structural glass items in the cabin area of an executive... standards that the Administrator considers necessary to establish a level of safety equivalent to that...

77 FR 40255 - Special Conditions: Boeing, Model 737-800; Large Non-Structural Glass in the Passenger Compartment

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-09

...-0499; Special Conditions No. 25-466-SC] Special Conditions: Boeing, Model 737-800; Large Non-Structural... with the installation of large non-structural glass items in the cabin area of an executive interior... Administrator considers necessary to establish a level of safety equivalent to that established by the existing...

Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing.

PubMed

Robinson, Paulette M; Smith, Tyler S; Patel, Dilan; Dave, Meera; Lewin, Alfred S; Pi, Liya; Scott, Edward W; Tuli, Sonal S; Schultz, Gregory S

2012-12-13

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas. Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.

Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells.

PubMed

Sheng, Xiu-Zhen; Wang, Mu; Xing, Jing; Zhan, Wen-Bin

2012-08-13

In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

PubMed

Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

2017-11-01

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

Microparticles prepared with 50-190kDa chitosan as promising non-toxic carriers for pulmonary delivery of isoniazid.

PubMed

Oliveira, Paula M; Matos, Breno N; Pereira, Priscilla A T; Gratieri, Taís; Faccioli, Lucia H; Cunha-Filho, Marcílio S S; Gelfuso, Guilherme M

2017-10-15

Chitosan biocompatibility and mucoadhesiveness make it an ideal polymer for antituberculotic drugs microcapsulation for pulmonary delivery. Yet, previous study indicated toxicity problems to J-774.1-cells treated with some medium molecular weight (190-310kDa) chitosan microparticles. As polymer molecular weight is a crucial factor to be considered, this paper describes the preparation and characterization of chitosan (50-190kDa) microparticles containing isoniazid (INH). Cytotoxicity assays were also performed on murine peritoneal (J-774.1) and alveolar (AMJ2-C11) macrophages cell lines, followed by cytokines detection from AMJ2-C11 cells. Spray-drying process produced mucoadhesive microparticles from 3.2μm to 3.9μm, entrapping more than 89% of the drug and preserving their chemical stability. Drug release behavior could be controlled by the use of cross-linked or uncross-linked chitosan, the latter leading to a rapid drug release. Mucoadhesive potential of the microparticles was characterized following in vitro and ex vivo assays. Finally, a significant reduction on toxicity against peritoneal macrophages and no toxic effect on alveolar macrophages with use of such microparticles were observed. In conclusion, 50-190kDa chitosan microparticles may act as promising non-cytotoxic carriers for pulmonary delivery of INH showing marked alveoli macrophage activation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation, purification and characterisation of selenium-containing polysaccharides and proteins in selenium-enriched Radix puerariae.

PubMed

Zou, Ye; Zhao, Ting; Mao, Guanghua; Zhang, Min; Zheng, Daheng; Feng, Weiwei; Wang, Wei; Wu, Xiangyang; Yang, Liuqing

2014-01-30

Selenium (Se) is an essential dietary mineral and Radix puerariae (RP) (the dried root of Pueraria lobata Willd.) is a botanical supplement widely used as a nutraceutical. Food enriched with Se provides a feasible and economic approach for production of organic Se compounds. However, little is known about Se-enriched RP and the structure of Se-containing polysaccharides and proteins derived from Se-enriched RP. The organic form of Se accounted for 82.42% of total content. Purification by DEAE-52 and Sephadex G-100 column chromatography yielded three single fractions--RP-SeP-11, RP-SeP-22 and RP-SeP-33--with Se contents of 0.9562 × 10⁻³, 0.6113 × 10⁻³ and 0.3827 × 10⁻³ g kg⁻¹, respectively. RP-SeP-11 (3.5 kDa) was made of glucose, RP-SeP-22 (19.6 kDa) was composed of xylose and glucose, and RP-SeP-33 (97.9 kDa) was made up galactose, mannose and glucose. Two Se-containing proteins were obtained with Se content of 3.175 × 10⁻³ and 4.328 × 10⁻³ g kg⁻¹, respectively. One appeared as three subunits with molecular masses of 43.0, 29.0 and 17.8 kDa while the other appeared as two subunits with molecular masses of 43.0 and 26.3 kDa. The results provide a basis for promoting the utilisation of RP resources enriched with Se as a promising tool for the food industry and are significant for its contribution to Se biochemistry in plants. © 2013 Society of Chemical Industry.

Modification of linear prepolymers to tailor heterogeneous network formation through photo-initiated Polymerization-Induced Phase Separation

PubMed Central

Szczepanski, Caroline R.; Stansbury, Jeffrey W.

2015-01-01

Polymerization-induced phase separation (PIPS) was studied in ambient photopolymerizations of triethylene glycol dimethacrylate (TEGDMA) modified by poly(methyl methacrylate) (PMMA). The molecular weight of PMMA and the rate of network formation (through incident UV-irradiation) were varied to influence both the promotion of phase separation through increases in overall free energy, as well as the extent to which phase development occurs during polymerization through diffusion prior to network gelation. The overall free energy of the polymerizing system increases with PMMA molecular weight, such that PIPS is promoted thermodynamically at low loading levels (5 wt%) of a higher molecular weight PMMA (120 kDa), while a higher loading level (20 wt%) is needed to induce PIPS with lower PMMA molecular weight (11 kDa), and phase separation was not promoted at any loading level tested of the lowest molecular weight PMMA (1 kDa). Due to these differences in overall free energy, systems modified by PMMA (11 kDa) underwent phase separation via Nucleation and Growth, and systems modified by PMMA (120 kDa), followed the Spinodal Decomposition mechanism. Despite differences in phase structure, all materials form a continuous phase rich in TEGDMA homopolymer. At high irradiation intensity (Io=20mW/cm2), the rate of network formation prohibited significant phase separation, even when thermodynamically preferred. A staged curing approach, which utilizes low intensity irradiation (Io=300µW/cm2) for the first ~50% of reaction to allow phase separation via diffusion, followed by a high intensity flood-cure to achieve a high degree of conversion, was employed to form phase-separated networks with reduced polymerization stress yet equivalent final conversion and modulus. PMID:26190865

Solution Structure of the 128 kDa Enzyme I Dimer from Escherichia coli and Its 146 kDa Complex with HPr Using Residual Dipolar Couplings and Small- and Wide-Angle X-ray Scattering

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

Schwieters, Charles D.; Suh, Jeong-Yong; Grishaev, Alexander

2010-09-17

The solution structures of free Enzyme I (EI, {approx}128 kDa, 575 x 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr ({approx}146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS datamore » that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C{sub 2} symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large ({approx}70-90{sup o}) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.« less

Separation of water-soluble polysaccharides from Cyclocarya paliurus by ultrafiltration process.

PubMed

Xie, Jian-Hua; Shen, Ming-Yue; Nie, Shao-Ping; Zhao, Qiang; Li, Chang; Xie, Ming-Yong

2014-01-30

In this study, ultrafiltration membrane process was employed to separate polysaccharides from Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) to simulate industrial production. Meanwhile, the molecular weight distribution of C. paliurus polysaccharides was investigated by gel permeation chromatography. Four fractions were obtained and named as CPPS-A, CPPS-B, CPPS-C and CPPS-D, respectively. CPPS-A and CPPS-B contained approximately 69.5% and 12.7% of polysaccharides, whose molecular weight were in the range of 100-300 kDa and 120 kDa, respectively. CPPS-C was comprised of two polysaccharides with average molecular weight of 40 kDa and 15 kDa. Results showed that ultrafiltration resulted in the removal of parts of small molecule weight polysaccharides, the increase of proportion of high molecule weight ones and the obvious improvement of quality of products. Compared with ethanol precipitation and gel permeation chromatography techniques, ultrafiltration showed many advantages, and also provided theoretical support for industrial manufacturing of C. paliurus polysaccharides in separation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

PubMed

Chen, Yeming; Zhao, Luping; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

2014-01-29

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2.

PubMed

Li, Na; Yan, Yunhuan; Zhang, Angke; Gao, Jiming; Zhang, Chong; Wang, Xue; Hou, Gaopeng; Zhang, Gaiping; Jia, Jinbu; Zhou, En-Min; Xiao, Shuqi

2016-12-13

Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Racking tests of non-structural building partitions (The behavior of architectural (nonstructural) building components during earthquakes)

NASA Astrophysics Data System (ADS)

Rihal, S. S.

1980-12-01

The effects of inter-story displacement (drift) during simllated earthquake conditions are reported. The correlation between inter-story relative displacement and building partition behavior, the threshold levels of partition damage, and the fundamental characteristics of non-structural building partitions (stiffness, energy absorption capacity, and strength) under horizontal racking actions were investigated. Parameters in this study consist of geometry of partition configuration and placement of gypsum wallboard panels.

Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

PubMed

Becker, S; Klenk, H D; Mühlberger, E

1996-11-01

The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

Qualitative and Quantitative Analysis of Proteome and Peptidome of Human Follicular Fluid Using Multiple Samples from Single Donor with LC-MS and SWATH Methodology.

PubMed

Lewandowska, Aleksandra E; Macur, Katarzyna; Czaplewska, Paulina; Liss, Joanna; Łukaszuk, Krzysztof; Ołdziej, Stanisław

2017-08-04

Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small-scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient-specific. Ultrafiltration was used to fractionate hFF to high-molecular-weight (HMW) proteome (>10 kDa) and low-molecular-weight (LMW) peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, from which 59 were never reported before as hFF components. 55 (45 not reported before) proteins were found by analyzing LMW fraction, 67 (14 not reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 11 proteins varied substantially among hFF samples from single donors, and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

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

Noisakran, Sansanee; Medical Molecular Biology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Adulyadejvikrom Building; Sengsai, Suchada

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometrymore » (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.« less

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

PubMed

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

2012-02-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

PubMed Central

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

2012-01-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings.

PubMed

Sadeghipour, Hamid Reza; Bhatla, Satish Chander

2002-10-01

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.

A Bayesian framework based on a Gaussian mixture model and radial-basis-function Fisher discriminant analysis (BayGmmKda V1.1) for spatial prediction of floods

NASA Astrophysics Data System (ADS)

Tien Bui, Dieu; Hoang, Nhat-Duc

2017-09-01

In this study, a probabilistic model, named as BayGmmKda, is proposed for flood susceptibility assessment in a study area in central Vietnam. The new model is a Bayesian framework constructed by a combination of a Gaussian mixture model (GMM), radial-basis-function Fisher discriminant analysis (RBFDA), and a geographic information system (GIS) database. In the Bayesian framework, GMM is used for modeling the data distribution of flood-influencing factors in the GIS database, whereas RBFDA is utilized to construct a latent variable that aims at enhancing the model performance. As a result, the posterior probabilistic output of the BayGmmKda model is used as flood susceptibility index. Experiment results showed that the proposed hybrid framework is superior to other benchmark models, including the adaptive neuro-fuzzy inference system and the support vector machine. To facilitate the model implementation, a software program of BayGmmKda has been developed in MATLAB. The BayGmmKda program can accurately establish a flood susceptibility map for the study region. Accordingly, local authorities can overlay this susceptibility map onto various land-use maps for the purpose of land-use planning or management.

Seminar Proceedings Implementation of Nonstructural Measures Held at Ft. Belvoir, Virginia on 15, 16 and 17 November 1983

DTIC Science & Technology

1983-11-01

S-Approximate Household inventory item average chance of being moved (%) High Electric toaster Vacuum cleaner 80 Colour television Medium Record...most rtadily moved are small items of electrical. I equipment and valuable items such as colour televisions. However, many respondents reported that...WESSEX WATER AUTHORITY, "Somerset Land Drainage District, land drainage sur ey report", Wessex Water Authority, Bridgwater, England, 1979. .34 "* • I.U

U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5' splice sites.

PubMed Central

Förch, Patrik; Merendino, Livia; Martínez, Concepción; Valcárcel, Juan

2003-01-01

The splicing factor U2AF(65), U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3' splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF(65) can also promote the recruitment of U1 snRNP to weak 5' splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF(65) is critical for U1 recruitment, and we discuss the role of its RNA-RNA annealing activity in this novel function of U2AF(65). PMID:12558503

Small-molecule inhibitors of hepatitis C virus (HCV) non-structural protein 5A (NS5A): a patent review (2010-2015).

PubMed

Ivanenkov, Yan A; Aladinskiy, Vladimir A; Bushkov, Nikolay A; Ayginin, Andrey A; Majouga, Alexander G; Ivachtchenko, Alexandre V

2017-04-01

Non-structural 5A (NS5A) protein has achieved a considerable attention as an attractive target for the treatment of hepatitis C (HCV). A number of novel NS5A inhibitors have been reported to date. Several drugs having favorable ADME properties and mild side effects were launched into the pharmaceutical market. For instance, daclatasvir was launched in 2014, elbasvir is currently undergoing registration, ledipasvir was launched in 2014 as a fixed-dose combination with sofosbuvir (NS5B inhibitor). Areas covered: Thomson integrity database and SciFinder database were used as a valuable source to collect the patents on small-molecule NS5A inhibitors. All the structures were ranked by the date of priority. Patent holder and antiviral activity for each scaffold claimed were summarized and presented in a convenient manner. A particular focus was placed on the best-in-class bis-pyrrolidine-containing NS5A inhibitors. Expert opinion: Several first generation NS5A inhibitors have recently progressed into advanced clinical trials and showed superior efficacy in reducing viral load in infected subjects. Therapy schemes of using these agents in combination with other established antiviral drugs with complementary mechanisms of action can address the emergence of resistance and poor therapeutic outcome frequently attributed to antiviral drugs.

Low molecular weight chitosan conjugated with folate for siRNA delivery in vitro: optimization studies

PubMed Central

Fernandes, Julio C; Qiu, Xingping; Winnik, Francoise M; Benderdour, Mohamed; Zhang, Xiaoling; Dai, Kerong; Shi, Qin

2012-01-01

The low transfection efficiency of chitosan is one of its drawbacks as a gene delivery carrier. Low molecular weight chitosan may help to form small-sized polymer-DNA or small interfering RNA (siRNA) complexes. Folate conjugation may improve gene transfection efficiency because of the promoted uptake of folate receptor-bearing cells. In the present study, chitosan was conjugated with folate and investigated for its efficacy as a delivery vector for siRNA in vitro. We demonstrate that the molecular weight of chitosan has a major influence on its biological and physicochemical properties, and very low molecular weight chitosan (below 10 kDa) has difficulty in forming stable complexes with siRNA. In this study, chitosan 25 kDa and 50 kDa completely absorbed siRNA and formed nanoparticles (≤220 nm) at a chitosan to siRNA weight ratio of 50:1. The introduction of a folate ligand onto chitosan decreased nanoparticle toxicity. Compared with chitosan-siRNA, folate-chitosan-siRNA nanoparticles improved gene silencing transfection efficiency. Therefore, folate-chitosan shows potential as a viable candidate vector for safe and efficient siRNA delivery. PMID:23209368

The role of nuclear localization signal in parvovirus life cycle.

PubMed

Liu, Peng; Chen, Shun; Wang, Mingshu; Cheng, Anchun

2017-04-14

Parvoviruses are small, non-enveloped viruses with an approximately 5.0 kb, single-stranded DNA genome. Usually, the parvovirus capsid gene contains one or more nuclear localization signals (NLSs), which are required for guiding the virus particle into the nucleus through the nuclear pore. However, several classical NLSs (cNLSs) and non-classical NLSs (ncNLSs) have been identified in non-structural genes, and the ncNLSs can also target non-structural proteins into the nucleus. In this review, we have summarized recent research findings on parvovirus NLSs. The capsid protein of the adeno-associated virus has four potential nuclear localization sequences, named basic region 1 (BR), BR2, BR3 and BR4. BR3 was identified as an NLS by fusing it with green fluorescent protein. Moreover, BR3 and BR4 are required for infectivity and virion assembly. In Protoparvovirus, the canine parvovirus has a common cNLS located in the VP1 unique region, similar to parvovirus minute virus of mice (MVM) and porcine parvovirus. Moreover, an ncNLS is found in the C-terminal region of MVM VP1/2. Parvovirus B19 also contains an ncNLS in the C-terminal region of VP1/2, which is essential for the nuclear transport of VP1/VP2. Approximately 1 or 2 cNLSs and 1 ncNLS have been reported in the non-structural protein of bocaviruses. Understanding the role of the NLS in the process of parvovirus infection and its mechanism of nuclear transport will contribute to the development of therapeutic vaccines and novel antiviral medicines.

Non-Structural Proteins of Arthropod-Borne Bunyaviruses: Roles and Functions

PubMed Central

Eifan, Saleh; Schnettler, Esther; Dietrich, Isabelle; Kohl, Alain; Blomström, Anne-Lie

2013-01-01

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod. PMID:24100888

Response of a 2-story test-bed structure for the seismic evaluation of nonstructural systems

NASA Astrophysics Data System (ADS)

Soroushian, Siavash; Maragakis, E. "Manos"; Zaghi, Arash E.; Rahmanishamsi, Esmaeel; Itani, Ahmad M.; Pekcan, Gokhan

2016-03-01

A full-scale, two-story, two-by-one bay, steel braced-frame was subjected to a number of unidirectional ground motions using three shake tables at the UNR-NEES site. The test-bed frame was designed to study the seismic performance of nonstructural systems including steel-framed gypsum partition walls, suspended ceilings and fire sprinkler systems. The frame can be configured to perform as an elastic or inelastic system to generate large floor accelerations or large inter story drift, respectively. In this study, the dynamic performance of the linear and nonlinear test-beds was comprehensively studied. The seismic performance of nonstructural systems installed in the linear and nonlinear test-beds were assessed during extreme excitations. In addition, the dynamic interactions of the test-bed and installed nonstructural systems are investigated.

Kinetics of Plasma Viremia and Soluble Nonstructural Protein 1 Concentrations in Dengue: Differential Effects According to Serotype and Immune Status

PubMed Central

Duyen, Huynh T. L.; Ngoc, Tran V.; Hang, Vu T. T.; Kieu, Nguyen T. T.; Young, Paul R.; Farrar, Jeremy J.; Simmons, Cameron P.; Wolbers, Marcel; Wills, Bridget A.

2011-01-01

We describe the magnitude and kinetics of plasma viremia and nonstructural protein 1 (sNS1) levels in sequential samples from 167 children with acute dengue, enrolled early in a community study in Vietnam. All children recovered fully, and only 5 required hospitalization. Among those with dengue virus type 1 (DENV-1), plasma viremia was significantly greater in primary (49) than secondary (44) infections and took longer to resolve. In primary DENV-2 and 3 infections, viremia was significantly lower than among primary DENV-1 infections. Concentrations of sNS1 were significantly higher for DENV-1 than for DENV-2 after adjusting for viremia, with marked differences in the kinetic profiles between primary and secondary infections. Secondary infection and higher viremia were independent predictors of more severe thrombocytopenia, and higher viremia was associated with a small increase in hemoconcentration. Our findings identify clear serotype and immune-status related effects on the dynamics of dengue viremia and sNS1 responses, together with associations with important clinical parameters. PMID:21335562

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

PubMed Central

Weinberg, A; Holt, S C

1991-01-01

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 FIG. 11 FIG. 12 FIG. 13 FIG. 14 PMID:1938897

Non-structural Components influencing Hospital Disaster Preparedness in Malaysia

NASA Astrophysics Data System (ADS)

Samsuddin, N. M.; Takim, R.; Nawawi, A. H.; Rosman, M. R.; SyedAlwee, S. N. A.

2018-04-01

Hospital disaster preparedness refers to measures taken by the hospital’s stakeholders to prepare, reduce the effects of disaster and ensure effective coordination during incident response. Among the measures, non-structural components (i.e., medical laboratory equipment & supplies; architectural; critical lifeline; external; updated building document; and equipment & furnishing) are critical towards hospital disaster preparedness. Nevertheless, over the past few years these components are badly affected due to various types of disasters. Hence, the objective of this paper is to investigate the non-structural components influencing hospital’s disaster preparedness. Cross-sectional survey was conducted among thirty-one (31) Malaysian hospital’s employees. A total of 6 main constructs with 107 non-structural components were analysed and ranked by using SPSS and Relative Importance Index (RII). The results revealed that 6 main constructs (i.e. medical laboratory equipment & supplies; architectural; critical lifeline; external; updated building document; and equipment & furnishing) are rated as ‘very critical’ by the respondents. Among others, availability of medical laboratory equipment and supplies for diagnostic and equipment was ranked first. The results could serve as indicators for the public hospitals to improve its disaster preparedness in terms of planning, organising, knowledge training, equipment, exercising, evaluating and corrective actions through non-structural components.

Impact of diatomite on the slightly polluted algae-containing raw water treatment process using ozone oxidation coupled with polyaluminum chloride coagulation.

PubMed

Hu, Wenchao; Wu, Chunde; Jia, Aiyin; Zhang, Zhilin; Chen, Fang

2014-01-01

The impact of adding diatomite on the treatment performance of slightly polluted algae-containing raw water using ozone pre-oxidation and polyaluminum chloride (PAC) coagulation was investigated. Results demonstrated that the addition of diatomite is advantageous due to reduction of the PAC dose (58.33%) and improvement of the removal efficiency of algae, turbidity, and dissolved organic matter (DOM) in raw water. When the ozone concentration was 1.0 mg L⁻¹ and the PAC dosage was 2.5 mg L⁻¹, the removal rates of algae, turbidity, UV254, and TOC were improved by 6.39%, 7.06%, 6.76%, and 4.03%, respectively, with the addition of 0.4 g L⁻¹ diatomite. It has been found that the DOM presented in the Pearl River raw water mainly consisted of small molecules (<1 kDa) and large ones (> 50 kDa). After adding diatomite (0.4 g L⁻¹), the additional removal of 5.77% TOC and 14.82% UV254 for small molecules (<1 kDa) of DOM, and 8.62% TOC and 7.33% UV254 for large ones (>50 kDa) could be achieved, respectively, at an ozone concentration of 1.0 mg L⁻¹ and a PAC dose of 2.5 mg L⁻¹. The growth of anabaena flos-aquae (A.F.) was observed by an atomic force microscope (AFM) before and after adding diatomite. AFM images demonstrate that diatomite may have a certain adsorption on A.F.

Comparison of nonstructural carbohydrates across cranberry cultivars

USDA-ARS?s Scientific Manuscript database

explain low fruit set and biennial bearing tendencies of cranberry (Vaccinium macrocarpon). Yet, comparisons of nonstructural carbohydrate concentrations during critical phenological stages across cultivars that differ in biennial bearing tendencies and return bloom potential are lacking, particular...

A user exposure based approach for non-structural road network vulnerability analysis

PubMed Central

Jin, Lei; Wang, Haizhong; Yu, Le; Liu, Lin

2017-01-01

Aiming at the dense urban road network vulnerability without structural negative consequences, this paper proposes a novel non-structural road network vulnerability analysis framework. Three aspects of the framework are mainly described: (i) the rationality of non-structural road network vulnerability, (ii) the metrics for negative consequences accounting for variant road conditions, and (iii) the introduction of a new vulnerability index based on user exposure. Based on the proposed methodology, a case study in the Sioux Falls network which was usually threatened by regular heavy snow during wintertime is detailedly discussed. The vulnerability ranking of links of Sioux Falls network with respect to heavy snow scenario is identified. As a result of non-structural consequences accompanied by conceivable degeneration of network, there are significant increases in generalized travel time costs which are measurements for “emotionally hurt” of topological road network. PMID:29176832

Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas

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

Gaudino, G.; Cirillo, D.; Naldini, L.

1988-04-01

It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. The authors have shown that the neuropeptide bombesin leads to the activation of a protein-tyrosine kinase that phosphorylates a 115-kDa protein (p115) associated with the bombesin receptor complex in mouse Swiss 3T3 fibroblasts. They now report that phosphotyrosine antibodies recognize a 115-kDa protein, phosphorylated on tyrosine, in four human small cell lung carcinoma cell lines producing bombesin but not in a nonproducer variant line. p115 from detergent-treated small cell lung carcinoma cells binds to bombesin-Sepharose and can be phosphorylatedmore » on tyrosine in the presence of radiolabeled ATP and Mn{sup 2+}. As for the p115 immunoprecipitated from mouse fibroblast, the small cell lung carcinoma p115 can be phosphorylated in an immunocomplex kinase assay. However, the latter does not require the presence of exogenous bombesin for activity. Binding data, obtained by using radiolabeled ligand, suggest receptor occupancy in the cell lines producing bombesin. These observations are consistent with the hypothesis that proliferation in some human small cell lung carcinoma lines is under autocrine control, regulated through activation of bombesin receptors.« less

Gastrointestinal Endogenous Protein-Derived Bioactive Peptides: An in Vitro Study of Their Gut Modulatory Potential

PubMed Central

Dave, Lakshmi A.; Hayes, Maria; Mora, Leticia; Montoya, Carlos A.; Moughan, Paul J.; Rutherfurd, Shane M.

2016-01-01

A recently proposed paradigm suggests that, like their dietary counterparts, digestion of gastrointestinal endogenous proteins (GEP) may also produce bioactive peptides. With an aim to test this hypothesis, in vitro digests of four GEP namely; trypsin (TRYP), lysozyme (LYS), mucin (MUC), serum albumin (SA) and a dietary protein chicken albumin (CA) were screened for their angiotensin-I converting (ACE-I), renin, platelet-activating factor-acetylhydrolase (PAF-AH) and dipeptidyl peptidase-IV inhibitory (DPP-IV) and antioxidant potential following simulated in vitro gastrointestinal digestion. Further, the resultant small intestinal digests were enriched to obtain peptides between 3–10 kDa in size. All in vitro digests of the four GEP were found to inhibit ACE-I compared to the positive control captopril when assayed at a concentration of 1 mg/mL, while the LYS < 3-kDa permeate fraction inhibited renin by 40% (±1.79%). The LYS < 10-kDa fraction inhibited PAF-AH by 39% (±4.34%), and the SA < 3-kDa fraction inhibited DPP-IV by 45% (±1.24%). The MUC < 3-kDa fraction had an ABTS-inhibition antioxidant activity of 150 (±24.79) µM trolox equivalent and the LYS < 10-kDa fraction inhibited 2,2-Diphenyl-1-picrylhydrazyl (DPPH) by 54% (±1.62%). Moreover, over 190 peptide-sequences were identified from the bioactive GEP fractions. The findings of the present study indicate that GEP are a significant source of bioactive peptides which may influence gut function. PMID:27043546

The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity.

PubMed

Heide, Heinrich; Kalisz, Henryk M; Follmann, Hartmut

2004-02-01

A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants. The structure of the 28 kDa protein, which had been envisaged to represent a precursor, or fusion product of the two more specialized, common chloroplast thioredoxins f and m has now been determined by amino acid sequencing. Although it exhibits virtually all the properties and enzyme-modulating activities of a thioredoxin proper this algal protein, surprisingly, does not belong to the thioredoxin family of small redox proteins but is identical with OEE (oxygen evolving enhancer) protein 1, an auxiliary component of the photosystem II manganese cluster. Extracts of Chlorella vulgaris and Chlamydomonas reinhardtii also contain heat-stable protein fractions of 23-26 kDa capable of specifically stimulating chloroplast fructosebisphosphatase in vitro. In contrast, OEE protein 1 from spinach is not able to modulate FbPase or NADP malate dehydrogenase from spinach chloroplasts. A dual function of the OEE protein in algal photosynthesis is envisaged.

The minimal structure containing the band 3 anion transport site. A 35Cl NMR study.

PubMed

Falke, J J; Kanes, K J; Chan, S I

1985-10-25

35Cl NMR, which enables observation of chloride binding to the anion transport site on band 3, is used in the present study to determine the minimal structure containing the intact transport site. Removal of cytoskeletal and other nonintegral membrane proteins, or removal of the 40-kDa cytoskeletal domain of band 3, each leave the transport site intact. Similarly, cleavage of the 52-kDa transport domain into 17- and 35-kDa fragments by chymotrypsin leaves the transport site intact. Extensive proteolysis by papain reduces the integral red cell membrane proteins to their transmembrane segments. Papain treatment removes approximately 60% of the extramembrane portion of the transport domain and produces small fragments primarily in the range 3-7 kDa, with 5 kDa being most predominant. Papain treatment damages, but does not destroy, chloride binding to the transport site; thus, the minimal structure containing the transport site is composed solely of transmembrane segments. In short, the results are completely consistent with a picture in which the transport site is buried in the membrane where it is protected from proteolysis; the transmembrane segments that surround the transport site are held together by strong attractive forces within the bilayer; and the transport site is accessed by solution chloride via an anion channel leading from the transport site to the solution.

Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

NASA Astrophysics Data System (ADS)

Carlsohn, Elisabet; Ångström, Jonas; Emmett, Mark R.; Marshall, Alan G.; Nilsson, Carol L.

2004-05-01

Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: [alpha] (26.5 kDa) and [beta] (61.7 kDa). Three ([alpha][beta]) heterodimers form a trimeric ([alpha][beta])3 assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four ([alpha][beta])3 units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in [alpha], [beta], and native complex) were assigned. Molecular modeling of urease [alpha][beta] complex and trimeric urease units ([alpha][beta])3 revealed a linkage site between the [alpha]-subunit and the [beta]-subunit, and an internal cross-linkage in the [beta]-subunit.

Removal of dissolved organic matter in municipal effluent with ozonation, slow sand filtration and nanofiltration as high quality pre-treatment option for artificial groundwater recharge.

PubMed

Linlin, Wu; Xuan, Zhao; Meng, Zhang

2011-04-01

In the paper the combination process of ozonation, slow sand filtration (SSF) and nanofiltration (NF) was investigated with respect to dissolved organic matter (DOM) removal as high quality pre-treatment option for artificial groundwater recharge. With the help of ozonation leading to breakdown of the large organic molecules, SSF preferentially removes soluble microbial by-product-like substances and DOM with molecular weight (MW) less than 1.0 kDa. NF, however, removes aromatic, humic acid-like and fulvic acid-like substances efficiently and specially removes DOM with MW above 1.0 kDa. The residual DOM of the membrane permeate is dominated by small organics with MW 500 Da, which can be further reduced by the aquifer treatment, despite of the very low concentration. Consequently, the O(3)/SSF/NF system offers a complementary process in DOM removal. Dissolved organic carbon (DOC) and trihalomethane formation potential (THMFP) can be reduced from 6.5±1.1 to 0.7±0.3 mg L(-1) and from 267±24 to 52±6 μg L(-1), respectively. The very low DOC concentration of 0.6±0.2 mg L(-1) and THMFP of 44±4 μg L(-1) can be reached after the aquifer treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Stover composition in maize and sorghum reveals remarkable genetic variation and plasticity for carbohydrate accumulation

DOE PAGES

Sekhon, Rajandeep S.; Breitzman, Matthew W.; Silva, Renato R.; ...

2016-06-08

Carbohydrates stored in vegetative organs, particularly stems, of grasses are a very important source of energy. We examined carbohydrate accumulation in adult sorghum and maize hybrids with distinct phenology and different end uses (grain, silage, sucrose or sweetness in stalk juice, and biomass). Remarkable variation was observed for nonstructural carbohydrates and structural polysaccharides during three key developmental stages both between and within hybrids developed for distinct end use in both species. At the onset of the reproductive phase (average 65 days after planting, DAP), a wide range for accumulation of non-structural carbohydrates (free glucose and sucrose combined), was observed inmore » internodes of maize (11-24%) and sorghum (7-36%) indicating substantial variation for transient storage of excess photosynthate during periods of low grain or vegetative sink strength. Remobilization of these reserves for supporting grain fill or vegetative growth was evident from lower amounts in maize (8-19%) and sorghum (9-27%) near the end of the reproductive period (average 95 DAP). At physiological maturity of grain hybrids (average 120 DAP), amounts of these carbohydrates were generally unchanged in maize (9-21%) and sorghum (16-27%) suggesting a loss of photosynthetic assimilation due to weakening sink demand. Nonetheless, high amounts of non-structural carbohydrates at maturity even in grain maize and sorghum (15-18%) highlight the potential for developing dual-purpose (grain/stover) crops. For both species, the amounts of structural polysaccharides in the cell wall, measured as monomeric components (glucose and pentose), decreased during grain fill but remained unchanged thereafter with maize biomass possessing slightly higher amounts than sorghum. In conclusion, availability of carbohydrates in maize and sorghum highlights the potential for developing energy-rich dedicated biofuel or dual-purpose (grain/stover) crops.« less

Stover composition in maize and sorghum reveals remarkable genetic variation and plasticity for carbohydrate accumulation

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

Sekhon, Rajandeep S.; Breitzman, Matthew W.; Silva, Renato R.

Carbohydrates stored in vegetative organs, particularly stems, of grasses are a very important source of energy. We examined carbohydrate accumulation in adult sorghum and maize hybrids with distinct phenology and different end uses (grain, silage, sucrose or sweetness in stalk juice, and biomass). Remarkable variation was observed for nonstructural carbohydrates and structural polysaccharides during three key developmental stages both between and within hybrids developed for distinct end use in both species. At the onset of the reproductive phase (average 65 days after planting, DAP), a wide range for accumulation of non-structural carbohydrates (free glucose and sucrose combined), was observed inmore » internodes of maize (11-24%) and sorghum (7-36%) indicating substantial variation for transient storage of excess photosynthate during periods of low grain or vegetative sink strength. Remobilization of these reserves for supporting grain fill or vegetative growth was evident from lower amounts in maize (8-19%) and sorghum (9-27%) near the end of the reproductive period (average 95 DAP). At physiological maturity of grain hybrids (average 120 DAP), amounts of these carbohydrates were generally unchanged in maize (9-21%) and sorghum (16-27%) suggesting a loss of photosynthetic assimilation due to weakening sink demand. Nonetheless, high amounts of non-structural carbohydrates at maturity even in grain maize and sorghum (15-18%) highlight the potential for developing dual-purpose (grain/stover) crops. For both species, the amounts of structural polysaccharides in the cell wall, measured as monomeric components (glucose and pentose), decreased during grain fill but remained unchanged thereafter with maize biomass possessing slightly higher amounts than sorghum. In conclusion, availability of carbohydrates in maize and sorghum highlights the potential for developing energy-rich dedicated biofuel or dual-purpose (grain/stover) crops.« less

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

PubMed Central

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

2011-01-01

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

Phenology, growth and physiological adjustments of oil palm (Elaeis guineensis) to sink limitation induced by fruit pruning

PubMed Central

Legros, S.; Mialet-Serra, I.; Caliman, J.-P.; Siregar, F. A.; Clement-Vidal, A.; Fabre, D.; Dingkuhn, M.

2009-01-01

Background and Aims Despite its simple architecture and small phenotypic plasticity, oil palm has complex phenology and source–sink interactions. Phytomers appear in regular succession but their development takes years, involving long lag periods between environmental influences and their effects on sinks. Plant adjustments to resulting source–sink imbalances are poorly understood. This study investigated oil palm adjustments to imbalances caused by severe fruit pruning. Methods An experiment with two treatments (control and complete fruit pruning) during 22 months in 2006–2008) and six replications per treatment was conducted in Indonesia. Phenology, growth of above-ground vegetative and reproductive organs, leaf morphology, inflorescence sex differentiation, dynamics of non-structural carbohydrate reserves and light-saturated net photosynthesis (Amax) were monitored. Key Results Artificial sink limitation by complete fruit pruning accelerated development rate, resulting in higher phytomer, leaf and inflorescence numbers. Leaf size and morphology remained unchanged. Complete fruit pruning also suppressed the abortion of male inflorescences, estimated to be triggered at about 16 months before bunch maturity. The number of female inflorescences increased after an estimated lag of 24–26 months, corresponding to time from sex differentiation to bunch maturity. The most important adjustment process was increased assimilate storage in the stem, attaining nearly 50 % of dry weight in the stem top, mainly as starch, whereas glucose, which in controls was the most abundant non-structural carbohydrate stored in oil palm, decreased. Conclusions The development rate of oil palm is in part controlled by source–sink relationships. Although increased rate of development and proportion of female inflorescences constituted observed adjustments to sink limitation, the low plasticity of plant architecture (constant leaf size, absence of branching) limited compensatory growth. Non-structural carbohydrate storage was thus the main adjustment process. PMID:19748908

Antioxidant activity and oxidative stress protection of duck proteins hydrolysates in SK-N-SH cells.

PubMed

Guo, Yuxing; Pan, Daodong; Wu, Zhen; Zhao, Chuanchuan; Cao, Jinxuan

2013-02-26

Studies have found that natural antioxidants, which are free-radical scavengers, can reduce the risk of diseases caused by free radicals. This work investigated the antioxidant properties of duck proteins hydrolysates. The free-radical scavenging function of CP-1 (M(r) > 10 kDa), CP-2 (5 kDa < M(r) < 10 kDa) and CP-3 (M(r) < 5 kDa), obtained through ultrafiltration and gel filtration were evaluated. The results showed that the lower molecular weight fraction exhibited a stronger free-radical scavenging ability. The highest free-radical scavenging activity was detected in the fraction of p4 purified from CP-3 using Sephadex G-15 column chromatography. The 50% inhibitory value (IC(50)) of p4 for scavenging radicals of superoxide, hydroxyl and 1,1-diphenyl-2-pycrylhydrazyl (DPPH) were, respectively, 0.97 mg mL(-1), 0.84 mg mL(-1) and 1.84 mg mL(-1). Furthermore, the p4 fraction at a concentration of 10 μg mL(-1) increased cell viability from 84.8% to 94% under antioxidative stress in neuroblastoma SK-N-SH cells.

Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381.

PubMed Central

Washington, O R; Deslauriers, M; Stevens, D P; Lyford, L K; Haque, S; Yan, Y; Flood, P M

1993-01-01

Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381. Images PMID:8094377

Purification and characterization of charantin, a napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds.

PubMed

Parkash, A; Ng, T B; Tso, W W

2002-05-01

A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono S and gel filtration on Superdex 75. The N-terminal sequence of charantin exhibited marked similarity to that of the 7.8-kDa napin-like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 400 nm, a potency lower than that of the previously reported small ribosome-inactivating protein gamma-momorcharin (IC50 = 55 nm) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N-glycosidase assay, yielding a band similar to that formed by the small ribosome-inactivating proteins gamma-momorcharin and luffin S.

Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains.

PubMed

Setoh, Yin Xiang; Prow, Natalie A; Rawle, Daniel J; Tan, Cindy Si En; Edmonds, Judith H; Hall, Roy A; Khromykh, Alexander A

2015-06-01

A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent. © 2015 The Authors.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

PubMed Central

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; Kota, Krishna P.; Whitehouse, Chris A.; Bavari, Sina

2016-01-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection. PMID:26837067

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

PubMed

Mudhasani, Rajini; Tran, Julie P; Retterer, Cary; Kota, Krishna P; Whitehouse, Chris A; Bavari, Sina

2016-02-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

DOE PAGES

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; ...

2016-02-02

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.« less

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

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

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.« less

Dietary starch breakdown product sensing mobilizes and apically activates α-glucosidases in small intestinal enterocytes.

PubMed

Chegeni, Mohammad; Amiri, Mahdi; Nichols, Buford L; Naim, Hassan Y; Hamaker, Bruce R

2018-02-20

Dietary starch is finally converted to glucose for absorption by the small intestine mucosal α-glucosidases (sucrase-isomaltase [SI] and maltase-glucoamylase), and control of this process has health implications. Here, the molecular mechanisms were analyzed associated with starch-triggered maturation and transport of SI. Biosynthetic pulse-chase in Caco-2 cells revealed that the high MW SI species (265 kDa) induced by maltose (an α-amylase starch digestion product) had a higher rate of early trafficking and maturation compared with a glucose-induced SI (245 kDa). The maltose-induced SI was found to have higher affinity to lipid rafts, which are associated with enhanced targeting to the apical membrane and higher activity. Accordingly, in situ maltose-hydrolyzing action was enhanced in the maltose-treated cells. Thus, starch digestion products at the luminal surface of small intestinal enterocytes are sensed and accelerate the intracellular processing of SI to enhance starch digestion capacity in the intestinal lumen.-Chegeni, M., Amiri, M., Nichols, B. L., Naim, H. Y., Hamaker, B. R. Dietary starch breakdown product sensing mobilizes and apically activates α-glucosidases in small intestinal enterocytes.

Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

PubMed Central

Crichton, Paul G.; Harding, Marilyn; Ruprecht, Jonathan J.; Lee, Yang; Kunji, Edmund R. S.

2013-01-01

Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein. PMID:23744064

Detection of disease specific sialoglycoconjugate specific antibodies in bronchoalveolar lavage fluid of non-small cell lung cancer patients.

PubMed

Mehta, Sangeeta; Chhetra, Rakhee; Srinivasan, Radhika; Sharma, Suresh C; Behera, Digambar; Ghosh, Sujata

2010-07-01

In this study, we report the presence of significantly higher level of GM3 specific IgG antibodies (IgG(TL)) in the bronchoalveolar lavage fluid obtained from tumor bearing lung of non-small cell lung cancer (NSCLC) patients as compared to other non-neoplastic controls. The antibodies were isolated using DEAE-cellulose anion exchange chromatography and molecular weight of the subunits of IgG(TL) was confirmed in SDS-PAGE. IgG(TL) revealed high specificity to GM3 and the IgG distribution was confined to IgG1. Furthermore, IgG(TL) showed strong reactivity with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. A 66 kDa membrane glycoprotein of NSCLC cell lines was found to interact specifically with IgG(TL), the intensity of which was drastically reduced in presence of GM3. Further, binding of Maackia amurensis agglutinin [specific for NeuAcalpha(2-->3)Gal unit , the same disaccharide unit also known to be present in GM3] to the 66 kDa band confirmed it to be a sialoglycoprotein in nature. IgG(TL) could not show any reactivity to alkaline borohydrate treated or periodate oxidised membrane fractions, suggesting the probable involvement of the carbohydrate moiety of the 66 kDa glycoprotein in the interaction with IgG(TL). Thus, the 66 kDa sialoglycoprotein seems to be the NSCLC specific sialoglycoconjugate. Taken together, IgG(TL) antibodies may have the potential to serve as a unique probe for detail investigation of NSCLC specific cell surface sialoglycoconjugate. Further, due to high specificity of IgG(TL) to GM3, it may be possible to develop a simple alternative diagnostic approach (GM3-ELISA) for NSCLC.

Monoclonal antibody against a serotype antigen of Porphyromonas (Bacteroides) endodontalis and characteristics of the antigen.

PubMed Central

Hanazawa, S; Sagiya, T; Amano, S; Nishikawa, H; Kitano, S

1990-01-01

Recent studies have demonstrated the presence of three serotypes (O1K1, O1K2, and O1K-) of Porphyromonas (Bacteroides) endodontalis. In the present study, a hybridoma cell line producing monoclonal antibody (BEE11) specific for serotype O1K1 of P. endodontalis was established. The specificity of the antibody was evaluated by enzyme-linked immunosorbent assay and immunoslot blot analysis. BEE11 antibody reacted with strains ATCC 35406, HG 400, and HG 421 of the bacterium. However, it did not react with HG 422 or HG 948. Also, the antibody did not react with any of the black-pigmented Bacteroides strains tested. Although the antibody reacted with total cell envelope and capsule materials, it did not do so with lipopolysaccharide. The antibody reacted with antigen material having a molecular mass of 110 kilodaltons (kDa), as judged from fractionation by Superose 12 prep gel chromatography. When the peak fraction from the Superose 12 column was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the reactivity was detected as a single band at an apparent molecular mass of about 52 kDa. The antigen material purified partially by high-performance liquid chromatography was sensitive to trypsin, V8 protease, and heating to 80 degrees C but not to neuraminidase. Therefore, the present study shows that BEE11 antibody recognizes a serotype antigen of P. endodontalis which may be a dimer consisting of monomers having molecular masses of approximately 52 kDa and sensitivity to proteases and heat. Images PMID:2370106

Production and characterization of monoclonal antibodies specific to pangasius catfish, basa, and tra.

PubMed

Gajewski, K G; Chen, Y-T; Hsieh, Y-H P

2009-04-01

Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

The effect of polymer size and charge of molecules on permeation through synovial membrane and accumulation in hyaline articular cartilage.

PubMed

Sterner, B; Harms, M; Wöll, S; Weigandt, M; Windbergs, M; Lehr, C M

2016-04-01

The treatment of joint related diseases often involves direct intra-articular injections. For rational development of novel delivery systems with extended residence time in the joint, detailed understanding of transport and retention phenomena within the joint is mandatory. This work presents a systematic study on the in vitro permeation, penetration and accumulation of model polymers with differing charges and molecular weights in bovine joint tissue. Permeation experiments with bovine synovial membrane were performed with PEG polymers (6-200 kDa) and methylene blue in customized diffusion chambers. For polyethylene glycol, 2-fold (PEG 6 kDa), 3-fold (PEG 10 kDa) and 13-fold (PEG 35 kDa) retention by the synovial membrane in reference to the small molecule methylene blue was demonstrated. No PEG 200 kDa was found in the acceptor in detectable amounts after 48 h. This showed the potential for a distinct extension of joint residence times by increasing molecular weights. In addition, experiments with bovine cartilage tissue were conducted. The ability for positively charged, high molecular weight chitosans and HEMA-Co-TMAP (HCT) polymers (up to 233 kDa) to distribute throughout the entire cartilage matrix was demonstrated. In contrast, a distribution into cartilage was not observed for neutral PEG polymers (6-200 kDa). Furthermore, the positive charge density of different compounds (chitosan, HEMA-Co-TMAP, methylene blue, MSC C1 (neutral NCE) and MSC D1 (positively charged NCE) was found to correlate with their accumulation in bovine cartilage tissue. In summary, the results offer pre-clinical in vitro data, indicating that the modification of molecular size and charge of a substance has the potential to decelerate its clearance through the synovial membrane and to promote accumulation inside the cartilage matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells.

PubMed

Kawano, M; Matsushima, K; Oppenheim, J J

1987-08-01

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.

Upper Mississippi River and Illinois Waterways : non-structural measures cost-benefit study

DOT National Transportation Integrated Search

2003-05-01

These analyses support the U.S. Army Corps of Engineers study of navigation in the Upper Mississippi River (UMR) and Illinois Waterway (IWW) and address the need to examine the potential of non-structural measures to improve efficiency in th...

Nonstructural damages of reinforced concrete buildings due to 2015 Ranau earthquake

NASA Astrophysics Data System (ADS)

Adiyanto, Mohd Irwan; Majid, Taksiah A.; Nazri, Fadzli Mohamed

2017-07-01

On 15th June 2016 a moderate earthquake with magnitude Mw5.9 was occurred in Sabah, Malaysia. Specifically, the epicentre was located at 16 km northwest of Ranau. Less than two days after the first event, a reconnaissance mission took action to investigate the damages on buildings. Since the reinforced concrete buildings in Ranau were designed based on gravity and wind load only, a lot of minor to severe damages was occurred. This paper presents the damages on the nonstructural elements of reinforced concrete buildings due to Ranau earthquake. The assessment was conducted via in-situ field investigation covering the visual observation, taking photo, and interview with local resident. Based on in-situ field investigation, there was a lot of damages occurred on the nonstructural elements like the brick walls. Such damages cannot be neglected since it can cause injury and fatality to the victims. Therefore, it can be concluded that the installation of nonstructural elements should be reviewed for the sake of safety.

Differential permeability of the blood-testis barrier during reinitiation of spermatogenesis in adult male rats.

PubMed

Haverfield, Jenna T; Meachem, Sarah J; Nicholls, Peter K; Rainczuk, Katarzyna E; Simpson, Evan R; Stanton, Peter G

2014-03-01

The blood-testis barrier (BTB) sequesters meiotic spermatocytes and differentiating spermatids away from the vascular environment. We aimed to assess whether meiosis and postmeiotic differentiation could occur when the BTB is permeable. Using a model of meiotic suppression and reinitiation, BTB function was assessed using permeability tracers of small, medium, and large (0.6-, 70-, and 150-kDa) sizes to emulate blood- and lymphatic-borne factors that could cross the BTB. Adult rats (n = 9/group) received the GnRH antagonist acyline (10 wk) to suppress gonadotropins, followed by testosterone (24cm Silastic implant), for 2, 4, 7, 10, 15, and 35 days. In acyline-suppressed testes, all tracers permeated the seminiferous epithelium. As spermatocytes up to diplotene stage XIII reappeared, both the 0.6- and 70-kDa tracers, but not 150 kDa, permeated around these cells. Intriguingly, the 0.6- and 70-kDa tracers were excluded from pachytene spermatocytes at stages VII and VIII but not in subsequent stages. The BTB became progressively impermeable to the 0.6- and 70-kDa tracers as stages IV-VII round spermatids reappeared in the epithelium. This coincided with the appearance of the tight junction protein, claudin-12, in Sertoli cells and at the BTB. We conclude that meiosis can occur when the BTB is permeable to factors up to 70 kDa during the reinitiation of spermatogenesis. Moreover, BTB closure corresponds with the presence of particular pachytene spermatocytes and round spermatids. This research has implications for understanding the effects of BTB dynamics in normal spermatogenesis and also potentially in states where spermatogenesis is suppressed, such as male hormonal contraception or infertility.

The effect of seaweed Ecklonia maxima extract and mineral nitrogen on fodder grass chemical composition.

PubMed

Ciepiela, Grażyna Anna; Godlewska, Agnieszka; Jankowska, Jolanta

2016-02-01

The objective of this study was to determine the effect of the biostimulant Kelpak and different nitrogen rates on cellulose, hemicellulose and lignin contents as well as non-structural carbohydrates in orchard grass and Braun's festulolium. The experiment was a split-plot arrangement with three replicates. It was set up at the experimental facility of the University of Natural Sciences and Humanities, Siedlce, in late April 2009. The following factors were examined: biostimulant with the trade name Kelpak SL applied at 2 dm(3) ha(-1) and a control-no biostimulant; nitrogen application rates 50 and 150 kg ha(-1) and a control (0 kg ha(-1)); pure stands of grass species grown in monoculture--orchard grass (Dactylis glomerata), cv. Amila,-Braun's festulolium (Festulolium braunii), cv. Felopa. Kelpak significantly increased non-structural carbohydrates, and increasing nitrogen rates reduced the concentration of these components in plants. Increasing nitrogen rates significantly decreased cellulose, hemicellulose, lignin and non-structural carbohydrate contents. Compared with orchard grass, Braun's festulolium proved to be of a higher nutritional value due to lower cellulose, hemicellulose and lignin contents and more non-structural carbohydrates. The aforementioned contents in the grasses differed significantly depending on the cut. Most cellulose and non-structural carbohydrates were determined in second-cut grass whereas most hemicellulose and lignin in second-cut grass.

Ultratight crystal packing of a 10 kDa protein

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

Trillo-Muyo, Sergio; Jasilionis, Andrius; Domagalski, Marcin J.

2013-03-01

The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

Effects of autoclaving and high pressure on allergenicity of hazelnut proteins

PubMed Central

2012-01-01

Background Hazelnut is reported as a causative agent of allergic reactions. However it is also an edible nut with health benefits. The allergenic characteristics of hazelnut-samples after autoclaving (AC) and high-pressure (HHP) processing have been studied and are also presented here. Previous studies demonstrated that AC treatments were responsible for structural transformation of protein structure motifs. Thus, structural analyses of allergen proteins from hazelnut were carried out to observe what is occurring in relation to the specific-IgE recognition of the related allergenic proteins. The aims of this work are to evaluate the effect of AC and HHP processing on hazelnut in vitro allergenicity using human-sera and to analyse the complexity of hazelnut allergen-protein structures. Methods Hazelnut-samples were subjected to AC and HHP processing. The specific IgE- reactivity was studied in 15 allergic clinic-patients via western blotting analyses. A series of homology-based-bioinformatics 3D-models (Cora 1, Cora 8, Cora 9 and Cora 11) were generated for the antigens included in the study to analyse the co mplexity of their protein structure. This study is supported by the Declaration of Helsinki and subsequent ethical guidelines. Results A severe reduction in vitro in allergenicity to hazelnut after AC processing was observed in the allergic clinic-patients studied. The specific-IgE binding of some of the described immunoreactive hazelnut protein-bands: Cora 1 ~18KDa, Cora 8 ~9KDa, Cora 9 ~35-40KDa and Cora 11 ~47-48 KDa decreases. Furthermore a relevant glycosylation was assigned and visualized via structural analysis of proteins (3D-modelling) for the first time in the protein-allergen Cora 11 showing a new role which could open a new door for allergenicity-unravellings. Conclusion Hazelnut allergenicity-studies in vivo via Prick-Prick and other means using AC processing are crucial to verify the data we observed via in vitro analyses. Glycosylation studies provided us with clues to elucidate, in the near future, mechanisms of the structures that contribute to hazelnut allergenicity, which thus, in turn, help alleviate food allergens. PMID:22616776

Rapid Analysis of Nonstructural Carbohydrate Components in Grass Forage Using Microplate Enzymatic Assays

USDA-ARS?s Scientific Manuscript database

Measurements of nonstructural carbohydrates (NSC) in plant tissues are important to estimate plant organ resources available for plant growth and stress tolerance or for feed value to grazing animals. A popular commercially available assay kit used to detect glucose with a light sensitive dye reacti...

Non-structural Carbohydrate Reserves of Eelgrass Zostera marina

Treesearch

William C. Dennison; Kenneth A. Moore

1996-01-01

The high minimum light requirement of eelgrass Zostera marina L. suggests that this species has difficulty in maintaining a positive carbon balance except under high fight conditions. The carbon balance of Z. marina can be studied by following seasonal changes in non-structural carbohydrate (NSC) reserves, however, little is...

Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

PubMed

Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

2003-05-01

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

Carboxymethylated degraded polysaccharides from Enteromorpha prolifera: Preparation and in vitro antioxidant activity.

PubMed

Shi, Mei-Jia; Wei, Xiaoyi; Xu, Jie; Chen, Bing-Jie; Zhao, De-Yin; Cui, Shuai; Zhou, Tao

2017-01-15

In order to improve the bioactivities of the polysaccharide from Enteromorpha prolifera (PE), crude PE (Mw 1400kDa) was degraded to low molecular weight polysaccharide (44kDa) in the presence of hydrogen peroxide/ascorbic acid, followed by carboxymethylation. The reaction conditions for carboxymethylation of degraded polysaccharide (DPE) were optimized by Response Surface Methodology. The carboxymethyled degraded polysaccharide (CDPE) obtained under optimized conditions, with a degree of carboxymethylation of 0.849, was characterized by FT-IR and (13)C NMR. The molecular weight of CDPE was measured to be 53.7kDa. CDPE was evaluated for its antioxidant activity by determining the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals, and by determining the ferric reducing power. The antioxidant activity of CDPE was found to be greatly improved in comparison with degraded polysaccharide (DPE) and crude polysaccharide from Enteromorpha prolifera (PE). Copyright © 2016 Elsevier Ltd. All rights reserved.

Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

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

Coseno,M.; Martin, G.; Berger, C.

Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

Crystal structure of the 25 kDa subunit of human cleavage factor Im

PubMed Central

Coseno, Molly; Martin, Georges; Berger, Christopher; Gilmartin, Gregory; Keller, Walter; Doublié, Sylvie

2008-01-01

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing. PMID:18445629

Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa.

PubMed

Turgeon, B; Saba-El-Leil, M K; Meloche, S

2000-02-15

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

The Impact of Structured and Nonstructured Work Experiences on College Students' Attitudes, Values, and Academic Performance.

ERIC Educational Resources Information Center

Heller, Barbara R.; Heinemann, Harry N.

1987-01-01

A study of 353 college students compared effects of structured and nonstructured forms of work on career and school goals, attitudes toward work, and work-related values. Results indicate that working during college seems to relate to attitudes toward work, education, and student life. (CH)

Non-structural carbohydrates in woody plants compared among laboratories

Treesearch

Audrey G. Quentin; Elizabeth A. Pinkard; Michael G. Ryan; David T. Tissue; L. Scott Baggett; Henry D. Adams; Pascale Maillard; Jacqueline Marchand; Simon M. Landhausser; Andre Lacointe; Yves Gibon; William R. L. Anderegg; Shinichi Asao; Owen K. Atkin; Marc Bonhomme; Caroline Claye; Pak S. Chow; Anne Clement-Vidal; Noel W. Davies; L. Turin Dickman; Rita Dumbur; David S. Ellsworth; Kristen Falk; Lucía Galiano; Jose M. Grunzweig; Henrik Hartmann; Gunter Hoch; Sharon Hood; Joanna E. Jones; Takayoshi Koike; Iris Kuhlmann; Francisco Lloret; Melchor Maestro; Shawn D. Mansfield; Jordi Martinez-Vilalta; Mickael Maucourt; Nathan G. McDowell; Annick Moing; Bertrand Muller; Sergio G. Nebauer; Ulo Niinemets; Sara Palacio; Frida Piper; Eran Raveh; Andreas Richter; Gaelle Rolland; Teresa Rosas; Brigitte Saint Joanis; Anna Sala; Renee A. Smith; Frank Sterck; Joseph R. Stinziano; Mari Tobias; Faride Unda; Makoto Watanabe; Danielle A. Way; Lasantha K. Weerasinghe; Birgit Wild; Erin Wiley; David R. Woodruff

2016-01-01

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also...

Reuse of textile effluent treatment plant sludge in building materials.

PubMed

Balasubramanian, J; Sabumon, P C; Lazar, John U; Ilangovan, R

2006-01-01

This study examines the potential reuse of textile effluent treatment plant (ETP) sludge in building materials. The physico-chemical and engineering properties of a composite textile sludge sample from the southern part of India have been studied. The tests were conducted as per Bureau of Indian Standards (BIS) specification codes to evaluate the suitability of the sludge for structural and non-structural application by partial replacement of up to 30% of cement. The cement-sludge samples failed to meet the required strength for structural applications. The strength and other properties met the Bureau of Indian Standards for non-structural materials such as flooring tiles, solid and pavement blocks, and bricks. Results generally meet most ASTM standards for non-structural materials, except that the sludge-amended bricks do not meet the Grade NW brick standard. It is concluded that the substitution of textile ETP sludge for cement, up to a maximum of 30%, may be possible in the manufacturing of non-structural building materials. Detailed leachability and economic feasibility studies need to be carried out as the next step of research.

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

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

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains

PubMed Central

2017-01-01

The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell. PMID:28486476

Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.

PubMed

Laurie, Diane E; Splan, Rebecca K; Green, Kari; Still, Katherine M; McKown, Robert L; Laurie, Gordon W

2012-09-12

Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

Hard-to-cook bean (Phaseolus vulgaris L.) proteins hydrolyzed by alcalase and bromelain produced bioactive peptide fractions that inhibit targets of type-2 diabetes and oxidative stress.

PubMed

Oseguera-Toledo, Miguel E; Gonzalez de Mejia, Elvira; Amaya-Llano, Silvia L

2015-10-01

The objective was to evaluate the effect of bioactive peptide fractions from de-hulled hard-to-cook (HTC) bean on enzyme targets of type-2 diabetes and oxidative stress. Protein isolates from Pinto Durango and Negro 8025 beans were hydrolyzed (120min) with either alcalase® or bromelain and separated into five peptide fractions (<1, 1-3.5, 3.5-5, 5-10, and >10kDa) using an ultrafiltration membrane system. The <1kDa pinto Durango-bromelain fraction showed the best inhibition of α-amylase (49.9±1.4%), and the <1kDa pinto Durango-alcalase fraction inhibited both, α-glucosidase (76.4±0.5%), and dipeptidyl peptidase-IV (DPP-IV, 55.3±1.6%). Peptides LLSL, QQEG and NEGEAH were present in the most potent fractions. Hydrolysates and peptide fractions showed antioxidant capacity (ORAC: 159.6±2.9 to 932.6±1.1mmolTE/g) and nitric oxide inhibition (57.5±0.9 to 68.3±4.2%). Hydrolysates and fractions <1 and 1-3kDa were able to increase glucose-stimulated insulin secretion from iNS-1E cells up to 57% compared to glucose control. Hydrolysates from HTC beans inhibited enzymes related to diabetes management, being the smallest peptides (<1kDa) the most potent. HTC bean could be a source of protein to produce bioactive peptides with potential antidiabetic properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

The effect of pressure and mobile phase velocity on the retention properties of small analytes and large biomolecules in ultra-high pressure liquid chromatography.

PubMed

Fekete, Szabolcs; Veuthey, Jean-Luc; McCalley, David V; Guillarme, Davy

2012-12-28

A possible complication of ultra-high pressure liquid chromatography (UHPLC) is related to the effect of pressure and mobile phase velocity on the retention properties of the analytes. In the present work, numerous model compounds have been selected including small molecules, peptides, and proteins (such as monoclonal antibodies). Two instrumental setups were considered to attain elevated pressure drops, firstly the use of a post-column restrictor capillary at low mobile phase flow rate (pure effect of pressure) and secondly the increase of mobile phase flow rate without restrictor (i.e. a combined effect of pressure and frictional heating). In both conditions, the goal was to assess differences in retention behaviour, depending on the type or character of the analyte. An important conclusion is that the effect of pressure and mobile phase velocity on retention varied in proportion with the size of the molecule and in some cases showed very different behaviour. In isocratic mode, the pure effect of pressure (experiments with a post-column restrictor capillary) induces an increase in retention by 25-100% on small molecules (MW<300 g/mol), 150% for peptides (~1.3 kDa), 800% for insulin (~6 kDa) and up to >3000% for myoglobin (~17 kDa) for an increase in pressure from 100 bar up to 1100 bar. The important effect observed for the isocratic elution of proteins is probably related to conformational changes of the protein in addition to the effect of molecular size. Working in gradient elution mode, the pressure related effects on retention were found to be less pronounced but still present (an increase of apparent retention factor between 0.2 and 2.5 was observed). Copyright © 2012 Elsevier B.V. All rights reserved.

Ocular tissue distribution and pharmacokinetic study of a small 13kDa domain antibody after intravitreal, subconjuctival and eye drop administration in rabbits.

PubMed

Gough, Gerald; Szapacs, Matthew; Shah, Tejash; Clements, Peter; Struble, Craig; Wilson, Robert

2018-02-01

Domain antibodies (dAb's) comprise the smallest functional unit of human IgG and can be targeted to a range of different soluble cytokine and receptor targets in the eye. In particular their small size may offer advantage for ocular tissue penetration and distribution. To investigate this we used a 13kDa tool molecule to undertake a preliminary short term ocular tissue distribution and pharmacokinetic study in the rabbit eye. The dAb was administered by the intravitreal or subconjunctival route or, as topical eye drops for up to five days and dAb concentrations measured in vitreous, aqueous, conjunctiva, choroid-RPE, retina, iris, sclera, and ciliary body. The observed elimination half-live of the dAb (~3 days) in vitreous showed a similar elimination rate to that of a much larger (∼50kDa) Fab fragment whilst the half-life following subconjunctival administration was ∼24 h and, after eye drop dosing the dAb was detectable in aqueous and conjunctiva. These preliminary data show that the intravitreal half-life of dAb's are similar to much larger antibody fragments, offering the potential to deliver significantly more drug to target on a molar basis with a single intravitreal injection potentially enabling dosing frequencies of once a month or less. Subconjunctival injection may provide short duration therapeutic levels of dAb to the anterior and posterior chamber whilst topical eye drop delivery of dAbs may be useful in front-of-eye disease. These data indicate that small domain antibodies may have utility in ophthalmology. Further studies are warranted. Copyright © 2017 Elsevier Ltd. All rights reserved.

A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

PubMed

Topilina, Natalya I; Ermolenkov, Vladimir V; Sikirzhytski, Vitali; Higashiya, Seiichiro; Lednev, Igor K; Welch, John T

2010-07-01

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non-chondrodystrophoid (greyhound) canine intervertebral disc.

PubMed

Melrose, J; Taylor, T K; Ghosh, P

1997-06-01

Trypsin inhibitory proteins of low buoyant density (p < or = 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter-alpha-trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti-human alpha1-proteinase inhibitor (alpha-1-PI), alpha2-macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti-BPTI detected an inhibitor species of approximately 85-90 kDa; anti-ITI detected species of 120-250 kDa; biotinylated trypsin detected species of 60-110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti-BPTI.

ADP-ribosylation of transducin by pertussis toxin

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

Watkins, P.A.; Burns, D.L.; Kanaho, Y.

1985-11-05

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is (TSP)ADP-ribosylated by pertussis toxin and (TSP)NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1more » molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and TS kDa. The amino terminus of both 38- and TS-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The TS-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of (TSP)ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased (TSP)ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed (TSP)ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma.« less

Ambient ozone effects on gas exchange and total non-structural carbohydrate levels in cutleaf coneflower (Rudbeckia laciniata L.) growing in Great Smoky Mountains National Park

USDA-ARS?s Scientific Manuscript database

Ozone-sensitive and -tolerant individuals of the perennial herbaceous cutleaf coneflower (Rudbeckia laciniata L.) were compared for their gas exchange characteristics and total non-structural carbohydrates in the Great Smoky Mountains National Park USA. Net photosynthesis decreased with increased f...

Distribution and mixing of old and new nonstructural carbon in two temperate trees

Treesearch

Andrew D. Richardson; Mariah S. Carbone; Brett A. Huggett; Morgan E. Furze; Claudia I. Czimczik; Jennifer C. Walker; Xiaomei Xu; Paul G. Schaberg; Paula Murakami

2015-01-01

We know surprisingly little about whole-tree nonstructural carbon (NSC; primarily sugars and starch) budgets. Even less well understood is the mixing between recent photosynthetic assimilates (new NSC) and previously stored reserves. And, NSC turnover times are poorly constrained. We characterized the distribution of NSC in the stemwood, branches, and roots of two...

Age, allocation and availability of nonstructural carbon in mature red maple trees

Treesearch

Mariah S. Carbone; Claudia I. Czimczik; Trevor F. Keenan; Paula F. Murakami; Neil Pederson; Paul G. Schaberg; Xiaomei Xu; Andrew D. Richardson

2013-01-01

The allocation of nonstructural carbon (NSC) to growth, metabolism and storage remains poorly understood, but is critical for the prediction of stress tolerance and mortality.We used the radiocarbon (14C) ‘bomb spike’ as a tracer of substrate and age of carbon in stemwood NSC, CO2 emitted by stems, tree...

The effect of solids retention times on the characterization of extracellular polymeric substances and soluble microbial products in a submerged membrane bioreactor.

PubMed

Duan, Liang; Song, Yonghui; Yu, Huibin; Xia, Siqing; Hermanowicz, Slawomir W

2014-07-01

In this study, the effect of solids retention times (SRTs) on extracellular polymeric substances (EPS) and soluble microbial products (SMPs) were investigated in a membrane bioreactor (MBR) at SRTs of 10, 5 and 3 days. The results showed that more carbohydrates and proteins were accumulated at short SRT, which can due to the higher biomass activity in the reactor. The molecular weight (MW) distribution analysis suggested that macromolecules (MW>30 kDa) and small molecules (MW<1 kDa) were the dominant fraction of EPS and SMP, respectively. The reactor at shorter SRT had more small molecules and less macromolecules of carbohydrates. The MW distribution of total organic carbon (TOC) suggested that other organic moieties were exuded by microbes into the solution. The shorter SRT had more undefined microbial by-product-like substances and different O − H bonds in hydroxyl functional groups. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recombinant expression, isolation, and proteolysis of extracellular matrix-secreted phosphoprotein-24 kDa.

PubMed

Murray, Elsa J Brochmann; Murray, Samuel S; Simon, Robert; Behnam, Keyvan

2007-01-01

Secreted phosphoprotein-24 kDa (spp24) is an extracellular matrix protein first cloned from bone. Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a pro-osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin. A synthetic peptide corresponding to the TRH1 domain of spp18.5 and spp24 specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo. The parental protein, spp24, exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded. The availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure:function relationships. We describe here the expression of His(6)-tagged bovine spp24 (residues 24 to 203) in E. coli, its purification by high-resolution IMAC (immobilized metal affinity chromatography), and the characterization of the full-length recombinant 21.5 kDa protein and its two major 16 kDa and 14.5 kDa degradation products (spp24, residues 24 to 157, and spp24, residues 24 to 143) by mass spectroscopy. The recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, bypassing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.

Wind-Tunnel Evaluation of the Effect of Blade Nonstructural Mass Distribution on Helicopter Fixed-System Loads

NASA Technical Reports Server (NTRS)

Wilbur, Matthew L.; Yeager, William T., Jr.; Singleton, Jeffrey D.; Mirick, Paul H.; Wilkie, W. Keats

1998-01-01

This report provides data obtained during a wind-tunnel test conducted to investigate parametrically the effect of blade nonstructural mass on helicopter fixed-system vibratory loads. The data were obtained with aeroelastically scaled model rotor blades that allowed for the addition of concentrated nonstructural masses at multiple locations along the blade radius. Testing was conducted for advance ratios ranging from 0.10 to 0.35 for 10 blade-mass configurations. Three thrust levels were obtained at representative full-scale shaft angles for each blade-mass configuration. This report provides the fixed-system forces and moments measured during testing. The comprehensive database obtained is well-suited for use in correlation and development of advanced rotorcraft analyses.

pH : a key control of the nature and distribution of dissolved organic matter and associated trace metals in soil

NASA Astrophysics Data System (ADS)

Pédrot, M.; Dia, A.; Davranche, M.

2009-04-01

Dissolved organic matter is ubiquitous at the Earth's surface and plays a prominent role in controlling metal speciation and mobility from soils to hydrosystems. Humic substances (HS) are usually considered to be the most reactive fraction of organic matter. Humic substances are relatively small and formed by chemically diverse organic molecules, bearing different functional groups that act as binding sites for cations and mineral surfaces. Among the different environmental physicochemical parameters controlling the metal speciation, pH is likely to be the most important one. Indeed, pH affect the dissociation of functional groups, and thus can influence the HS structure, their ability to complex metals, their solubility degree allowing the formation of aggregates at the mineral surface. In this context, soil/water interactions conducted through batch system experiments, were carried out with a wetland organic-rich soil to investigate the effect of pH on the release of dissolved organic carbon (DOC) and associated trace elements. The pH was regulated between 4 and 7.5 using an automatic pH stat titrator. Ultrafiltration experiments were performed to separate the dissolved organic pool following decreasing pore sizes (30 kDa, 5 kDa and 2 kDa with 1 Da = 1 g.mol-1). The pH increase induced a significant DOC release, especially in heavy organic molecules (size >5 kDa) with a high aromaticity (>30 %). These were probably humic acids (HA). This HA release influenced (i) directly the trace element concentrations in soil solution since HA were enriched in several trace elements such as Th, REE, Y, U, Cr and Cu; and (ii) indirectly by the breaking of clay-humic complexes releasing Fe- and Al-rich nanoparticles associated with V, Pb and Ti. By contrast, at acid pH, most HS were complexed onto mineral surfaces. They also sequestered iron nanoparticles. Therefore, at low pH, most part of DOC molecules had a size < 5 kDa and lower aromaticity. Thus, the DOC was mostly composed of simple organic compounds little complexing. Consequently, the soil solution was depleted in trace elements such as Th, REE, Y, U, Cr, Cu, Al, Fe, V, Pb and Ti, but also enriched in Ca, Sr, Ba, Mn, Mg, Co, Zn and in a lesser proportion in Rb, Li and Ni. The aromaticity in the fractions <5 kDa was higher than in the fractions <30 kDa or <0.2 µm. Complementary experiments were performed to understand the HS size distribution and aromaticity according to pH and ionic strength .The molecular size and shape of HS is usually explained by two concepts: (i) the macropolymeric structure with heavy organic molecules considered to be flexible linear polyelectrolytes and (ii) the supramolecular structure with an association of a complex mixture of different molecules held together by dispersive weak forces. Ours results supported the HA supramolecular structure at neutral or basic pH conditions. But, at acid pH, a disruption of the humic supramolecular associations involved the release of small organic molecules with a high aromaticity. Moreover, this aromaticity variation can be due also to the presence of fulvic acids in the fractions <5 kDa and a mixture of heavy organic molecules little complexing in the fractions >5 kDa. These latter molecules displayed a low aromaticity decreasing the global aromaticity of the fractions <30 kDa and <0.2 µm. To summarize, these new data demonstrated that the DOC and trace element concentrations of the soil solutions were strongly controlled by pH. This parameter influenced the nature and the size of the DOC as well as, the trace element concentrations in the soil solutions, with a decreasing contribution of HA when pH decreased. This pH dependence is a key issue of concern since local (human pressure) and/or global (climatic) warning result in pH water changes.

Grand Lake Saint Marys, Ohio, Survey Report for Flood Control and Allied Purposes. Volume 1.

DTIC Science & Technology

1981-08-01

nuprotected shoreline have reduced the lake depth. A range of structural and, nanatructural flood damage reduction1 usasures wee exmined. Nonstructural masaes ...24 330 Apr 1972 872.67 32 310 Apr 1938 872.42 19 550 Feb 1950 872.42 24 520 Apr 1978 872.17 37 380 Jan 1949 872.08 19 260 Apr 1957 872.08 23 550 Jun...1958 871.92 11 380 May 1933 871.92 5 490 Nov 1972 871.92 9 510 16 . ..... Ie Flood Damages The areas under consideration include Beaver Creek

T-cell suppression and selective in vivo activation of TH2 subpopulation by the Entamoeba histolytica 220-kilodalton lectin.

PubMed Central

Talamás-Rohana, P; Schlie-Guzmán, M A; Hernández-Ramírez, V I; Rosales-Encina, J L

1995-01-01

A 220-kDa surface protein (L220) with lectin activity from Entamoeba histolytica trophozoites has been characterized previously (J. L. Rosales-Encina, I. Meza, A. López-de-León, P. Talamás-Rohana, and M. Rojkind, J. Infect. Dis. 156:790-797, 1987). This molecule is involved in the adhesion process (I. Meza, F. Cázares, J. L. Rosales-Encina, P. Talamás-Rohana, and M. Rojkind, J. Infect. Dis. 156:798-805, 1987) and is very immunogenic. In this work, we studied both the humoral and the cellular immune responses to L220. We compared L220 with L220-derived components, such as a fusion peptide (M-11) and chemically obtained peptides (by treating the 220-kDa molecule with N-chlorosuccinimide-urea). Spleen cells from L220-immunized mice were unable to proliferate in vitro when stimulated with the protein. However, a proliferative response was obtained when mice were immunized with the L220-derived fusion peptide or the cleaved lectin. To find out if there was a correlation between the observed responses and TH1 or TH2 activation, we analyzed patterns of cytokine secretion (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon). Cells from mice immunized with peptides that induced cell proliferation (100, 80, and 47 kDa) with the peptides (P < 0.01) and with the intact molecule secreted IL-2 and gamma interferon, showing a TH1-subset pattern. Conversely, cells from mice immunized with the intact 220-kDa molecule secreted IL-4 and IL-10, typical of a TH2 subpopulation; however, antibodies from each group recognized the 220-kDa molecule as determined by Western blotting (immunoblotting). These results suggest that various epitopes in the 220-kDa molecule generate different response patterns, suppressing or activating T-cell responses. PMID:7558304

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes.

PubMed

Crosslin, James M; Hamm, Philip B; Kirk, William W; Hammond, Rosemarie W

2010-04-01

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

Cloning and expression of a small heat and salt tolerant protein (Hsp22) from Chaetomium globosum.

PubMed

Aggarwal, Rashmi; Gupta, Sangeeta; Sharma, Sapna; Banerjee, Sagar; Singh, Priyanka

2012-11-01

The present study reports molecular characterization of small heat shock protein gene in Indian isolates of Chaetomium globosum, C. perlucidum, C. reflexum, C. cochlioides and C. cupreum. Six isolates of C. globosum and other species showed a band of 630bp using specific primers. Amplified cDNA product of C. globosum (Cg 1) cloned and sequenced showed 603bp open reading frame encoding 200 amino-acids. The protein sequence had a molecular mass of 22 kDa and was therefore, named Hsp22. BlastX analysis revealed that the gene codes for a protein homologous to previously characterized Hsp22.4 gene from C. globosum (AAR36902.1, XP 001229241.1) and shared 95% identity in amino acid sequence. It also showed varying degree of similarities with small Hsp protein from Neurospora spp. (60%), Myceliophthora sp. (59%), Glomerella sp. (50%), Hypocrea sp. (52%), and Fusarium spp. (51%). This gene was further cloned into pET28a (+) and transformed E. coli BL21 cells were induced by IPTG, and the expressed protein of 30 kDa was analyzed by SDS-PAGE. The IPTG induced transformants displayed significantly greater resistance to NaCl and Na2CO3 stresses.

Limited overlap between phylogenetic HIV and hepatitis C virus clusters illustrates the dynamic sexual network structure of Dutch HIV-infected MSM.

PubMed

Vanhommerig, Joost W; Bezemer, Daniela; Molenkamp, Richard; Van Sighem, Ard I; Smit, Colette; Arends, Joop E; Lauw, Fanny N; Brinkman, Kees; Rijnders, Bart J; Newsum, Astrid M; Bruisten, Sylvia M; Prins, Maria; Van Der Meer, Jan T; Van De Laar, Thijs J; Schinkel, Janke

2017-09-24

MSM are at increased risk for infection with HIV-1 and hepatitis C virus (HCV). Is HIV/HCV coinfection confined to specific HIV transmission networks? A HIV phylogenetic tree was constructed for 5038 HIV-1 subtype B polymerase (pol) sequences obtained from MSM in the AIDS therapy evaluation in the Netherlands cohort. We investigated the existence of HIV clusters with increased HCV prevalence, the HIV phylogenetic density (i.e. the number of potential HIV transmission partners) of HIV/HCV-coinfected MSM compared with HIV-infected MSM without HCV, and the overlap in HIV and HCV phylogenies using HCV nonstructural protein 5B sequences from 183 HIV-infected MSM with acute HCV infection. Five hundred and sixty-three of 5038 (11.2%) HIV-infected MSM tested HCV positive. Phylogenetic analysis revealed 93 large HIV clusters (≥10 MSM), 370 small HIV clusters (2-9 MSM), and 867 singletons with a median HCV prevalence of 11.5, 11.6, and 9.3%, respectively. We identified six large HIV clusters with elevated HCV prevalence (range 23.5-46.2%). Median HIV phylogenetic densities for MSM with HCV (3, interquartile range 1-7) and without HCV (3, interquartile range 1-8) were similar. HCV phylogeny showed 12 MSM-specific HCV clusters (clustersize: 2-39 HCV sequences); 12.7% of HCV infections were part of the same HIV and HCV cluster. We observed few HIV clusters with elevated HCV prevalence, no increase in the HIV phylogenetic density of HIV/HCV-coinfected MSM compared to HIV-infected MSM without HCV, and limited overlap between HIV and HCV phylogenies among HIV/HCV-coinfected MSM. Our data do not support the existence of MSM-specific sexual networks that fuel both the HIV and HCV epidemic.

Development and application of a time-history analysis for rotorcraft dynamics based on a component approach

NASA Technical Reports Server (NTRS)

Sopher, R.; Hallock, D. W.

1985-01-01

A time history analysis for rotorcraft dynamics based on dynamical substructures, and nonstructural mathematical and aerodynamic components is described. The analysis is applied to predict helicopter ground resonance and response to rotor damage. Other applications illustrate the stability and steady vibratory response of stopped and gimballed rotors, representative of new technology. Desirable attributes expected from modern codes are realized, although the analysis does not employ a complete set of techniques identified for advanced software. The analysis is able to handle a comprehensive set of steady state and stability problems with a small library of components.

Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes

NASA Technical Reports Server (NTRS)

Sukharev, Sergei

2002-01-01

The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.

Production performance and milk fatty acids profile in grazing dairy cows offered ground corn or liquid molasses as the sole supplemental nonstructural carbohydrate source

USDA-ARS?s Scientific Manuscript database

The objective of this study was to compare the effects of corn meal or liquid molasses fed as the sole supplemental nonstructural carbohydrate source on milk yield and composition, milk fatty acids, and N use efficiency in grazing dairy cows. Ten multiparous organically-certified Jersey cows averagi...

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

Direct Replacement of Arbitrary Grid-Overlapping by Non-Structured Grid

NASA Technical Reports Server (NTRS)

Kao, Kai-Hsiung; Liou, Meng-Sing

1994-01-01

A new approach that uses nonstructured mesh to replace the arbitrarily overlapped structured regions of embedded grids is presented. The present methodology uses the Chimera composite overlapping mesh system so that the physical domain of the flowfield is subdivided into regions which can accommodate easily-generated grid for complex configuration. In addition, a Delaunay triangulation technique generates nonstructured triangular mesh which wraps over the interconnecting region of embedded grids. It is designed that the present approach, termed DRAGON grid, has three important advantages: eliminating some difficulties of the Chimera scheme, such as the orphan points and/or bad quality of interpolation stencils; making grid communication in a fully conservative way; and implementation into three dimensions is straightforward. A computer code based on a time accurate, finite volume, high resolution scheme for solving the compressible Navier-Stokes equations has been further developed to include both the Chimera overset grid and the nonstructured mesh schemes. For steady state problems, the local time stepping accelerates convergence based on a Courant - Friedrichs - Leury (CFL) number near the local stability limit. Numerical tests on representative steady and unsteady supersonic inviscid flows with strong shock waves are demonstrated.

Molecular characterization of a 40 kDa OmpC-like porin from Serratia marcescens.

PubMed

Hutsul, J A; Worobec, E

1994-02-01

An oligonucleotide that encodes the N-terminal portion of a 41 kDa porin of Serratia marcescens was used to probe S. marcescens UOC-51 genomic DNA. An 11 kb EcoRI fragment which hybridized with the oligonucleotide was subcloned into Escherichia coli, examined for expression, and sequenced. The product expressed by the cloned gene was 40 kDa. The nucleotide sequence has an ORF of 1.13 kb. When the deduced amino acid sequence was aligned and compared to other enterobacterial porins the cloned S. marcescens porin most closely resembled E. coli OmpC. Although we did not detect osmoregulation or thermoregulation of any porins in S. marcescens UOC-51, sequences analogous to the E. coli osmoregulator OmpR-binding regions are seen upstream to the cloned gene. We examined the regulation of the S. marcescens porin in E. coli and found that its expression increased in a high salt environment. A micF gene, whose transcriptional product functions to inhibit synthesis of OmpF by hybridizing with the ompF transcript, was also seen upstream of the S. marcescens ompC. An alignment with the E. coli micF gene revealed that the functional region of the S. marcescens micF gene is conserved. Based on the results obtained we have determined that S. marcescens UOC-51 produces a 40 kDa porin similar to the E. coli OmpC porin.

Asp- and Glu-specific Novel Dipeptidyl Peptidase 11 of Porphyromonas gingivalis Ensures Utilization of Proteinaceous Energy Sources*

PubMed Central

Ohara-Nemoto, Yuko; Shimoyama, Yu; Kimura, Shigenobu; Kon, Asako; Haraga, Hiroshi; Ono, Toshio; Nemoto, Takayuki K.

2011-01-01

Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp22. A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the kcat/Km value was higher for Asp than Glu. Those activities were lost by substitution of Ser652 in P. endodontalis and Ser655 in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg670 is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg670 interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods. PMID:21896480

Asp- and Glu-specific novel dipeptidyl peptidase 11 of Porphyromonas gingivalis ensures utilization of proteinaceous energy sources.

PubMed

Ohara-Nemoto, Yuko; Shimoyama, Yu; Kimura, Shigenobu; Kon, Asako; Haraga, Hiroshi; Ono, Toshio; Nemoto, Takayuki K

2011-11-04

Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp(22). A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the k(cat)/K(m) value was higher for Asp than Glu. Those activities were lost by substitution of Ser(652) in P. endodontalis and Ser(655) in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg(670) is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg(670) interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods.

Family Ophioviridae

USDA-ARS?s Scientific Manuscript database

The Ophioviridae is a family of filamentous plant viruses, with a single stranded negative, and possibly ambisense, RNA genome of 11.3–12.5 kb in length divided into 3–4 segments, each separately encapsidated by a single coat protein (43–50 kDa). Virions are naked filamentous nucleocapsids, in shape...

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity.

PubMed

Scirè, Andrea; Tanfani, Fabio; Bertoli, Enrico; Furlani, Emiliano; Nadozie, Hope-Onyekwere N; Cerutti, Helena; Cortelazzo, Alessio; Bini, Luca; Guerranti, Roberto

2011-07-15

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds. Copyright © 2011 Elsevier GmbH. All rights reserved.

Paranodal permeability in `myelin mutants'

PubMed Central

Shroff, S.; Mierzwa, A.; Scherer, S.S.; Peles, E.; Arevalo, J.C.; Chao, M.V.; Rosenbluth, J.

2011-01-01

Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three `myelin mutant' mice, Caspr-null, cst-null and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3kDa, 10kDa), which penetrate most fibers, and to larger tracers (40kDa, 70kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of transverse bands in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of transverse bands. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of transverse bands but does depend on the length of the paranode and, in turn, on the length of `pathway 3', the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath. PMID:21618613

Synthesis and Characterization of a Poly(ethylene glycol)-Poly(simvastatin) Diblock Copolymer

PubMed Central

Asafo-Adjei, Theodora A.; Dziubla, Thomas D.; Puleo, David A.

2014-01-01

Biodegradable polyesters are commonly used as drug delivery vehicles, but their role is typically passive, and encapsulation approaches have limited drug payload. An alternative drug delivery method is to polymerize the active agent or its precursor into a degradable polymer. The prodrug simvastatin contains a lactone ring that lends itself to ring-opening polymerization (ROP). Consequently, simvastatin polymerization was initiated with 5 kDa monomethyl ether poly(ethylene glycol) (mPEG) and catalyzed via stannous octoate. Melt condensation reactions produced a 9.5 kDa copolymer with a polydispersity index of 1.1 at 150 °C up to a 75 kDa copolymer with an index of 6.9 at 250 °C. Kinetic analysis revealed first-order propagation rates. Infrared spectroscopy of the copolymer showed carboxylic and methyl ether stretches unique to simvastatin and mPEG, respectively. Slow degradation was demonstrated in neutral and alkaline conditions. Lastly, simvastatin, simvastatin-incorporated molecules, and mPEG were identified as the degradation products released. The present results show the potential of using ROP to polymerize lactone-containing drugs such as simvastatin. PMID:25431653

A proteomic analysis of Pakistan Daboia russelii russelii venom and assessment of potency of Indian polyvalent and monovalent antivenom.

PubMed

Mukherjee, Ashis K; Kalita, Bhargab; Mackessy, Stephen P

2016-07-20

To address the dearth of knowledge on the biochemical composition of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV), the venom proteome has been analyzed and several biochemical and pharmacological properties of the venom were investigated. SDS-PAGE (reduced) analysis indicated that proteins/peptides in the molecular mass range of ~56.0-105.0kDa, 31.6-51.0kDa, 15.6-30.0kDa, 9.0-14.2kDa and 5.6-7.2kDa contribute approximately 9.8%, 12.1%, 13.4%, 34.1% and 30.5%, respectively of Pakistan RVV. Proteomics analysis of gel-filtration peaks of RVV resulted in identification of 75 proteins/peptides which belong to 14 distinct snake venom protein families. Phospholipases A2 (32.8%), Kunitz type serine protease inhibitors (28.4%), and snake venom metalloproteases (21.8%) comprised the majority of Pakistan RVV proteins, while 11 additional families accounted for 6.5-0.2%. Occurrence of aminotransferase, endo-β-glycosidase, and disintegrins is reported for the first time in RVV. Several of RVV proteins/peptides share significant sequence homology across Viperidae subfamilies. Pakistan RVV was well recognized by both the polyvalent (PAV) and monovalent (MAV) antivenom manufactured in India; nonetheless, immunological cross-reactivity determined by ELISA and neutralization of pro-coagulant/anticoagulant activity of RVV and its fractions by MAV surpassed that of PAV. The study establishes the proteome profile of the Pakistan RVV, thereby indicating the presence of diverse proteins and peptides that play a significant role in the pathophysiology of RVV bite. Further, the proteomic findings will contribute to understand the variation in venom composition owing to different geographical location and identification of pharmacologically important proteins in Pakistan RVV. Copyright © 2016. Published by Elsevier B.V.

Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12).

PubMed

Salwan, Richa; Sharma, Vivek; Pal, Mohinder; Kasana, Ramesh Chand; Yadav, Sudesh Kumar; Gulati, Arvind

2018-02-01

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323bp ORF encoding 441 amino acids protein with molecular weight 47.2kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1-21 amino acids, pre-peptide 22-143 amino acids, peptidase S8 domain 144-434 amino acids, and pro-peptide 435-441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed ∼50kDa and ∼40kDa bands, respectively. The pre-propeptide ∼11kDa removed from Alp1 resulted in mature protein of ∼35kDa with 1738Umg -1 specific activity. The recombinant protease was optimally active at 40°C and pH 9, and stable over 10-70°C and 6-12pH. The activity at low-temperature and alkaline pH was supported by high R/(R+K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry. Copyright © 2017. Published by Elsevier B.V.

Detection of Prosecretory Mitogen Lacritin in Nonprimate Tears Primarily as a C-Terminal-Like Fragment

PubMed Central

Laurie, Diane E.; Splan, Rebecca K.; Green, Kari; Still, Katherine M.; McKown, Robert L.; Laurie, Gordon W.

2012-01-01

Purpose. Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Methods. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Results. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal–reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Conclusions. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair. PMID:22871838

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

PubMed

Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

2016-04-04

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia.

PubMed

Gallage, Nethaji J; Jørgensen, Kirsten; Janfelt, Christian; Nielsen, Agnieszka J Z; Naake, Thomas; Dunski, Eryk; Dalsten, Lene; Grisoni, Michel; Møller, Birger Lindberg

2018-02-01

Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast.

The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia

PubMed Central

Gallage, Nethaji J; JØrgensen, Kirsten; Janfelt, Christian; Nielsen, Agnieszka J Z; Naake, Thomas; Duński, Eryk; Dalsten, Lene; Grisoni, Michel; MØller, Birger Lindberg

2018-01-01

Abstract Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast. PMID:29186560

Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China.

PubMed

Huang, Jianying; Zhang, Zhenjie; Zhang, Yiqi; Yang, Yong; Zhao, Jinfeng; Wang, Rongjun; Jian, Fuchun; Ning, Changshen; Zhang, Wanyu; Zhang, Longxian

2018-04-12

Little is known about the prevalence and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in deer in China. In this study, 662 fecal samples were collected from 11 farms in Henan and Jilin Provinces between July 2013 and August 2014, and were screened for the presence of Cryptosporidium and G. duodenalis with genotyping and subtyping methods. Cryptosporidium spp. and G. duodenalis were detected in 6.80% (45/662) and 1.21% (5/662) of samples, respectively. Six Cryptosporidium species/genotypes were identified based on the small subunit ribosomal ribonucleic acid (SSU rRNA) gene: C. parvum (n = 11); C. andersoni (n = 5); C. ubiquitum (n = 3); C. muris (n = 1); C. suis-like (n = 1); and Cryptosporidium deer genotype (n = 24). When five of the 11 C. parvum isolates were subtyped by sequencing the 60 kDa glycoprotein (gp60) gene, zoonotic subtypes IIaA15G2R2 (n = 4) and IIdA19G1 (n = 1) were found. According to a subtype analysis, three C. ubiquitum isolates belonged to XIIa subtype 2. In contrast, only assemblage E was detected in the five Giardia-positive samples with small subunit ribosomal ribonucleic acid (SSU rRNA) gene sequencing. To our knowledge, this is the first study to report C. andersoni, as well as C. parvum zoonotic subtypes IIaA15G2R2 and IIdA19G1 in cervids. These data, though limited, suggest that cervids may be a source of zoonotic Cryptosporidium and Giardia. Cervids in the present study are likely to be of low zoonotic potential to humans, and more molecular epidemiological studies are required to clarify the prevalence and public health significance of Cryptosporidium and G. duodenalis in cervids throughout China.

Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

PubMed

Shimojo, M; Ricketts, M L; Petrelli, M D; Moradi, P; Johnson, G D; Bradwell, A R; Hewison, M; Howie, A J; Stewart, P M

1997-03-01

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the interaction between the MR and aldosterone or cortisol is in part a nuclear event.

Targeting GH-1 splicing as a novel pharmacological strategy for growth hormone deficiency type II.

PubMed

Miletta, Maria Consolata; Flück, Christa E; Mullis, Primus-E

2017-01-15

Isolated growth hormone deficiency type II (IGHD II) is a rare genetic splicing disorder characterized by reduced growth hormone (GH) secretion and short stature. It is mainly caused by autosomal dominant-negative mutations within the growth hormone gene (GH-1) which results in missplicing at the mRNA level and the subsequent loss of exon 3, producing the 17.5-kDa GH isoform: a mutant and inactive GH protein that reduces the stability and the secretion of the 22-kDa GH isoform, the main biologically active GH form. At present, patients suffering from IGHD II are treated with daily injections of recombinant human GH (rhGH) in order to reach normal height. However, this type of replacement therapy, although effective in terms of growth, does not prevent the toxic effects of the 17.5-kDa mutant on the pituitary gland, which may eventually lead to other hormonal deficiencies. As the severity of the disease inversely correlates with the 17.5-kDa/22-kDa ratio, increasing the inclusion of exon 3 is expected to ameliorate disease symptoms. This review focuses on the recent advances in experimental and therapeutic strategies applicable to treat IGHD II in clinical and preclinical contexts. Several avenues for alternative IGHD II therapy will be discussed including the use of small interfering RNA (siRNA) and short hairpin RNA (shRNA) constructs that specifically target the exon 3-deleted transcripts as well as the application of histone deacetylase inhibitors (HDACi) and antisense oligonucleotides (AONs) to enhance full-length GH-1 transcription, correct GH-1 exon 3 splicing and manipulate GH pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

PubMed

Gupta, R; Jentoft, N

1992-02-15

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.

The effect of carbohydrate accumulation and nitrogen deficiency on feedback regulation of photosynthesis in beech (Fagus sylvatica) under elevated CO2 concentration

NASA Astrophysics Data System (ADS)

Klem, K.; Urban, O.; Holub, P.; Rajsnerova, P.

2012-04-01

One of the main manifestations of global change is an increase in atmospheric CO2 concentration. Elevated concentration of CO2 has stimulating effect on plant photosynthesis and consequently also on the productivity. Long-term studies, however, show that this effect is progressively reduced due to feedback regulation of photosynthesis. The main causes of this phenomenon are considered as two factors: i) increased biomass production consumes a larger amount of nitrogen from the soil and this leads to progressive nitrogen limitation of photosynthesis, particularly at the level of the enzyme Rubisco, ii) the sink capacity is genetically limited and elevated CO2 concentration leads to increased accumulation of carbohydtrates (mainly sucrose, which is the main transport form of assimilates) in leaves. Increased concentrations of carbohydrates leads to a feedback regulation of photosynthesis by both, long-term feedback regulation of synthesis of the enzyme Rubisco, and also due to reduced capacity to produce ATP in the chloroplasts. However, mechanisms for interactive effects of nitrogen and accumulation of non-structural carbohydrates are still not well understood. Using 3-year-old Fagus sylvatica seedlings we have explored the interactive effects of nitrogen nutrition and sink capacity manipulation (sucrose feeding) on the dynamics of accumulation of non-structural carbohydrates and changes in photosynthetic parameters under ambient (385 μmol (CO2) mol-1) and elevated (700 μmol(CO2) mol-1) CO2 concentration. Sink manipulation by sucrose feeding led to a continuous increase of non-structural carbohydrates in leaves, which was higher in nitrogen fertilized seedlings. The accumulation of non-structural carbohydrates was also slightly stimulated by elevated CO2 concentration. Exponential decay (p <0.01) was observed in CO2 assimilation rate and stomatal conductance when the content of non-structural carbohydrates increased. However, this relationship was modified by the nitrogen content. Accumulation of non-structural carbohydrates had relatively smaller effect on actual quantum yield of photosystem II. Both, CO2 assimilation rate and the actual quantum yield of photosystem II decreased more rapidly during sink manipulation in elevated concentrations of CO2 than in ambient. Application of chlorophyll fluorescence imaging enabled us to evaluate changes in spatial distribution of feedback regulation of photosynthesis on the leaf-level. We can conclude that the accumulation of non-structural carbohydrates down-regulates photosynthesis mainly through the stomatal conductance, and this effect is further modified by nitrogen content.

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan.

PubMed

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

2011-09-14

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.

The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress.

PubMed

von Einem, Jens; Schumacher, Daniel; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2006-03-01

The equine herpesvirus 1 (EHV-1) alpha-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11deltaETIF) was constructed and analyzed. After transfection of vL11deltaETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11deltaETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11deltaETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11deltaETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11deltaETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.

Analysis of protein profiles in diabetic rat blood plasma that induced by alloxan

NASA Astrophysics Data System (ADS)

Hidayati, Dewi; Abdulgani, Nurlita; Setiyawan, Hengki; Trisnawati, Indah; Ashuri, Nova Maulidina; Sa'adah, Noor Nailis

2017-06-01

Proteomics is the study to identify the proteins involved in physiological metabolic pathway. The protein profiles of blood plasma from alloxan-induced diabetic rats has investigated using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Data were analyzed descriptively based on variations of the type and intensity of the protein. There were identified the similarity of protein variant between diabetic and control rats included ankyrin (200kDa), IgG (150kDa), nephrin (136 kDa), IDE (112 kDA), albumin (66 kDa), prealbumin (55 kDA), CICP (43 kDa), ApoA-V (39 kDa), GAPDH (35 kDa), C-RP (27,1 kDa), leptin (16 kDa) and apelin (13 kDa). However, the apelin profile at diabetic rats shows the higher intensity than control.

Effects of non-structural components and soil-structure interaction on the seismic response of framed structures

NASA Astrophysics Data System (ADS)

Ditommaso, Rocco; Auletta, Gianluca; Iacovino, Chiara; Nigro, Antonella; Carlo Ponzo, Felice

2017-04-01

In this paper, several nonlinear numerical models of reinforced concrete framed structures have been defined in order to evaluate the effects of non-structural elements and soil-structure interaction on the elastic dynamic behaviour of buildings. In the last few years, many and various studies have highlighted the significant effects derived from the interaction between structural and non-structural components on the main dynamic characteristics of a building. Usually, structural and non-structural elements act together, adding both masses and stiffness. The presence of infill panels is generally neglected in the design process of structural elements, although these elements can significantly increase the lateral stiffness of a structure leading to a modification in the dynamic properties. Particularly, at the Damage Limit State (where an elastic behaviour is expected), soil-structure interaction effects and non-structural elements may further affect the elastic natural period of buildings, changing the spectral accelerations compared with those provided by seismic codes in case of static analyses. In this work, a parametric study has been performed in order to evaluate the elastic fundamental period of vibration of buildings as a function of structural morphology (height, plan area, ratio between plan dimensions), infills presence and distribution and soil characteristics. Acknowledgements This study was partially funded by the Italian Department of Civil Protection within the project DPC-RELUIS 2016 - RS4 ''Seismic observatory of structures and health monitoring'' and by the "Centre of Integrated Geomorphology for the Mediterranean Area - CGIAM" within the Framework Agreement with the University of Basilicata "Study, Research and Experimentation in the Field of Analysis and Monitoring of Seismic Vulnerability of Strategic and Relevant Buildings for the purposes of Civil Protection and Development of Innovative Strategies of Seismic Reinforcement".

Development of a peptide ELISA to discriminate vaccine-induced immunity from natural infection of hepatitis A virus in a phase IV study.

PubMed

Ye, C; Luo, J; Wang, X; Xi, J; Pan, Y; Chen, J; Yang, X; Li, G; Sun, Q; Yang, J

2017-11-01

Hepatitis A virus (HAV) is a highly infectious agent that causes acute liver disease. The infection can trigger the production of antibodies against the structural and non-structural proteins of HAV. Nonetheless, vaccination with an HAV vaccine leads to the production of a primary antibody against the structural proteins. Because the non-structural proteins are only produced during active virus replication, there is no or very little antibody production against the non-structural proteins. However, the current commercial immunoassay cannot distinguish between antibodies produced during natural infection and those from vaccination against HAV. In our study, six immune-dominant epitopes from the non-structural proteins were designed, synthesized, linked together and cloned into pGEX-5X-1 plasmid. The recombinant protein was expressed in E. coli and purified by Ni 2+ -coated magnetic agarose beads. Then the purified recombinant protein was used as an ELISA antigen to detect antibodies for HAV non-structural proteins in serum samples. Seventy-seven attenuated and 89 inactivated vaccinated samples collected from our previous phase IV study of HAV vaccines were detected by peptide ELISA developed in this study. The mean OD 450 value for the vaccination samples and acute infection samples were 0.529 (0.486 for the attenuated group and 0.567 for the inactivated group) and 1.187, respectively. According to the receiver operating characteristic (ROC) curve, the sensitivity and specificity of the peptide ELISA were 93.80% and 91.00%, respectively. This peptide ELISA was confirmed to discriminate vaccine-induced immunity from natural infection of HAV in a phase IV study with high sensitivity and specificity.

The system of sulfated galactans from the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta): Location and structural analysis.

PubMed

Estevez, José M; Ciancia, Marina; Cerezo, Alberto S

2008-09-05

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low Mr (6-11.4kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium Mr (18-45kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which "pure" κ/ι-carrageenans were isolated. Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures. Alkaline treatment and alkaline dialysis of the main extracts gave "pure" κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low Mr agarans or agaran-dl-hybrids. Copyright © 2008 Elsevier Ltd. All rights reserved.

Patterns of lignin degradation and oxidative enzyme secretion by different wood- and litter-colonizing basidiomycetes and ascomycetes grown on beech-wood.

PubMed

Liers, Christiane; Arnstadt, Tobias; Ullrich, René; Hofrichter, Martin

2011-10-01

The degradation of lignocellulose and the secretion of extracellular oxidoreductases were investigated in beech-wood (Fagus sylvatica) microcosms using 11 representative fungi of four different ecophysiological and taxonomic groups causing: (1) classic white rot of wood (e.g. Phlebia radiata), (2) 'nonspecific' wood rot (e.g. Agrocybe aegerita), (3) white rot of leaf litter (Stropharia rugosoannulata) or (4) soft rot of wood (e.g. Xylaria polymorpha). All strong white rotters produced manganese-oxidizing peroxidases as the key enzymes of ligninolysis (75-2200 mU g(-1)), whereas lignin peroxidase activity was not detectable in the wood extracts. Interestingly, activities of two recently discovered peroxidases - aromatic peroxygenase and a manganese-independent peroxidase of the DyP-type - were detected in the culture extracts of A. aegerita (up to 125 mU g(-1)) and Auricularia auricula-judae (up to 400 mU g(-1)), respectively. The activity of classic peroxidases correlated to some extent with the removal of wood components (e.g. Klason lignin) and the release of small water-soluble fragments (0.5-1.0 kDa) characterized by aromatic constituents. In contrast, laccase activity correlated with the formation of high-molecular mass fragments (30-200 kDa). The differences observed in the degradation patterns allow to distinguish the rot types caused by basidiomycetes and ascomycetes and may be suitable for following the effects of oxidative key enzymes (ligninolytic peroxidases vs. laccases, role of novel peroxidases) during wood decay. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Localization and quantitative co-localization of enamelin with amelogenin.

PubMed

Gallon, Victoria; Chen, Lisha; Yang, Xiudong; Moradian-Oldak, Janet

2013-08-01

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular and catalytic properties of 2,4'-dihydroxyacetophenone dioxygenase from Burkholderia sp. AZ11.

PubMed

Enya, Mayu; Aoyagi, Keiko; Hishikawa, Yoshihiro; Yoshimura, Azusa; Mitsukura, Koichi; Maruyama, Kiyofumi

2012-01-01

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 µM and 6.28 µmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.

Interaction of foot-and-mouth disease virus non-structural protein 3A with host protein DCTN3 is important for viral virulence in cattle

USDA-ARS?s Scientific Manuscript database

Non-structural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular ...

Water stress, shoot growth and storage of non-structural carbohydrates along a tree height gradient in a tall conifer

Treesearch

David R. Woodruff; Frederick C. Meinzer

2011-01-01

We analyzed concentrations of starch, sucrose, glucose and fructose in upper branch wood, foliage and trunk sapwood of Douglas-fir trees in height classes ranging from ~2 to ~57 m. Mean concentrations of non-structural carbohydrates (NSC) for all tissues were highest in the tallest height class and lowest in the lowest height class, and height-related trends in NSC...

Development of hazard-compatible building fragility and vulnerability models

USGS Publications Warehouse

Karaca, E.; Luco, N.

2008-01-01

We present a methodology for transforming the structural and non-structural fragility functions in HAZUS into a format that is compatible with conventional seismic hazard analysis information. The methodology makes use of the building capacity (or pushover) curves and related building parameters provided in HAZUS. Instead of the capacity spectrum method applied in HAZUS, building response is estimated by inelastic response history analysis of corresponding single-degree-of-freedom systems under a large number of earthquake records. Statistics of the building response are used with the damage state definitions from HAZUS to derive fragility models conditioned on spectral acceleration values. Using the developed fragility models for structural and nonstructural building components, with corresponding damage state loss ratios from HAZUS, we also derive building vulnerability models relating spectral acceleration to repair costs. Whereas in HAZUS the structural and nonstructural damage states are treated as if they are independent, our vulnerability models are derived assuming "complete" nonstructural damage whenever the structural damage state is complete. We show the effects of considering this dependence on the final vulnerability models. The use of spectral acceleration (at selected vibration periods) as the ground motion intensity parameter, coupled with the careful treatment of uncertainty, makes the new fragility and vulnerability models compatible with conventional seismic hazard curves and hence useful for extensions to probabilistic damage and loss assessment.

Temporal change in molecular weight distribution of hot-water extractable organic nitrogen from cattle manure compost buried in soil using high-performance size exclusion chromatography with chemiluminescent nitrogen detection

NASA Astrophysics Data System (ADS)

Moriizumi, M.; Mutsunaga, T.

2012-04-01

The application of compost can improve the fertility of the agricultural soils. The compost organic nitrogen is absorbed by plants after degradation and mineralization. To investigate the degradation process of compost organic nitrogen in soil, we conducted soil burial test of compost and observed the molecular weight distribution of hot-water extractable organic nitrogen from the compost. The cattle manure compost (1g) was mixed with soil (25g), put into glass fiber-filter paper bag and buried in 15 cm under surface of the ground for 6 months. The soils used were Andosol, Gray Lowland soil, and Yellow soil without organic matter application for 25 years in Tsukuba, Japan. Organic matter was extracted from the buried sample with 80° C of water for 16 hours. The molecular weight distribution of the hot-water extractable organic matter (HWEOM) was measured by high-performance size exclusion chromatography and chemiluminescent nitrogen detection (HPSEC/CLND). In this system, N-containing compound eluted from a SEC column was introduced into a furnace at 1050° C, and N in the compound was oxidized to nitric oxide and then detected using a chemiluminescent reaction with ozone. The N chromatogram showed that N in the HWEOM from the soil with compost had various molecular weights ranging from 0.1 to 100 kDa. A void peak (>100 kDa), a broad peak around 30 kDa, and several sharp peaks less than 30 kDa were observed in the chromatogram. The broad peak (~ 30kDa) was likely to be derived from the compost, because it was not observed in the chromatogram of HWEOM from soil alone. The N intensities of all peaks decreased with burial time, especially, the broad peak (~30 kDa) intensity rapidly decreased by 10 - 50 % in only first 2 months. The decreasing rates of the broad peak were higher than that of the sharp peaks, indicating that the organic nitrogen with a larger molecular weight decomposed faster. The broad peak (~ 30 kDa) had visible (420nm) absorption and less fulvic acid like florescence (Ex340nm, Em440 nm). The several sharp peaks had small visible absorption and intense florescence. Further studies are needed to assign the chemical forms for each peak.

[A case of an allergic reaction due to Anisakis simplex possibly after the ingestion of squid--successful detection of four A. simplex allergens, Ani s 1, Ani s 2, Ani s 12 and troponin C-like protein].

PubMed

Iijima, Shigeruko; Moriyama, Tatsuya; Ichikawa, Hidetaka; Kobayashi, Yukihiro; Shiomi, Kazuo

2012-08-01

A 62-year-old man ingested dressed salmon and its roe (ikura) and grilled mackerel and one hour later further ingested raw tuna and squid as an evening meal at a bar. Soon after the ingestion of raw seafood, he showed wheals, loss of consciousness and low blood pressure. Specific serum IgE to the nematode Anisakis simplex was positive but those to some seafoods were negative. Moreover, a skin prick test using the crude extract was positive for A. simplex but negative for the seafoods, which he ingested on the day of the above episode. When the A. simplex extract was analyzed by IgE-binding immunoblot analysis using the patient serum, two highly intense protein bands were recognized at 18 and 17 kDa, one intense band at 35 kDa and two weak bands at 28 and 26 kDa. ELISA with 11 natural or recombinant A. simplex allergens (Ani s 1-6, 8, 9, 11 and 12 and troponin C-like protein) showed that the patient serum strongly reacted to Ani s 1 and Ani s 12 and weakly to Ani s 2 and troponin C-like protein. Based on these results, he was diagnosed as IgE-mediated A. simplex allergy due to four allergens (Ani s 1, Ani s 2, Ani s 12 and troponin C-like protein), possibly infested in the raw squid which he had ingested just before manifestation of allergic reactions.

Sequential Extraction Results in Improved Proteome Profiling of Medicinal Plant Pinellia ternata Tubers, Which Contain Large Amounts of High-Abundance Proteins

PubMed Central

An, SuFang; Gong, FangPing; Wang, Wei

2012-01-01

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae. PMID:23185632

Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

PubMed

Wu, Xiaolin; Xiong, Erhui; An, Sufang; Gong, Fangping; Wang, Wei

2012-01-01

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames.

PubMed Central

Stallmeyer, B; Drugeon, G; Reiss, J; Haenni, A L; Mendel, R R

1999-01-01

A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning. PMID:10053003

Tula hantavirus NSs protein accumulates in the perinuclear area in infected and transfected cells.

PubMed

Virtanen, Jussi Oskari; Jääskeläinen, Kirsi Maria; Djupsjöbacka, Janica; Vaheri, Antti; Plyusnin, Alexander

2010-01-01

The small RNA segment of some hantaviruses (family Bunyaviridae) encodes two proteins: the nucleocapsid protein and, in an overlapping reading frame, a non-structural (NSs) protein. The hantavirus NSs protein, like those of orthobunya- and phleboviruses, counteracts host innate immunity. Here, for the first time, the NSs protein of a hantavirus (Tula virus) has been observed in infected cells and shown to localize in the perinuclear area. Transiently expressed NSs protein showed similar localization, although the kinetics was slightly different, suggesting that to reach its proper location in the infected cell, the NSs protein does not have to cooperate with other viral proteins.

Diffusion profile of macromolecules within and between human skin layers for (trans)dermal drug delivery.

PubMed

Römgens, Anne M; Bader, Dan L; Bouwstra, Joke A; Baaijens, Frank P T; Oomens, Cees W J

2015-10-01

Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500 kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40 kDa than 500 kDa dextran, with median values of 23 and 9 µm(2)/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40 kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin. Copyright © 2015 Elsevier Ltd. All rights reserved.

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

Multiplex Detection of IgG and IgM to Rift Valley Fever Virus Nucleoprotein, Nonstructural Proteins, and Glycoprotein in Ovine and Bovine.

PubMed

Hossain, Mohammad M; Wilson, William C; Faburay, Bonto; Richt, Jürgen; McVey, David S; Rowland, Raymond R

2016-08-01

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were assembled into a multiplex and tested in serum samples from infected wild-type RVFV or MP12, a modified live virus vaccine. As expected, the N protein was immunodominant and the best target for early detection of infection. Antibody activity against the other targets was also detected. The experimental results demonstrate the capabilities of FMIA for the detection of antibodies to RVFV structural and nonstructural proteins, which can be applied to future development and validation of diagnostic tests that can be used to differentiate vaccinated from infected animals.

Determination of prohibited, small peptides in urine for sports drug testing by means of nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass spectrometry.

PubMed

Thomas, Andreas; Walpurgis, Katja; Krug, Oliver; Schänzer, Wilhelm; Thevis, Mario

2012-10-12

In the present study, a screening assay was developed comprising 11 prohibited peptides (<1.5 kDa) that are sufficiently purified from urine using weak cation exchange with subsequent determination of all substances by means of nanoUHPLC separation coupled to high resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH), Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin, Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable to further target analytes or metabolites. The method was validated with a main focus on qualitative result interpretation considering the parameters specificity, linearity (0-500 pg/mL), recovery (45-95%), precision (<20% at 100 pg/mL), limits of detection (2-10 pg/mL), robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion study urine samples for LHRH, Desmopressin and GHRP-2. Copyright © 2012 Elsevier B.V. All rights reserved.

Evidence for a repair enzyme complex involving ERCC1 and complementing activities of ERCC4, ERCC11 and xeroderma pigmentosum group F.

PubMed Central

van Vuuren, A J; Appeldoorn, E; Odijk, H; Yasui, A; Jaspers, N G; Bootsma, D; Hoeijmakers, J H

1993-01-01

Nucleotide excision repair (NER), one of the major cellular DNA repair systems, removes a wide range of lesions in a multi-enzyme reaction. In man, a NER defect due to a mutation in one of at least 11 distinct genes, can give rise to the inherited repair disorders xeroderma pigmentosum (XP), Cockayne's syndrome or PIBIDS, a photosensitive form of the brittle hair disease trichothiodystrophy. Laboratory-induced NER-deficient mutants of cultured rodent cells have been classified into 11 complementation groups (CGs). Some of these have been shown to correspond with human disorders. In cell-free extracts prepared from rodent CGs 1-5 and 11, but not in a mutant from CG6, we find an impaired repair of damage induced in plasmids by UV light and N-acetoxy-acetylaminofluorene. Complementation analysis in vitro of rodent CGs is accomplished by pairwise mixing of mutant extracts. The results show that mutants from groups 2, 3, 5 and XP-A can complement all other CGs tested. However, selective non-complementation in vitro was observed in mutual mixtures of groups 1, 4, 11 and XP-F, suggesting that the complementing activities involved somehow affect each other. Depletion of wild-type human extracts from ERCC1 protein using specific anti-ERCC1 antibodies concomitantly removed the correcting activities for groups 4, 11 and XP-F, but not those for the other CGs. Furthermore, we find that 33 kDa ERCC1 protein sediments as a high mol. wt species of approximately 120 kDa in a native glycerol gradient.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8253091

Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8+ T cell response.

PubMed

Culshaw, Abigail; Ladell, Kristin; Gras, Stephanie; McLaren, James E; Miners, Kelly L; Farenc, Carine; van den Heuvel, Heleen; Gostick, Emma; Dejnirattisai, Wanwisa; Wangteeraprasert, Apirath; Duangchinda, Thaneeya; Chotiyarnwong, Pojchong; Limpitikul, Wannee; Vasanawathana, Sirijitt; Malasit, Prida; Dong, Tao; Rossjohn, Jamie; Mongkolsapaya, Juthathip; Price, David A; Screaton, Gavin R

2017-11-01

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8 + T cell populations specific for variants of the nonstructural protein epitope NS3 133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3 133 -DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2 + TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2 + TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

PubMed

Ikigai, H; Ono, T; Nakae, T; Otsuru, H; Shimamura, T

1999-01-08

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

Elucidating Adsorptive Fractions of Natural Organic Matter on Carbon Nanotubes.

PubMed

Ateia, Mohamed; Apul, Onur G; Shimizu, Yuta; Muflihah, Astri; Yoshimura, Chihiro; Karanfil, Tanju

2017-06-20

Natural organic matter (NOM) is a heterogeneous mixture of organic compounds that is omnipresent in natural waters. To date, the understanding of the adsorption of NOM components by carbon nanotubes (CNTs) is limited because of the limited number of comprehensive studies in the literature examining the adsorption of NOM by CNTs. In this study, 11 standard NOM samples from various sources were characterized, and their adsorption behaviors on four different CNTs were examined side-by-side using total organic carbon, fluorescence, UV-visible spectroscopy, and high-performance size-exclusion chromatography (HPSEC) analysis. Adsorption was influenced by the chemical properties of the NOM, including aromaticity, degree of oxidation, and carboxylic acidity. Fluorescence excitation-emission matrix (EEM) analysis showed preferential adsorption of decomposed and terrestrial-derived NOM compared to freshly produced and microbial-derived NOM. HPSEC analysis revealed preferential adsorption of fractions in the molecular weight range of 0.5-2 kDa for humic acids but in the molecular weight range of 1-3 kDa for all fulvic acids and reverse-osmosis isolates. However, the smallest characterized fraction (MW < 0.4 kDa) in all samples did not adsorb on the CNTs.

A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea.

PubMed

Lucchesi, P M; Parma, A E

1999-11-30

Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library. Although there are references of transcription of leptospiral genes in E. coli from their own leptospiral promoters, in this recombinant construction the leptospiral DNA was located under the control of lacZ promoter since no expression could be detected in the absence of IPTG. This clone, isolated by expression screening with polyclonal serum raised against equine corneal proteins, encodes a 90 kDa protein of L. interrogans which crossreacts with equine cornea as proved Western-blotting. Antibodies directed against this leptospiral protein strongly recognised a 66 kDa equine corneal protein, one of those recognised by an anti-equine cornea serum. Our findings suggest that an immune response to 90 kDa protein participates in pathogenesis of equine uveitis.

Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae.

PubMed

Fotso Fotso, Aurélien; Mediannikov, Oleg; Nappez, Claude; Azza, Saïd; Raoult, Didier; Drancourt, Michel

2016-01-01

Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field. © The American Society of Tropical Medicine and Hygiene.

A novel Amoeba proteus 120 kDa actin-binding protein with only 1 filamin repeat and a coiled-coil region.

PubMed

Sobczak, Magdalena; Kocik, Elzbieta; Redowicz, Maria Jolanta

2007-02-01

A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ~95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1-104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209-324), and a central region (residues 344-577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.

Dark respiration rate increases with plant size in saplings of three temperate tree species despite decreasing tissue nitrogen and nonstructural carbohydrates.

PubMed

Machado, José-Luis; Reich, Peter B

2006-07-01

In shaded environments, minimizing dark respiration during growth could be an important aspect of maintaining a positive whole-plant net carbon balance. Changes with plant size in both biomass distribution to different tissue types and mass-specific respiration rates (R(d)) of those tissues would have an impact on whole-plant respiration. In this paper, we evaluated size-related variation in R(d), biomass distribution, and nitrogen (N) and total nonstructural carbohydrate (TNC) concentrations of leaves, stems and roots of three cold-temperate tree species (Abies balsamea (L.) Mill, Acer rubrum L. and Pinus strobus L.) in a forest understory. We sampled individuals varying in age (6 to 24 years old) and in size (from 2 to 500 g dry mass), and growing across a range of irradiances (from 1 to 13% of full sun) in northern Minnesota, USA. Within each species, we found small changes in R(d), N and TNC when comparing plants growing across this range of light availability. Consistent with our hypotheses, as plants grew larger, whole-plant N and TNC concentrations in all species declined as a result of a combination of changes in tissue N and shifts in biomass distribution patterns. However, contrary to our hypotheses, whole-plant and tissue R(d) increased with plant size in the three species.

Biscarbamate Cross-Linked Low-Molecular-Weight Polyethylenimine for Delivering Anti-chordin siRNA into Human Mesenchymal Stem Cells for Improving Bone Regeneration.

PubMed

Wang, Chuandong; Yuan, Weien; Xiao, Fei; Gan, Yaokai; Zhao, Xiaotian; Zhai, Zhanjing; Zhao, Xiaoying; Zhao, Chen; Cui, Penglei; Jin, Tuo; Chen, Xiaodong; Zhang, Xiaoling

2017-01-01

Small-interfering RNA (siRNA) provides a rapid solution for drug design and provides new methods to develop customizable medicines. Polyethyleneimine 25 kDa (PEI25kDa) is an effective transfection agent used in siRNA delivery. However, the lack of degradable linkage causes undesirable toxicity, hindering its clinical application. We designed a low-molecular-weight cross-linked polyethylenimine named PEI-Et (Mn:1220, Mw:2895) by using degradable ethylene biscarbamate linkage with lower cytotoxicity and higher knockdown efficiency than PEI25kDa in delivery Chordin siRNA to human bone mesenchymal stem cells (hBMSCs). Suppression of Chordin by using anti-Chordin siRNA delivered by PEI-Et improved bone regeneration in vitro and in vivo associated with the bone morphogenetic protein-2 (BMP-2) mediated smad1/5/8 signaling pathway. Results of this study suggest that Chordin siRNA can be potentially used to improve osteogenesis associated with the BMP-2-mediated Smad1/5/8 signaling pathway and biodegradable biscarbamate cross-linked low-molecular-weight polyethylenimine (PEI-Et) is a therapeutically feasible carrier material to deliver anti-Chordin siRNA to hBMSCs.

The Development of Nonstructural Alternatives.

DTIC Science & Technology

1979-05-01

particular appeal and benefits for those concerned with environmental issues. However, on reflection it is clear that, almost by definition, easily...peculiar to nonstructural alternatives (that is, the social costs are borne primaqrilv by th,, )ent ficiaries) makes them appealing from the national...Costs Of prId-nt ! loo41p l jotve iopment lie basically in the costs of elevated 111’Z ’’l’i. Pie 1land reqat reti c,- snob dovelIopmeot is loss

The influence of a yoga exercise program for young adults with intellectual disabilities.

PubMed

Hawkins, Brent L; Stegall, Joanna B; Weber, Madalyn F; Ryan, Joseph B

2012-07-01

Individuals with intellectual disabilities (ID) have an increased risk of obesity and are significantly less likely to engage in physical activity compared to their nondisabled peers. A growing body of research supports the physical and mental health benefits of yoga. While the benefits of yoga have been studied across a host of populations with varying ages and physical disabilities, no studies could be identified investigating the benefits of yoga for young adults with ID. This study investigated the impact of participating in yoga classes on the amount of exercise behavior and perception of physical exertion when compared to non-structured exercise sessions between two young adults with ID in a post-secondary education setting. A single subject multiple baseline research design was implemented across two young adults with mild ID to determine the effects of a yoga exercise class on frequency of exercise behavior and perception of physical exertion when compared to non-structured exercise sessions. Partial interval recording, the Eston-Parfitt curvilinear rating of perceived exertion scale, and the physical activity enjoyment scale were implemented to collect data on dependent variables and consumer satisfaction during each non-structured exercise session and each yoga class. indicated that percentage of exercise behavior and perceived exertion levels during yoga group exercise sharply increased with large effect sizes when compared to non-structured exercise sessions.

A perspective on targeting non-structural proteins to combat neglected tropical diseases: Dengue, West Nile and Chikungunya viruses.

PubMed

Bhakat, Soumendranath; Karubiu, Wilson; Jayaprakash, Venkatesan; Soliman, Mahmoud E S

2014-11-24

Neglected tropical diseases are major causes of fatality in poverty stricken regions across Africa, Asia and some part of America. The combined potential health risk associated with arthropod-borne viruses (arboviruses); Dengue virus (DENV), West Nile Virus (WNV) and Chikungunya Virus (CHIKV) is immense. These arboviruses are either emerging or re-emerging in many regions with recent documented outbreaks in the United States. Despite several recent evidences of emergence, currently there are no approved drugs or vaccines available to counter these diseases. Non-structural proteins encoded by these RNA viruses are essential for their replication and maturation and thus may offer ideal targets for developing antiviral drugs. In recent years, several protease inhibitors have been sourced from plant extract, synthesis, computer aided drug design and high throughput screening as well as through drug reposition based approaches to target the non-structural proteins. The protease inhibitors have shown different levels of inhibition and may thus provide template to develop selective and potent drugs against these devastating arboviruses. This review seeks to shed light on the design and development of antiviral drugs against DENV, WNV and CHIKV to date. To the best of our knowledge, this review provides the first comprehensive update on the development of protease inhibitors targeting non-structural proteins of three most devastating arboviruses, DENV, WNV and CHIKV. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Association of Urinary N-Domain Angiotensin I-Converting Enzyme with Plasma Inflammatory Markers and Endothelial Function

PubMed Central

Fernandes, Fernanda B; Plavnik, Frida L; Teixeira, Andressa MS; Christofalo, Dejaldo MJ; Ajzen, Sergio A; Higa, Elisa MS; Ronchi, Fernanda A; Sesso, Ricardo CC; Casarini, Dulce E

2008-01-01

The aim of this study was to investigate the association between urinary 90 kDa N-domain Angiotensin I-converting enzyme (ACE) form with C-reactive protein (CRP) and homocysteine plasma levels (Hcy), urinary nitric oxide (NOu), and endothelial function (EF) in normotensive subjects. Forty healthy subjects were evaluated through brachial Doppler US to test the response to reactive hyperemia and a panel of blood tests to determine CRP and Hcy levels, NOu, and urinary ACE. They were divided into groups according to the presence (ACE90+) or absence (ACE90–) of the 90 kDa ACE, the presence (FH+) or absence (FH–) of family history of hypertension, and the presence or absence of these two variables FH+/ACE90+ and FH–/ACE90–. We found an impaired endothelial dilatation in subjects who presented the 90 kDa N-domain ACE as follows: 11.4% ± 5.3% in ACE90+ compared with 17.6% ± 7.1% in ACE90– group and 12.4% ± 5.6% in FH+/ACE90+ compared with 17.7% ± 6.2% in FH–/ACE90– group, P < 0.05. Hcy and CRP levels were statistically significantly lower in FH+/ACE90+ than in FH–/ACE90– group, as follows: 10.0 ± 2.3 μM compared with 12.7 ± 1.5 μM, and 1.3 ± 1.8 mg/L compared with 3.6 ± 2.0 mg/L, respectively. A correlation between flow-mediated dilatation (FMD) and CRP, Hcy, and NOu levels was not found. Our study suggests a reduction in the basal NO production confirmed by NOu analysis in subjects with the 90 kDa N-domain ACE isoform alone or associated with a family history of hypertension. Our data suggest that the presence of the 90 kDa N-domain ACE itself may have a negative impact on flow-mediated dilatation stimulated by reactive hyperemia. PMID:18475311

Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients

PubMed Central

Aw-Yong, Kam Leng; Sam, I-Ching; Koh, Mia Tuang

2016-01-01

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates. PMID:27806091

Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.

PubMed

Rurek, Michal

2010-08-18

Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress. All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls--from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes. All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye mitochondria. Some small thermolabile dehydrin-like proteins were induced under stress conditions applied and therefore they are likely to be involved in stress response.

Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress

PubMed Central

2010-01-01

Background Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress. Results All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls - from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes. Conclusions All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye mitochondria. Some small thermolabile dehydrin-like proteins were induced under stress conditions applied and therefore they are likely to be involved in stress response. PMID:20718974

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

PubMed Central

Grandi, Paola; Dang, Tam; Pané, Nelly; Shevchenko, Andrej; Mann, Matthias; Forbes, Douglass; Hurt, Ed

1997-01-01

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates. PMID:9348540

Bee Pollen-Induced Anaphylaxis: A Case Report and Literature Review.

PubMed

Choi, Jeong Hee; Jang, Young Sook; Oh, Jae Won; Kim, Cheol Hong; Hyun, In Gyu

2015-09-01

Bee pollen is pollen granules packed by honey bees and is widely consumed as natural healthy supplements. Bee pollen-induced anaphylaxis has rarely been reported, and its allergenic components have never been studied. A 40-year-old male came to the emergency room with generalized urticaria, facial edema, dyspnea, nausea, vomiting, abdominal pain, and diarrhea 1 hour after ingesting one tablespoon of bee pollen. Oxygen saturation was 91%. His symptoms resolved after injection of epinephrine, chlorpheniramine, and dexamethasone. He had seasonal allergic rhinitis in autumn. Microscopic examination of the bee pollen revealed Japanese hop, chrysanthemum, ragweed, and dandelion pollens. Skin-prick with bee pollen extracts showed positive reactions at 0.1 mg/mL (A/H ratio > 3+). Serum specific IgE to ragweed was 25.2, chrysanthemum 20.6, and dandelion 11.4 kU/L; however, Japanese hop, honey-bee venom and yellow-jacket venom were negative (UniCAP®, Thermo Fisher Scientific, Uppsala, Sweden). Enzyme-linked immunosorbent assay (ELISA) confirmed serum specific IgE to bee-pollen extracts, and an ELISA inhibition assay for evaluation of cross-allergenicity of bee pollen and other weed pollens showed more than 90% of inhibition with chrysanthemum and dandelion and ~40% inhibition with ragweed at a concentration of 1 μg/mL. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblot analysis revealed 9 protein bands (11, 14, 17, 28, 34, 45, 52, 72, and 90 kDa) and strong IgE binding at 28-34 kDa, 45 and 52 kDa. In conclusion, healthcare providers should be aware of the potential risk of severe allergic reactions upon ingestion of bee pollen, especially in patients with pollen allergy.

Characterization, efficacy, pharmacokinetics, and biodistribution of 5kDa mPEG modified tetrameric canine uricase variant.

PubMed

Zhang, Chun; Fan, Kai; Luo, Hua; Ma, Xuefeng; Liu, Riyong; Yang, Li; Hu, Chunlan; Chen, Zhenmin; Min, Zhiqiang; Wei, Dongzhi

2012-07-01

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ɛ amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated. Approximately 9.4 5 kDa mPEG chains were coupled to each monomeric uricase and the main conjugates contained 7-11 mPEG chains per subunit. mPEG-UHC showed significantly therapeutic or preventive effect on uric acid nephropathy and acute urate arthritis based on three different animal models. The clearance rate from an intravenous injection of mPEG-UHC varied significantly between species, at 2.61 mL/h/kg for rats and 0.21 mL/h/kg for monkeys. The long elimination half-life of mPEG-UHC in non-human primate (191.48 h, intravenous injection) indicated the long-term effects in humans. Moreover, the acceptable bioavailability of mPEG-UHC after subcutaneous administration in monkeys (94.21%) suggested that subcutaneous injection may be regarded as a candidate administration route in clinical trails. Non-specific tissue distribution was observed after administration of (125)I-labeled mPEG-UHC in rats, and elimination by the kidneys into the urine is the primary excretion route. Copyright © 2012 Elsevier B.V. All rights reserved.

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells.

PubMed

Phung, Thuy Thi Bich; Sugamata, Ryuichi; Uno, Kazuko; Aratani, Yasuaki; Ozato, Keiko; Kawachi, Shoji; Thanh Nguyen, Liem; Nakayama, Toshinori; Suzuki, Kazuo

2011-12-01

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

The whispering gallery mode biosensor: label-free detection from virus to single protein

NASA Astrophysics Data System (ADS)

Holler, S.; Dantham, V. R.; Keng, D.; Kolchenko, V.; Arnold, S.; Mulroe, Brigid; Paspaley-Grbavac, M.

2014-08-01

The whispering gallery mode (WGM) biosensor is a micro-optical platform capable of sensitive label-free detection of biological particles. Described by the reactive sensing principle (RSP), this analytic formulation quantifies the response of the system to the adsorption of bioparticles. Guided by the RSP, the WGM biosensor enabling from detection of virus (e.g., Human Papillomavirus, HPV) to the ultimate goal of single protein detection. The latter was derived from insights into the RSP, which resulted in the development of a hybrid plasmonic WGM biosensor, which has recently demonstrated detection of individual protein cancer markers. Enhancements from bound gold nanoparticles provide the sensitivity to detect single protein molecules (66 kDa) with good signal-to-noise (S/N > 10), and project that detection of proteins as small as 5 kDa.

Entericidin Is Required for a Probiotic Treatment (Enterobacter sp. Strain C6-6) To Protect Trout from Cold-Water Disease Challenge

PubMed Central

Schubiger, Carla B.; Orfe, Lisa H.; Sudheesh, Ponnerassery S.; Cain, Kenneth D.; Shah, Devendra H.

2014-01-01

Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens. PMID:25381243

Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model.

PubMed

Bell, Angela D; Hurtig, Mark B; Quenneville, Eric; Rivard, Georges-Étienne; Hoemann, Caroline D

2017-10-01

Objective This study tested the hypothesis that presolidified chitosan-blood implants are retained in subchondral bone channels perforated in critical-size sheep cartilage defects, and promote bone repair and hyaline-like cartilage resurfacing versus blood implant. Design Cartilage defects (10 × 10 mm) with 3 bone channels (1 drill, 2 Jamshidi biopsy, 2 mm diameter), and 6 small microfracture holes were created bilaterally in n = 11 sheep knee medial condyles. In one knee, 10 kDa chitosan-NaCl/blood implant (presolidified using recombinant factor VIIa or tissue factor), was inserted into each drill and Jamshidi hole. Contralateral knee defects received presolidified whole blood clot. Repair tissues were assessed histologically, biochemically, biomechanically, and by micro-computed tomography after 1 day ( n = 1) and 6 months ( n = 10). Results Day 1 defects showed a 60% loss of subchondral bone plate volume fraction along with extensive subchondral hematoma. Chitosan implant was resident at day 1, but had no effect on any subsequent repair parameter compared with blood implant controls. At 6 months, bone defects exhibited remodeling and hypomineralized bone repair and were partly resurfaced with tissues containing collagen type II and scant collagen type I, 2-fold lower glycosaminoglycan and fibril modulus, and 4.5-fold higher permeability compared with intact cartilage. Microdrill holes elicited higher histological ICRS-II overall assessment scores than Jamshidi holes (50% vs. 30%, P = 0.041). Jamshidi biopsy holes provoked sporadic osteonecrosis in n = 3 debrided condyles. Conclusions Ten kilodalton chitosan was insufficient to improve repair. Microdrilling is a feasible subchondral marrow stimulation surgical approach with the potential to elicit poroelastic tissues with at least half the compressive modulus as intact articular cartilage.

Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model

PubMed Central

Bell, Angela D.; Hurtig, Mark B.; Quenneville, Eric; Rivard, Georges-Étienne; Hoemann, Caroline D.

2016-01-01

Objective This study tested the hypothesis that presolidified chitosan-blood implants are retained in subchondral bone channels perforated in critical-size sheep cartilage defects, and promote bone repair and hyaline-like cartilage resurfacing versus blood implant. Design Cartilage defects (10 × 10 mm) with 3 bone channels (1 drill, 2 Jamshidi biopsy, 2 mm diameter), and 6 small microfracture holes were created bilaterally in n = 11 sheep knee medial condyles. In one knee, 10 kDa chitosan–NaCl/blood implant (presolidified using recombinant factor VIIa or tissue factor), was inserted into each drill and Jamshidi hole. Contralateral knee defects received presolidified whole blood clot. Repair tissues were assessed histologically, biochemically, biomechanically, and by micro–computed tomography after 1 day (n = 1) and 6 months (n = 10). Results Day 1 defects showed a 60% loss of subchondral bone plate volume fraction along with extensive subchondral hematoma. Chitosan implant was resident at day 1, but had no effect on any subsequent repair parameter compared with blood implant controls. At 6 months, bone defects exhibited remodeling and hypomineralized bone repair and were partly resurfaced with tissues containing collagen type II and scant collagen type I, 2-fold lower glycosaminoglycan and fibril modulus, and 4.5-fold higher permeability compared with intact cartilage. Microdrill holes elicited higher histological ICRS-II overall assessment scores than Jamshidi holes (50% vs. 30%, P = 0.041). Jamshidi biopsy holes provoked sporadic osteonecrosis in n = 3 debrided condyles. Conclusions Ten kilodalton chitosan was insufficient to improve repair. Microdrilling is a feasible subchondral marrow stimulation surgical approach with the potential to elicit poroelastic tissues with at least half the compressive modulus as intact articular cartilage. PMID:28934884

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

PubMed Central

Culver, J N; Lehto, K; Close, S M; Hilf, M E; Dawson, W O

1993-01-01

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8446627

Molecular and functional characterization of the American cockroach, Periplaneta americana, Rab5: the first exopterygotan low molecular weight ovarian GTPase during oogenesis.

PubMed

Elmogy, Mohamed; Mohamed, Amr A; Tufail, Muhammad; Uno, Tomohide; Takeda, Makio

2017-05-26

The small Rab GTPases are key regulators of membrane vesicle trafficking. Ovaries of Periplaneta americana (Linnaeus) (Blattodea: Blattidae) have small molecular weight GTP/ATP-binding proteins during early and late vitellogenic periods of oogenesis. However, the identification and characterization of the detected proteins have not been yet reported. Herein, we cloned a cDNA encoding Rab5 from the American cockroach, P. americana, ovaries (PamRab5). It comprises 796 bp, encoding a protein of 213 amino acid residues with a predicted molecular weight of 23.5 kDa. PamRab5 exists as a single-copy gene in the P. americana genome, as revealed by Southern blot analysis. An approximate 2.6 kb ovarian mRNA was transcribed especially at high levels in the previtellogenic ovaries, detected by Northern blot analysis. The muscle and head tissues also showed high levels of PamRab5 transcript. PamRab5 protein was localized, via immunofluorescence labeling, to germline-derived cells of the oocytes, very early during oocyte differentiation. Immunoblotting detected a ∼25 kDa signal as a membrane-associated form revealed after application of detergent in the extraction buffer, and 23 kDa as a cytosolic form consistent with the predicted molecular weight from amino acid sequence in different tissues including ovary, muscles and head. The PamRab5 during late vitellogenic periods is required to regulate the endocytotic machinery during oogenesis in this cockroach. This is the first report on Rab5 from a hemimetabolan, and presents an inaugural step in probing the molecular premises of insect oocyte endocytotic trafficking important for oogenesis and embryonic development. © 2017 Institute of Zoology, Chinese Academy of Sciences.

The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells*

PubMed Central

Striz, Anneliese C.; Tuma, Pamela L.

2016-01-01

A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5′ nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface. PMID:26957544

Synthesis and Preclinical Evaluation of Sulfonamido-based [11C-Carbonyl]-Carbamates and Ureas for Imaging Monoacylglycerol Lipase

PubMed Central

Wang, Lu; Mori, Wakana; Cheng, Ran; Yui, Joji; Hatori, Akiko; Ma, Longle; Zhang, Yiding; Rotstein, Benjamin H.; Fujinaga, Masayuki; Shimoda, Yoko; Yamasaki, Tomoteru; Xie, Lin; Nagai, Yuji; Minamimoto, Takafumi; Higuchi, Makoto; Vasdev, Neil; Zhang, Ming-Rong; Liang, Steven H.

2016-01-01

Monoacylglycerol lipase (MAGL) is a 33 kDa member of the serine hydrolase superfamily that preferentially degrades 2-arachidonoylglycerol (2-AG) to arachidonic acid in the endocannabinoid system. Inhibition of MAGL is not only of interest for probing the cannabinoid pathway but also as a therapeutic and diagnostic target for neuroinflammation. Limited attempts have been made to image MAGL in vivo and a suitable PET ligand for this target has yet to be identified and is urgently sought to guide small molecule drug development in this pathway. Herein we synthesized and evaluated the physiochemical properties of an array of eleven sulfonamido-based carbamates and ureas with a series of terminal aryl moieties, linkers and leaving groups. The most potent compounds were a novel MAGL inhibitor, N-((1-(1H-1,2,4-triazole-1-carbonyl)piperidin-4-yl) methyl)-4-chlorobenzenesulfonamide (TZPU; IC50 = 35.9 nM), and the known inhibitor 1,1,1,3,3,3-hexafluoropropan-2-yl 4-(((4-chlorophenyl)sulfonamido) methyl)piperidine-1-carboxylate (SAR127303; IC50 = 39.3 nM), which were also shown to be selective for MAGL over fatty acid amide hydrolase (FAAH), and cannabinoid receptors (CB1 & CB2). Both of these compounds were radiolabeled with carbon-11 via [11C]COCl2, followed by comprehensive ex vivo biodistribution and in vivo PET imaging studies in normal rats to determine their brain permeability, specificity, clearance and metabolism. Whereas TZPU did not show adequate specificity to warrant further evaluation, [11C]SAR127303 was advanced for preliminary PET neuroimaging studies in nonhuman primate. The tracer showed good brain permeability (ca. 1 SUV) and heterogeneous regional brain distribution which is consistent with the distribution of MAGL. PMID:27279908

Solubilization of adenylyl cyclase from human myometrium in a alphas-coupled form.

PubMed

Bajo, Ana M; Prieto, Juan C; Valenzuela, Pedro; Martinez, Pilar; Guijarro, Luis G

2003-08-01

Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.

An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

PubMed Central

Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

1998-01-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

PubMed

Bearden, S W; Staggs, T M; Perry, R D

1998-03-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Schmallenberg virus non-structural protein NSm: Intracellular distribution and role of non-hydrophobic domains.

PubMed

Kraatz, Franziska; Wernike, Kerstin; Reiche, Sven; Aebischer, Andrea; Reimann, Ilona; Beer, Martin

2018-03-01

Schmallenberg virus (SBV) induces fetal malformation, abortions and stillbirth in ruminants. While the non-structural protein NSs is a major virulence factor, the biological function of NSm, the second non-structural protein which consists of three hydrophobic transmembrane (I, III, V) and two non-hydrophobic regions (II, IV), is still unknown. Here, a series of NSm mutants displaying deletions of nearly the entire NSm or of the non-hydrophobic domains was generated and the intracellular distribution of NSm was assessed. SBV-NSm is dispensable for the generation of infectious virus and mutants lacking domains II - V showed growth properties similar to the wild-type virus. In addition, a comparable intracellular distribution of SBV-NSm was observed in mammalian cells infected with domain II mutants or wild-type virus. In both cases, NSm co-localized with the glycoprotein Gc in the Golgi compartment. However, domain IV-deletion mutants showed an altered distribution pattern and no co-localization of NSm and Gc. Copyright © 2018 Elsevier Inc. All rights reserved.

Ultrahigh-Resolution Differential Ion Mobility Separations of Conformers for Proteins above 10 kDa: Onset of Dipole Alignment?

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

Shvartsburg, Alexandre A.

2014-11-04

Biomacromolecules tend to assume numerous structures in solution or the gas phase. It has been possible to resolve disparate conformational families but not unique geometries within each, and drastic peak broadening has been the bane of protein analyses by chromatography, electrophoresis, and ion mobility spectrometry (IMS). The new differential IMS (FAIMS) approach using hydrogen-rich gases was recently found to separate conformers of a small protein ubiquitin with same peak width and resolving power up to ~400 as for peptides. Present work explores the reach of this approach for larger proteins, exemplified by cytochrome c and myoglobin. Resolution similar to thatmore » for ubiquitin was largely achieved with longer separations, while the onset of peak broadening and coalescence with shorter separations suggests the limitation of present technique to proteins under ~20 kDa. This capability may enable distinguishing whole proteins with differing residue sequences or localizations of posttranslational modifications. Small features at negative compensation voltages that markedly grow from cytochrome c to myoglobin indicate the dipole alignment of rare conformers in accord with theory, further supporting the concept of pendular macroions in FAIMS.« less

3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy

PubMed Central

Vahedi-Faridi, Ardeschir; Jastrzebska, Beata; Palczewski, Krzysztof; Engel, Andreas

2013-01-01

Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin. PMID:23267047

Phosphorylation of Tat-interactive protein 60 kDa by protein kinase C epsilon is important for its subcellular localisation.

PubMed

Sapountzi, Vasileia; Logan, Ian R; Nelson, Glyn; Cook, Susan; Robson, Craig N

2008-01-01

Tat-interactive protein 60 kDa is a nuclear acetyltransferase that both coactivates and corepresses transcription factors and has a definitive function in the DNA damage response. Here, we provide evidence that Tat-interactive protein 60 kDa is phosphorylated by protein kinase C epsilon. In vitro, protein kinase C epsilon phosphorylates Tat-interactive protein 60 kDa on at least two sites within the acetyltransferase domain. In whole cells, activation of protein kinase C increases the levels of phosphorylated Tat-interactive protein 60 kDa and the interaction of Tat-interactive protein 60 kDa with protein kinase C epsilon. A phosphomimetic mutant Tat-interactive protein 60 kDa has distinct subcellular localisation compared to the wild-type protein in whole cells. Taken together, these findings suggest that the protein kinase C epsilon phosphorylation sites on Tat-interactive protein 60 kDa are important for its subcellular localisation. Regulation of the subcellular localisation of Tat-interactive protein 60 kDa via phosphorylation provides a novel means of controlling Tat-interactive protein 60 kDa function.

Examining non-structural retrofitting status of teaching hospitals in Kerman against disasters.

PubMed

Moghadam, Mahmood Nekooi; Moradi, Seyed Mobin; Amiresmaili, Mohammadreza

2017-05-01

Continuous services provision of a hospital before and after a disaster is one of the most prominent issues that all people, especially the authorities must take into huge consideration. Concerning the experiences of previous earthquakes, the role and importance of nonstructural components becomes increasingly clear in the uninterrupted services of hospitals. In this study, non-structural retrofitting status of Kerman teaching hospitals was evaluated against natural disasters. This cross-sectional study was carried out in the second half of 2014 on the teaching hospitals in Kerman (Iran). The study population consisted of all Kerman teaching hospitals. The research instrument was World Health Organization/Pan American Health Organization (WHO/PAHO) standard checklist. Data analysis was carried out using descriptive statistics through SPSS 19. One hospital had a low retrofitting level, two hospitals had an average level and one had a high level. In the examined hospitals in this study, the medical gas section had the lowest preparedness against natural disasters, while the office, warehouse and furniture section had the highest resistance. Generally, the non-structural retrofitting status was 50% in one hospital and was between 65% and 85% in other hospitals. Generally, the retrofitting status of hospitals was not at the ideal condition, most hospitals were in average condition. Concerning the high risk of hospitals in disasters, it is necessary that senior executives and managers of Kerman Province and Kerman University of Medical Sciences take some measures to retrofit these buildings and to reduce the risk of vulnerability.

Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions.

PubMed

Read, G Sullivan; Patterson, Mary

2007-02-01

The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

Peptidases of the peripheral chemoreceptors: biochemical, immunological, in vitro hydrolytic studies and electron microscopic analysis of neutral endopeptidase-like activity of the carotid body.

PubMed

Kumar, G K

1997-02-14

The purposes of the present study are to identify and characterize the major peptidase(s) that may be involved in the inactivation of neuropeptides in the mammalian carotid body. Measurements of a number of peptidase activities in the cell-free extract of the cat carotid body using specific substrates and inhibitors indicated that the previously identified neutral endopeptidase (NEP)-like activity [Kumar et al., Brain Res., 517 (1990) 341-343] is the major peptidase in the chemoreceptor tissue. The NEP-like activity of the carotid body was further characterized using a monoclonal antibody to human neutral endopeptidase, EC 3.4.24.11. Immune blot analysis indicated strong immunoreactivity toward the cat and calf carotid bodies but a weak cross-reactivity with the rabbit carotid body. Furthermore, western blot analysis of the cat carotid body extract revealed the presence of a major 97-kDa protein and a minor 200-kDa protein. The 97-kDa NEP form of the carotid body was comparable to EC 3.4.24.11 and was consistent with its reported molecular weight suggesting NEP-like activity of the carotid body is structurally similar to the neutral endopeptidase, EC 3.4.24.11. In order to assess whether NEP is the primary peptide degrading activity in the cat carotid body in vitro hydrolysis studies using substance P (SP) as a model peptide were performed. HPLC analysis showed that SP is hydrolyzed maximally at pH 7.0 by carotid body peptidases with the formation of SP(1-7) and SP(1-8) as stable intermediates. Inhibitors specific to NEP also inhibited the SP-hydrolyzing activity of the carotid body. Analyses of the cell-free extracts showed the occurrence of both NEP and SP-hydrolyzing activities in the rabbit and rat carotid bodies although at 2- and 4-fold lower levels respectively than that observed in the cat carotid body. Immunoelectron microscopy showed that NEP-specific immunoreactivity is associated with the intercellular region between the type I cells and cell clusters of the carotid body. Taken together, the results from this investigation demonstrate that neutral endopeptidase (EC 3.4.24.11) is one of the major endopeptidases which mediates the degradation and inactivation of neuropeptides in the carotid body.

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

PubMed

Rostagno, A A; Schwarzbauer, J E; Gold, L I

1999-03-01

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

PubMed Central

Rostagno, A A; Schwarzbauer, J E; Gold, L I

1999-01-01

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction. PMID:10024513

Molecular cloning of a cDNA encoding the glycoprotein of hen oviduct microsomal signal peptidase.

PubMed Central

Newsome, A L; McLean, J W; Lively, M O

1992-01-01

Detergent-solubilized hen oviduct signal peptidase has been characterized previously as an apparent complex of a 19 kDa protein and a 23 kDa glycoprotein (GP23) [Baker & Lively (1987) Biochemistry 26, 8561-8567]. A cDNA clone encoding GP23 from a chicken oviduct lambda gt11 cDNA library has now been characterized. The cDNA encodes a protein of 180 amino acid residues with a single site for asparagine-linked glycosylation that has been directly identified by amino acid sequence analysis of a tryptic-digest peptide containing the glycosylated site. Immunoblot analysis reveals cross-reactivity with a dog pancreas protein. Comparison of the deduced amino acid sequence of GP23 with the 22/23 kDa glycoprotein of dog microsomal signal peptidase [Shelness, Kanwar & Blobel (1988) J. Biol. Chem. 263, 17063-17070], one of five proteins associated with this enzyme, reveals that the amino acid sequences are 90% identical. Thus the signal peptidase glycoprotein is as highly conserved as the sequences of cytochromes c and b from these same species and is likely to be found in a similar form in many, if not all, vertebrate species. The data also show conclusively that the dog and avian signal peptidases have at least one protein subunit in common. Images Fig. 1. PMID:1546959

A Structural Perspective on the Modulation of Protein-Protein Interactions with Small Molecules.

PubMed

Demirel, Habibe Cansu; Dogan, Tunca; Tuncbag, Nurcan

2018-05-31

Protein-protein interactions (PPIs) are the key components in many cellular processes including signaling pathways, enzymatic reactions and epigenetic regulation. Abnormal interactions of some proteins may be pathogenic and cause various disorders including cancer and neurodegenerative diseases. Although inhibiting PPIs with small molecules is a challenging task, it gained an increasing interest because of its strong potential for drug discovery and design. The knowledge of the interface as well as the structural and chemical characteristics of the PPIs and their roles in the cellular pathways are necessary for a rational design of small molecules to modulate PPIs. In this study, we review the recent progress in the field and detail the physicochemical properties of PPIs including binding hot spots with a focus on structural methods. Then, we review recent approaches for structural prediction of PPIs. Finally, we revisit the concept of targeting PPIs in a systems biology perspective and we refer to the non-structural approaches, usually employed when the structural information is not present. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The gamma subunit of transducin is farnesylated.

PubMed Central

Lai, R K; Perez-Sala, D; Cañada, F J; Rando, R R

1990-01-01

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification. Images PMID:2217200

Direct association of thioredoxin-1 (TRX) with macrophage migration inhibitory factor (MIF): regulatory role of TRX on MIF internalization and signaling.

PubMed

Son, Aoi; Kato, Noriko; Horibe, Tomohisa; Matsuo, Yoshiyuki; Mochizuki, Michika; Mitsui, Akira; Kawakami, Koji; Nakamura, Hajime; Yodoi, Junji

2009-10-01

Thioredoxin-1 (TRX) is a small (14 kDa) multifunctional protein with the redox-active site Cys-Gly-Pro-Cys. Macrophage migration inhibitory factor (MIF) is a 12 kDa cytokine belonging to the TRX family. Historically, when we purified TRX from the supernatant of ATL-2 cells, a 12 kDa protein was identified along with TRX, which was later proved to be MIF. Here, we show that TRX and MIF form a complex in the cell and the culture supernatant of ATL-2 cells. Using a BIAcore assay, we confirmed that TRX has a specific affinity with MIF. We also found that extracellular MIF was more effectively internalized into the ATL-2 cells expressing TRX on the cell surface, than the Jurkat T cells which do not express surface TRX. Moreover, anti-TRX antibody blocked the MIF internalization, suggesting that the cell surface TRX is involved in MIF internalization into the cells. Furthermore, anti-TRX antibody inhibited MIF-mediated enhancement of TNF-alpha production from macrophage RAW264.7 cells. These results suggest that the cell surface TRX serves as one of the MIF binding molecules or MIF receptor component and inhibits MIF-mediated inflammatory signals.

Bioavailability of riverine dissolved organic matter to phytoplankton in the marine coastal waters

NASA Astrophysics Data System (ADS)

Jurgensone, Iveta; Aigars, Juris

2012-07-01

Nutrient inputs from catchments with intensive agriculture are mostly dominated by inorganic nutrients, whereas the contribution of organic nutrients from catchments with natural forests can be considerable but there is a pooere understanding of this nutrient source. Consequently this study investigated spring, summer and autumn phytoplankton community responses to enrichment by riverine dissolved organic matter (DOM). Dissolved organic substances were extracted from the Daugava River, fractionated into three molecular size classes: 1) 5-100 kDa, 2) 100-1000 kDa, and 3) >1000 kDa, and added to a microcosm with natural assemblages from the Gulf of Riga. During the spring the phytoplankton community was dominated (97%) by diatoms and the species composition did not change over the course of the experiment. Specific species and functional groups of the summer and autumn phytoplankton communities responded positively to these treatments. Small-celled cyanobacteria and Monoraphidium contortum responded to almost all size fractions of DOM for the summer and autumn experiments. Oocystis spp. characteristic for the summer and Chaetoceros wighamii, Cyclotella spp., Thalassiosira baltica for the autumn responded to treatment by two and three size classes of organic substances, respectively, while Merismopedia spp. shifted from one food source to another during the summer experiment.

Dehaloperoxidase-Hemoglobin from Amphitrite ornata Is Primarily a Monomer in Solution

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

M Thompson; S Franzen; M Davis

2011-12-31

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 {angstrom}{sup 2}, an area significantly smaller than the typical value of 1200-2000 {angstrom}{sup 2} and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein,more » which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K{sub d} for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.« less

Passive immunization with Leptospira LPS-specific agglutinating but not non-agglutinating monoclonal antibodies protect guinea pigs from fatal pulmonary hemorrhages induced by serovar Copenhageni challenge.

PubMed

Challa, Sreerupa; Nally, Jarlath E; Jones, Carroll; Sheoran, Abhineet S

2011-06-15

Leptospira interrogans serovar Copenhageni causes pulmonary hemorrhages with respiratory failure, a major cause of death in leptospirosis patients. Protective immunity to Leptospira is known to correlate with the production of leptospiral lipopolysaccharide (L-LPS)-specific agglutinating antibodies. We generated L-LPS-specific mouse monoclonal antibodies (MAbs) and investigated if these MAbs can protect guinea pigs against fatal pulmonary hemorrhages caused by serovar Copenhageni. The MAbs L8H4 and L9B11 against 22kDa L-LPS agglutinated leptospires and completely protected guinea pigs from the development of fatal pulmonary hemorrhages by serovar Copenhageni, whereas the MAb L4C1 against 8kDa L-LPS neither agglutinated the bacteria nor protected the animals against the fatal pulmonary hemorrhages. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

USDA-ARS?s Scientific Manuscript database

Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

PubMed Central

Dhar, Jayeeta; Cuevas, Rolando A.; Goswami, Ramansu; Zhu, Jianzhong

2015-01-01

2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. PMID:26178980

Production and properties of the native Chromobacterium violaceum fucose-binding lectin (CV-IIL) compared to homologous lectins of Pseudomonas aeruginosa (PA-IIL) and Ralstonia solanacearum (RS-IIL).

PubMed

Zinger-Yosovich, Keren; Sudakevitz, Dvora; Imberty, Anne; Garber, Nachman C; Gilboa-Garber, Nechama

2006-02-01

Chromobacterium violaceum is a versatile, violet pigment (violacein)-producing beta-proteobacterium, confined to tropical and subtropical regions, dwelling in soil and water, like Pseudomonas aeruginosa and Ralstonia solanacearum. These three bacteria are saprophytes that occasionally become aggressive opportunistic pathogens virulently attacking animals (the first two) and plants (the third). The recent availability of their genome sequences enabled identification in the C. violaceum genome of an ORF (locus no. 1744) that is similar to those of P. aeruginosa and R. solanacearum lectins, PA-IIL and RS-IIL, respectively. A recombinant protein, CV-IIL, encoded by that ORF exhibited fucose>mannose-specific lectin activity resembling PA-IIL. This paper describes production and properties of the native CV-IIL, which, like PA-IIL and RS-IIL, is probably also a quorum-sensing-driven secondary metabolite, appearing concomitantly with violacein. Its formation is repressed in the CV026 mutant of C. violaceum, which lacks endogenous N-acylhomoserine lactone. The upstream extragenic sequence of its ORF contains a 20 bp sequence (5'-101-120) with partial similarities to the luxI-box and the related P. aeruginosa and R. solanacearum promoter boxes of quorum-sensing-controlled genes. The lectin level is augmented by addition of trehalose to the medium. The subunit size of CV-IIL (around 11.86 kDa) is similar to those of PA-IIL (11.73 kDa) and RS-IIL (11.60 kDa). Like PA-IIL, in the tetrameric form CV-IIL preferentially agglutinates alpha1-2 fucosylated H-positive human erythrocytes (regardless of their A, B or O type), as opposed to the O(h) Bombay type, but differs from it in having no interaction with rabbit erythrocytes and in displaying stronger affinity to l-galactose than to l-fucose. The greater similarity of CV-IIL to PA-IIL than to RS-IIL might be related to the selective adaptation of both C. violaceum and P. aeruginosa to animal tissues versus the preferential homing of R. solanacearum to plants.

High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids

PubMed Central

Nucci, Nathaniel V.; Valentine, Kathleen G.; Wand, A. Joshua

2014-01-01

High-resolution multi-dimensional solution NMR is unique as a biophysical and biochemical tool in its ability to examine both the structure and dynamics of macromolecules at atomic resolution. Conventional solution NMR approaches, however, are largely limited to examinations of relatively small (< 25 kDa) molecules, mostly due to the spectroscopic consequences of slow rotational diffusion. Encapsulation of macromolecules within the protective nanoscale aqueous interior of reverse micelles dissolved in low viscosity fluids has been developed as a means through which the ‘slow tumbling problem’ can be overcome. This approach has been successfully applied to diverse proteins and nucleic acids ranging up to 100 kDa, considerably widening the range of biological macromolecules to which conventional solution NMR methodologies may be applied. Recent advances in methodology have significantly broadened the utility of this approach in structural biology and molecular biophysics. PMID:24656086

Tryptic peptides of canine thyroglobulin reactive with sera of patients with canine hypothyroidism caused by autoimmune thyroiditis.

PubMed

Lee, J-Y; Uzuka, Y; Tanabe, S; Takasawa, T; Sarashina, T; Nachreiner, R F

2004-10-01

Canine thyroglobulin (cTg) was treated with trypsin at a ratio of trypsin to cTg of 1:100 (w/w). Tryptic peptides of cTg were analysed by Western immunoblotting for their reactivity to serum thyroglobulin autoantibodies (TgAA) from patients with TgAA-positive hypothyroidism and normal individuals. The sera of patients with TgAA-positive hypothyroidism reacted with several peptides: 43, 32.5 and 31 kDa; the sera of normal individuals did not bind these tryptic peptides. Some of the TgAA-positive sera of patients reacted with 25 kDa peptide in addition to three tryptic peptides above. This experiment was the first report about antigenic epitopes of cTg. These small tryptic peptides recognized by TgAA may be related with the induction of TgAA and may be useful as markers for autoimmune thyroid diseases in dog.

Single-protein detection in crowded molecular environments in cryo-EM images

PubMed Central

Rickgauer, J Peter; Grigorieff, Nikolaus; Denk, Winfried

2017-01-01

We present an approach to study macromolecular assemblies by detecting component proteins’ characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and—in the presence of protein background—a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material. DOI: http://dx.doi.org/10.7554/eLife.25648.001 PMID:28467302

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein

PubMed Central

Tabib-Salazar, Aline; Liu, Bing; Shadrin, Andrey; Burchell, Lynn; Wang, Zhexin; Wang, Zhihao; Goren, Moran G.; Yosef, Ido; Qimron, Udi; Severinov, Konstantin

2017-01-01

Abstract Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development. PMID:28486695

Possibility of the transformation of eEF-2 (100 kDa) to eEF-2 (65 kDa) in the peptide elongation process in vitro.

PubMed

Gajko, A; Sredzińska, K; Galasiński, W; Gindzieński, A

1999-02-16

Two active eEF-2 polypeptides of approximately 100 and 65 kDa were copurified from rat liver cells and separated. The fate of eEF-2 (100 kDa) during its binding to ribosomes and in the translocation step of the peptide elongation process was investigated. It was shown that eEF-2 (100 kDa) did not change its form during the process of binding to the ribosomes. In the postribosomal supernatant, obtained from the postincubation mixture of the elongation process, only eEF-2 (65 kDa) was found. These results suggest that the form of eEF-2 (100 kDa), when bound to the ribosome during the elongation process, is transformed to eEF-2 (65 kDa). Copyright 1999 Academic Press.

The husk fiber of Cocos nucifera L. (Palmae) is a source of anti-neoplastic activity.

PubMed

Koschek, P R; Alviano, D S; Alviano, C S; Gattass, C R

2007-10-01

In the present study, we investigated the in vitro anti-tumoral activities of fractions from aqueous extracts of the husk fiber of the typical A and common varieties of Cocos nucifera (Palmae). Cytotoxicity against leukemia cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2 x 10(4)/well) were incubated with 0, 5, 50 or 500 microg/mL high- or low-molecular weight fractions for 48 h, treated with MTT and absorbance was measured with an ELISA reader. The results showed that both varieties have almost similar antitumoral activity against the leukemia cell line K562 (60.1 +/- 8.5 and 47.5 +/- 11.9% for the typical A and common varieties, respectively). Separation of the crude extracts with Amicon membranes yielded fractions with molecular weights ranging in size from 1-3 kDa (fraction A) to 3-10 kDa (fraction B) and to more than 10 kDa (fraction C). Cells were treated with 500 microg/mL of these fractions and cytotoxicity was evaluated by MTT. Fractions ranging in molecular weight from 1-10 kDa had higher cytotoxicity. Interestingly, C. nucifera extracts were also active against Lucena 1, a multidrug-resistant leukemia cell line. Their cytotoxicity against this cell line was about 50% (51.9 +/- 3.2 and 56.3 +/- 2.9 for varieties typical A and common, respectively). Since the common C. nucifera variety is extensively cultured in Brazil and the husk fiber is its industrial by-product, the results obtained in the present study suggest that it might be a very inexpensive source of new antineoplastic and anti-multidrug resistant drugs that warrants further investigation.

Heparin and structurally related polymers attenuate eotaxin-1 (CCL11) release from human airway smooth muscle.

PubMed

Kanabar, V; Page, C P; Simcock, D E; Karner, C; Mahn, K; O'Connor, B J; Hirst, S J

2008-06-01

The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovis BCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

PubMed Central

Lefèvre, Philippe; Denis, Olivier; De Wit, Lucas; Tanghe, Audrey; Vandenbussche, Paul; Content, Jean; Huygen, Kris

2000-01-01

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test. PMID:10678905

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

PubMed Central

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

1988-01-01

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

Preclinical evaluation of biodegradable macromolecular contrast agents for magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Feng, Yi

Macromolecular contrast agents have been shown to be superior to small molecular weight contrast agents for MRI in blood pool imaging, tumor diagnosis and grading. However, none has been approved by the FDA because they circulate in the bloodstream much longer than small molecular weight contrast agents and result in high tissue accumulation of toxic Gd(III) ions. Biodegradable macromolecular contrast agents (BMCA) were invented to alleviate the toxic accumulation. They have a cleavable disulfide bond based backbone that can be degraded in vivo and excreted out of the body via renal filtration. Furthermore, the side chain of the backbone can be modified to achieve various degradation rates. Three BMCA, (Gd-DTPA)-cystamine copolymers (GDCC), Gd-DTPA cystine copolymers (GDCP), and Gd-DTPA cystine diethyl ester copolymers (GDCEP), were evaluated as blood pool contrast agents in a rat model. They have excellent blood pool enhancement, preferred pharmacokinetics, and only minimal long-term tissue retention of toxic Gd(III) ions. GDCC and GDCP, the lead agents with desired degradation rates, with molecular weights of 20 KDa and 70 KDa, were chosen for dynamic contrast enhanced MRI (DCE-MRI) to differentiate human prostate tumor models of different malignancy and growth rates. GDCC and GDCP could differentiate these tumor models, providing more accurate estimations of plasma volume, flow leakage rate, and permeability surface area product than a small molecular weight contrast agent Gd-DTPA-BMA when compared to the prototype macromolecular contrast agent albumin-Gd-DTPA. GDCC was favored for its neutral charge side chain and reasonable uptake rate by the tumors. GDCC with a molecular weight of 40 KDa (GDCC-40, above the renal filtration cutoff size) was used to assess the efficacy of two photothermal therapies (interstitial and indocyanine green enhanced). GDCC-40 provided excellent tumor enhancement shortly after its injection. Acute tumor response (4 hr) after therapies was revealed by DCE-MRI using GDCC-40. The region of the tumor with suspicious uptake of GDCC-40 could be correlated to the residual tumor. With only minimum tissue accumulation, BMCA have applications in blood pool imaging, cancer diagnosis, and efficacy assessment of anticancer treatment. Therefore, BMCA are promising for clinical applications.

Heterotetrameric composition of aquaporin-4 water channels.

PubMed

Neely, J D; Christensen, B M; Nielsen, S; Agre, P

1999-08-24

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.

Pore size assessment during corneal endothelial cells permeabilization by femtosecond laser activated carbon nanoparticles

NASA Astrophysics Data System (ADS)

Jumelle, C.; Mauclair, C.; Houzet, J.; Bernard, A.; He, Z.; Piselli, S.; Perrache, C.; Egaud, G.; Baubeau, E.; Gain, P.; Thuret, G.

2015-07-01

Corneal therapeutic molecules delivery represents a promising solution to maintain human corneal endothelial cells (HCECs) viability, but the difficulty is transport across cell membrane. A new delivery method published recently consists in ephemerally permeabilizing cell membranes using a photo-acoustic reaction produced by carbon nanoparticles (CNPs) and femtosecond laser (FsL). The aim of this work is to investigate the size of pores formed at cell membrane by this technique. To induce cell permeabilization, HCECs were put in contact with CNPs and irradiated with a 500 μm diameter Ti:Sa FsL focalized spot. Four sizes of marker molecules were delivered into HCECs to investigate pore sizes: calcein (1.2 nm), FITC-Dextran 4kDa (2.8 nm) and FITC-Dextran 70kDa (12 nm) and FITC-Dextran 2MDa (50 nm). Delivery of each molecule was assessed by flow cytometry, a technique able to measure their presence into cells. We showed that the delivery rate was dependent of their size. Calcein was delivered in 56.1±8.2% of HCECs, FITC-Dextran 4kDa in 42.2±3.5%, FITC-Dextran 70 kDa in 21.5±2.7% and finally FITC-Dextran 2MDa in 12.9±2.0%. It means that a large number of pores in the size ranging from 1.2 to 2.8 nm were formed. However, 12 nm and larger pores were almost half more infrequent. Pore sizes formed at cell membrane by the technique of cell permeabilization by FsL activated CNPs was investigated. The results indicated that the pore sizes are large enough for the efficient delivery of small, medium and big therapeutics molecules on HCECs by this technique.

Impact of large aggregated uricases and PEG diol on accelerated blood clearance of PEGylated canine uricase.

PubMed

Zhang, Chun; Fan, Kai; Ma, Xuefeng; Wei, Dongzhi

2012-01-01

Uricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated. Recombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC. The size of conjugates is important for triggering such phenomena and we speculate that 40-60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

Impact of Large Aggregated Uricases and PEG Diol on Accelerated Blood Clearance of PEGylated Canine Uricase

PubMed Central

Zhang, Chun; Fan, Kai; Ma, Xuefeng; Wei, Dongzhi

2012-01-01

Background Uricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated. Methods and Findings Recombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC. Conclusions The size of conjugates is important for triggering such phenomena and we speculate that 40–60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase. PMID:22745806

Retrieval Interference in Syntactic Processing: The Case of Reflexive Binding in English.

PubMed

Patil, Umesh; Vasishth, Shravan; Lewis, Richard L

2016-01-01

It has been proposed that in online sentence comprehension the dependency between a reflexive pronoun such as himself/herself and its antecedent is resolved using exclusively syntactic constraints. Under this strictly syntactic search account, Principle A of the binding theory-which requires that the antecedent c-command the reflexive within the same clause that the reflexive occurs in-constrains the parser's search for an antecedent. The parser thus ignores candidate antecedents that might match agreement features of the reflexive (e.g., gender) but are ineligible as potential antecedents because they are in structurally illicit positions. An alternative possibility accords no special status to structural constraints: in addition to using Principle A, the parser also uses non-structural cues such as gender to access the antecedent. According to cue-based retrieval theories of memory (e.g., Lewis and Vasishth, 2005), the use of non-structural cues should result in increased retrieval times and occasional errors when candidates partially match the cues, even if the candidates are in structurally illicit positions. In this paper, we first show how the retrieval processes that underlie the reflexive binding are naturally realized in the Lewis and Vasishth (2005) model. We present the predictions of the model under the assumption that both structural and non-structural cues are used during retrieval, and provide a critical analysis of previous empirical studies that failed to find evidence for the use of non-structural cues, suggesting that these failures may be Type II errors. We use this analysis and the results of further modeling to motivate a new empirical design that we use in an eye tracking study. The results of this study confirm the key predictions of the model concerning the use of non-structural cues, and are inconsistent with the strictly syntactic search account. These results present a challenge for theories advocating the infallibility of the human parser in the case of reflexive resolution, and provide support for the inclusion of agreement features such as gender in the set of retrieval cues.

Retrieval Interference in Syntactic Processing: The Case of Reflexive Binding in English

PubMed Central

Patil, Umesh; Vasishth, Shravan; Lewis, Richard L.

2016-01-01

It has been proposed that in online sentence comprehension the dependency between a reflexive pronoun such as himself/herself and its antecedent is resolved using exclusively syntactic constraints. Under this strictly syntactic search account, Principle A of the binding theory—which requires that the antecedent c-command the reflexive within the same clause that the reflexive occurs in—constrains the parser's search for an antecedent. The parser thus ignores candidate antecedents that might match agreement features of the reflexive (e.g., gender) but are ineligible as potential antecedents because they are in structurally illicit positions. An alternative possibility accords no special status to structural constraints: in addition to using Principle A, the parser also uses non-structural cues such as gender to access the antecedent. According to cue-based retrieval theories of memory (e.g., Lewis and Vasishth, 2005), the use of non-structural cues should result in increased retrieval times and occasional errors when candidates partially match the cues, even if the candidates are in structurally illicit positions. In this paper, we first show how the retrieval processes that underlie the reflexive binding are naturally realized in the Lewis and Vasishth (2005) model. We present the predictions of the model under the assumption that both structural and non-structural cues are used during retrieval, and provide a critical analysis of previous empirical studies that failed to find evidence for the use of non-structural cues, suggesting that these failures may be Type II errors. We use this analysis and the results of further modeling to motivate a new empirical design that we use in an eye tracking study. The results of this study confirm the key predictions of the model concerning the use of non-structural cues, and are inconsistent with the strictly syntactic search account. These results present a challenge for theories advocating the infallibility of the human parser in the case of reflexive resolution, and provide support for the inclusion of agreement features such as gender in the set of retrieval cues. PMID:27303315

Influence of dietary nonstructural carbohydrate concentration on growth performance and carcass characteristics of Holstein steers.

PubMed

Ramos-Aviña, Daniel; Plascencia, Alejandro; Zinn, Richard

2018-06-01

Since very little information exists about the topic; in this experiment we compare, in a long-term finishing program, the growth-performance responses and carcass characteristics of Holstein steers where non-structural carbohydrate concentration of the diet is reduced from 64% to 51% (dry matter basis). Sixty Holstein steer calves (129±2.2 kg) were blocked by initial weight into five groups and randomly assigned within weight groupings to 10 pens. Calves were fed with a steam-flaked corn-based finishing diets containing 51% higher fiber (HF) or 64% lower fiber (LF) nonstructural carbohydrates. Non-structural carbohydrates concentrations were manipulated substituting dried distiller grain with solubles and alfalfa hay for flaked corn. Cattle were weighed every 112 days and at the end of the experiment (day 308) when the cattle were harvested and carcass characteristics were evaluated. Steers fed the HF diet showed improvement (8.8%) in average daily gain (ADG) during the initial 112-d period. This effect was followed by a numerical trend for greater ADG throughout the remainder of the study so that overall ADG tended to be greater (4.9%, p = 0.06) for the HF than for LF. There were no treatment effects on dry matter intake. Gain efficiency and estimated dietary net energy (NE) were greater 8.3% and 5.2%, respectively for HF during the initial 112-d period. Overall (308-d) gain efficiency and estimated dietary NE were similar for both dietary treatments. However, due to differences in tabular dietary NE, the ratio of observed:expected dietary NE tended to be greater (4.1%, p = 0.06) for the HF vs LF diet. There were no treatment effects on carcass characteristics except for a tendency toward a slightly greater (0.5%, p = 0.09) estimated carcass yield. Reducing the non-structural carbohydrate concentration of a conventional steam-flaked corn-based growing finishing diet for Holstein steers can effectively enhance growth performance, particularly during the early growing and late finishing phases.

Poly(glycidyl ether)-Based Monolayers on Gold Surfaces: Control of Grafting Density and Chain Conformation by Grafting Procedure, Surface Anchor, and Molecular Weight.

PubMed

Heinen, Silke; Weinhart, Marie

2017-03-07

For a meaningful correlation of surface coatings with their respective biological response reproducible coating procedures, well-defined surface coatings, and thorough surface characterization with respect to layer thickness and grafting density are indispensable. The same applies to polymeric monolayer coatings which are intended to be used for, e.g., fundamental studies on the volume phase transition of surface end-tethered thermoresponsive polymer chains. Planar gold surfaces are frequently used as model substrates, since they allow a variety of straightforward surface characterization methods. Herein we present reproducible grafting-to procedures performed with thermoresponsive poly(glycidyl ether) copolymers composed of glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE). The copolymers feature different molecular weights (2 kDa, 9 kDa, 24 kDa) and are equipped with varying sulfur-containing anchor groups in order to achieve adjustable grafting densities on gold surfaces and hence control the tethered polymers' chain conformation. We determined "wet" and "dry" thicknesses of these coatings by QCM-D and ellipsometry measurements and deduced anchor distances and degrees of chain overlap of the polymer chains assembled on gold. Grafting under cloud point conditions allowed for higher degrees of chain overlap compared to grafting from a good solvent like ethanol, independent of the used sulfur-containing anchor group for polymers with low (2 kDa) and medium (9 kDa) molecular weights. By contrast, the achieved grafting densities and thus chain overlaps of surface-tethered polymers with high (24 kDa) molecular weights were identical for both grafting methods. Monolayers prepared from an ethanolic solution of poly(glycidyl ether)s equipped with sterically demanding disulfide-containing anchors revealed the lowest degrees of chain overlap. The ratio of the radius of gyration to the anchor distance (2 R g /l) of the latter coating was found to be lower than 1.4, indicating that the assembly was rather in the mushroom-like than in the brush regime. Polymer chains with thiol-containing anchors of different alkyl chain lengths (C 11 SH vs C 4 SH) formed assemblies with comparable degrees of chain overlap with 2 R g /l values above 1.4 and are thus in the brush regime. Molecular weights influenced the achievable degree of chain overlap on the surface. Coatings prepared with the medium molecular weight polymer (9 kDa) resulted in the highest chain packing density. Control of grafting density and thus chain overlap in different regimes (brush vs mushroom) on planar gold substrates are attainable for monolayer coatings with poly(GME-ran-EGE) by adjusting the polymer's molecular weight and anchor group as well as the conditions for the grafting-to procedure.

Examining non-structural retrofitting status of teaching hospitals in Kerman against disasters

PubMed Central

Moghadam, Mahmood Nekooi; Moradi, Seyed Mobin; Amiresmaili, Mohammadreza

2017-01-01

Background and objective Continuous services provision of a hospital before and after a disaster is one of the most prominent issues that all people, especially the authorities must take into huge consideration. Concerning the experiences of previous earthquakes, the role and importance of nonstructural components becomes increasingly clear in the uninterrupted services of hospitals. In this study, non-structural retrofitting status of Kerman teaching hospitals was evaluated against natural disasters. Methods This cross-sectional study was carried out in the second half of 2014 on the teaching hospitals in Kerman (Iran). The study population consisted of all Kerman teaching hospitals. The research instrument was World Health Organization/Pan American Health Organization (WHO/PAHO) standard checklist. Data analysis was carried out using descriptive statistics through SPSS 19. Results One hospital had a low retrofitting level, two hospitals had an average level and one had a high level. In the examined hospitals in this study, the medical gas section had the lowest preparedness against natural disasters, while the office, warehouse and furniture section had the highest resistance. Generally, the non-structural retrofitting status was 50% in one hospital and was between 65% and 85% in other hospitals. Conclusions Generally, the retrofitting status of hospitals was not at the ideal condition, most hospitals were in average condition. Concerning the high risk of hospitals in disasters, it is necessary that senior executives and managers of Kerman Province and Kerman University of Medical Sciences take some measures to retrofit these buildings and to reduce the risk of vulnerability. PMID:28713518

Non-structural protein P6 encoded by rice black-streaked dwarf virus is recruited to viral inclusion bodies by binding to the viroplasm matrix protein P9-1.

PubMed

Sun, Liying; Xie, Li; Andika, Ida Bagus; Tan, Zilong; Chen, Jianping

2013-08-01

Like other members of the family Reoviridae, rice black-streaked dwarf virus (RBSDV, genus Fijivirus) is thought to replicate and assemble within cytoplasmic viral inclusion bodies, commonly called viroplasms. RBSDV P9-1 is the key protein for the formation of viroplasms, but little is known about the other proteins of the viroplasm or the molecular interactions amongst its components. RBSDV non-structural proteins were screened for their association with P9-1 using a co-immunoprecipitation assay. Only P6 was found to directly interact with P9-1, an interaction that was confirmed by bimolecular fluorescence complementation assay in Spodoptera frugiperda (Sf9) cells. Immunoelectron microscopy showed that P6 and P9-1 co-localized in electron-dense inclusion bodies, indicating that P6 is a constituent of the viroplasm. In addition, non-structural protein P5 also localized to viroplasms and interacted with P6. In Sf9 cells, P6 was diffusely distributed throughout the cytoplasm when expressed alone, but localized to inclusions when co-expressed with P9-1, suggesting that P6 is recruited to viral inclusion bodies by binding to P9-1. P5 localized to the inclusions formed by P9-1 when co-expressed with P6 but did not when P6 was absent, suggesting that P5 is recruited to viroplasms by binding to P6. This study provides a model by which viral non-structural proteins are recruited to RBSDV viroplasms.

Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

PubMed

Freedman, John C; Li, Jihong; Uzal, Francisco A; McClane, Bruce A

2014-10-21

Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural host intestinal contents. These analyses demonstrated that (i) ETX processing in host intestinal contents occurs in an ordered, stepwise fashion, (ii) processing of prototoxin by host intestinal contents results in higher-molecular-mass material and 3 distinct ~27-kDa ETX species, and (iii) serine proteases, such as trypsin, chymotrypsin, and other proteases, including carboxypeptidases, play a role in the activation of ETX by intestinal contents. These studies provide new insights into the activation and processing of ETX and demonstrate that this process is more complicated than previously appreciated. Copyright © 2014 Freedman et al.

Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.

PubMed

Yamamoto, K; Ichinose, Y; Shinagawa, H; Makino, K; Nakata, A; Iwanaga, M; Honda, T; Miwatani, T

1990-12-01

Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.

Insulin stimulates the tyrosine phosphorylation of a 61-kilodalton protein in rat adipocytes.

PubMed

Mooney, R A; Bordwell, K L

1992-03-01

Insulin stimulated the tyrosine phosphorylation of a 61-kilodalton (kDa) protein in rat adipocytes prelabeled for 2 h with [32P]orthophosphate. Tyrosine phosphorylation of this 61-kDa protein displayed very similar insulin concentration dependency to receptor autophosphorylation and tyrosine phosphorylation of a high molecular mass receptor substrate of 160 kDa. Phosphorylation of the 61-kDa protein was very rapid with maximum labeling attained at 30 sec, paralleling that of the other two proteins. Phosphoamino acid analysis revealed that each of the insulin-responsive phosphoproteins contained phosphoserine as well as phosphotyrosine, though the ratio of two phosphoamino acids recovered from each protein differed. The 61-kDa protein yielded relatively equal proportions of phosphoserine and phosphotyrosine. In contrast, the insulin receptor yielded relatively more label on phosphotyrosine than phosphoserine, whereas label incorporated into the 160-kDa protein was recovered primarily on phosphoserine. Cleveland peptide maps using either Staphylococcus aureus V8 proteinase or chymotrypsin revealed no similarities between the 61-kDa protein and the other tyrosine phosphorylated proteins. With subcellular fractionation, the 160-kDa protein was found in equal proportions in the high speed pellet (100,000 g) and supernatant. The 61-kDa protein had a similar distribution to that of the 160-kDa protein but was also detected in the low speed pellet (10,000 g). The insulin receptor was localized to the low speed pellet. In summary, rat adipocytes contain an insulin-dependent phosphotyrosyl protein of 61 kDa which is distinct from the more prominent high molecular mass receptor substrate. This 61-kDa protein has characteristics consistent with it being a substrate for the insulin receptor tyrosine kinase.

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

PubMed

Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

Combined Use of Residual Dipolar Couplings and Solution X-ray Scattering To Rapidly Probe Rigid-Body Conformational Transitions in a Non-phosphorylatable Active-Site Mutant of the 128 kDa Enzyme I Dimer

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

Takayama, Yuki; Schwieters, Charles D.; Grishaev, Alexander

2012-10-23

The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidinemore » and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN{sup {alpha}/{beta}} subdomain and an aspartate (Asp129) on the EIN{sup {alpha}} subdomain results in a small ({approx}9{sup o}) reorientation of the EIN{sup {alpha}} and EIN{sup {alpha}/{beta}} subdomains that is in turn propagated to a larger reorientation ({approx}26{sup o}) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.« less

Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells.

PubMed

Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-06-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high-throughput proteomic assays, we have previously identified Bruton's tyrosine kinase (BTK) as a host protein that was uniquely upregulated in the plasma membrane of human immunodeficiency virus (HIV-1)-infected T cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant upregulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells; however, new BTK protein complexes were identified and distributed in both high molecular weight (∼600 kDa) and a small molecular weight complex (∼60-120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1-infected cells using small interfering RNA (siRNA) resulted in selective death of infected, but not uninfected, cells. Using BTK-specific antibody and small-molecule inhibitors including LFM-A13 and a FDA-approved compound, ibrutinib (PCI-32765), we have found that HIV-1-infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1-infected cells are sensitive to treatments targeting BTK expressed in infected cells.

Molecular detection and genetic analysis of Akabane virus genogroup Ib in small ruminants in Turkey.

PubMed

Şevik, Murat

2017-09-01

Akabane disease is a viral disease transmitted by biting midges and can cause teratogenic malformations in cattle, sheep and goats. Abortion outbreaks associated with arthrogryposis and cerebellar hypoplasia in two epidemiologically independent flocks were reported in the Mediterranean region of Turkey in 2015. Phylogenetic analysis based on nucleocapsid (N) gene sequences showed that the field isolates presented here belong to the genogroup Ib. The akabane virus (AKAV) field isolates analysed in this study displayed 6 new amino acid substitutions in N and non-structural protein chains compared with those of AKAV strains belonging to genogroup Ib. To the best of our knowledge, this is the first report on the presence of the AKAV genogroup Ib in Turkey.

Development of an indirect ELISA with epitope on nonstructural protein of Muscovy duck parvovirus for differentiating between infected and vaccinated Muscovy ducks.

PubMed

Yan, B; Ma, J-Z; Yu, T-F; Shao, S-L; Li, M; Fan, X-D

2014-12-01

The aim of this study was to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of Muscovy duck parvovirus (MDPV). Sera (100) from negative and vaccinated Muscovy ducks were compared with infected sera (240) to establish the cut-off value of this i-ELISA. There was a significant difference between the positive and negative populations (P < 0·05). The adoption of this positive-negative threshold value for this i-ELISA assay resulted in specificity of 98·0%. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus. In this study, we developed an i-ELISA based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of MDPV. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus. © 2014 The Society for Applied Microbiology.

Sequence, structure and function relationships in flaviviruses as assessed by evolutive aspects of its conserved non-structural protein domains.

PubMed

da Fonseca, Néli José; Lima Afonso, Marcelo Querino; Pedersolli, Natan Gonçalves; de Oliveira, Lucas Carrijo; Andrade, Dhiego Souto; Bleicher, Lucas

2017-10-28

Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed. Copyright © 2017 Elsevier Inc. All rights reserved.

Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

PubMed

Su, Qiudong; Guo, Minzhuo; Jia, Zhiyuan; Qiu, Feng; Lu, Xuexin; Gao, Yan; Meng, Qingling; Tian, Ruiguang; Bi, Shengli; Yi, Yao

2016-07-01

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

Studying the highly bent spectra of FR II-type radio galaxies with the KDA EXT model

NASA Astrophysics Data System (ADS)

Kuligowska, Elżbieta

2018-04-01

Context. The Kaiser, Dennett-Thorpe & Alexander (KDA, 1997, MNRAS, 292, 723) EXT model, that is, the extension of the KDA model of Fanaroff & Riley (FR) II-type source evolution, is applied and confronted with the observational data for selected FR II-type radio sources with significantly aged radio spectra. Aim. A sample of FR II-type radio galaxies with radio spectra strongly bent at their highest frequencies is used for testing the usefulness of the KDA EXT model. Methods: The dynamical evolution of FR II-type sources predicted with the KDA EXT model is briefly presented and discussed. The results are then compared to the ones obtained with the classical KDA approach, assuming the source's continuous injection and self-similarity. Results: The results and corresponding diagrams obtained for the eight sample sources indicate that the KDA EXT model predicts the observed radio spectra significantly better than the best spectral fit provided by the original KDA model.

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

PubMed Central

Modderman, P W; von dem Borne, A E; Sonnenberg, A

1994-01-01

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7514867

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

The sea urchin egg jelly coat consists of globular glycoproteins bound to a fibrous fucan superstructure.

PubMed

Bonnell, B S; Keller, S H; Vacquier, V D; Chandler, D E

1994-03-01

Intact egg jelly (EJ) coats surrounding eggs of the sea urchin Strongylocentrotus purpuratus were visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, rotary-shadowing technique. The hydrated EJ coat forms an extensive fibrous network that makes contact with the vitelline layer at the egg surface. Fibers are decorated along their length with particles, particle density being highest in the interior regions of the coat. The macromolecular components making up the EJ network were visualized by rotary-shadowing of mica-adsorbed EJ samples. Whole EJ coats solubilized in pH 5 sea-water and spread on the mica surface consist of complex networks of branching fibers decorated with large patches of amorphous material. As we have previously shown (Keller and Vacquier, 1994), EJ boiled in a dissolution buffer containing SDS and beta-mercaptoethanol and applied to a Sephacryl-500 gel filtration column can be separated into three fractions: a 380-kDa fucose sulfate polymer (FSP), which elutes in the void volume, and two column-included fractions consisting of intermediate (300 kDa) and low-molecular-weight (30- to 138-kDa) glycoproteins. Rotary-shadowing of the FSP fraction reveals branched fibrous components similar in appearance to that of solubilized whole EJ but devoid of any particulate decoration. In contrast, intermediate- and low-molecular-weight EJ components are strictly globular in appearance but are distinguishable on the basis of size. Ion-exchange purification of whole EJ yields two glycoproteins, of 82 and 138 kDa, having AR-inducing activity (Keller and Vacquier, 1994). Platinum replication shows these active components to be small spherical molecules about 8 nm in diameter. The above fractionation scheme requires harsh dissociation conditions. Indeed, if EJ is not boiled in SDS buffer before fractionation, the 300-kDa fraction and the FSP appear together in the void volume. Rotary-shadowing of this complex reveals a multistranded polymer, decorated with glycoproteins at specific kink points. Taken together, our data suggest that the EJ network is composed of a fucose sulfate polymer superstructure to which glycoproteins are bound.

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

PubMed

Chapanian, Rafi; Constantinescu, Iren; Rossi, Nicholas A A; Medvedev, Nadia; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

2012-11-01

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

PubMed Central

Nishikiori, Masaki; Dohi, Koji; Mori, Masashi; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

2006-01-01

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity. PMID:16912296

Effectivity of immunostimulant from Zoothamnium penaei protein membrane for decreasing the mortality rate of white shrimp (Litopenaeus vannamei) in traditional plus pond

NASA Astrophysics Data System (ADS)

Mahasri, G.; Kusdarwati, R.; Kismiyati; Rozi; Gustrifandi, H.

2018-04-01

The purpose of this research was to analys immunogenic membrane protein as immunostimulant development material to control the mortality of white shrimp in traditional plus pond. This research was designed to use explorative experiment and experimental laboratory methods which used completed random sampling design. Collected data was analyzed with analysis of variance for examination of survival rate (SR), total haemocyte count (THC) and differensial haemocyte Count (DHC). The research divided into 2 part of riset: (1) Identification, cultivation Zoothamnium penaei, analysed of membrane protein by SDS-PAGE, (2) Field test protein membran on Survival Rate level, immune response (THC and/or DHC level) and infestation of Zoothamnium penaei in traditional plus pond. The result showed that there were seven bands membrane protein of Zoothamnium penaei with molecular weight 38 kDa, 48 kDa, 67 kDa, 71 kDa, 77 kDa, 98 kDa dan 104 kDa by using SDS-PAGE. Immunogenicity tested decrease by using ELISA and western blotting there are only found three bands with molecular weight 38 kDa, 48 kDa dan 67 kDa. The membrane protein could increase the immun respons and decrease the mortality, by subsequenly, it could increase the survival rate from 17% until 68% and pressured the parasite infestation of white shrimp.

The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

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

Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

PubMed

Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

2009-01-01

The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

PubMed

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

PubMed

Tu, Mengyu; Liu, Fei; Chen, Shun; Wang, Mingshu; Cheng, Anchun

2015-11-01

Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

The KnowRISK project: Tools and strategies for risk communication and learning

NASA Astrophysics Data System (ADS)

Musacchio, Gemma; Amaral Ferreira, Mónica; Falsaperla, Susanna; Piangiamore, Giovanna Lucia; Pino, Nicola Alessandro; Solarino, Stefano; Crescimbene, Massimo; Eva, Elena; Reitano, Danilo; Þorvaldsdottir, Solveig; Sousa Silva, Delta; Rupakhety, Rajesh; Sousa Oliveira, Carlos

2016-04-01

Damage of non-structural elements of buildings (i.e. partitions, ceilings, cladding, electrical and mechanical systems and furniture) is known to cause injuries and human losses. Also it has a significant impact on earthquake resilience and is yet being worldwide underestimated. The project KnowRISK (Know your city, Reduce seISmic risK through non-structural elements) is financed by the European Commission to develop prevention measures that may reduce non-structural damage in urban areas. Pilot areas of the project are within the three European participating countries, namely Portugal, Iceland and Italy. They were chosen because they are prone to damage level 2 and 3 (EMS-98, European Macroseismic Scale) that typically affects non-structural elements. We will develop and test a risk communication strategy taking into account the needs of households and schools, putting into practice a portfolio of best practice to reduce the most common non-structural vulnerabilities. We will target our actions to different societal groups, considering their cultural background and social vulnerabilities, and implement a participatory approach that will promote engagement and interaction between the scientific community, practitioners and citizens to foster knowledge on everyone's own neighborhoods, resilience and vulnerability. A Practical Guide for citizens will highlight that low-cost actions can be implemented to increase safety of households, meant as being the places where the most vulnerable societal groups, including children and elderly people, spend much of their time. Since our actions towards communication will include education, we will define tools that allow a clear and direct understanding of elements exposed to risk. Schools will be one of our target societal groups and their central role played at the community level will ensure spreading and strengthening of the communication process. Schools are often located in old or re-adapted buildings, formerly used for other uses, or when the growing number of students or new needs require adapting the space to the necessities, often without taking enough care of safety. Moreover, in urban areas, schools may be hosted in tall buildings where the shaking of moderate-to-low magnitude earthquakes can cause damage level 2 and 3. Students will be involved into looking after their own situation in terms of non-structural vulnerability to promote education and prevention, while increasing resilience in terms of societal capacity to cope with future disasters. The actions will undergo specific effectiveness assessment with ex-ante and ex-post surveys. The results of this assessment will allow an evaluation of on-site risk communication activities, the comparability between pilot-areas achievements, and an opportunity for learning and guidance for future risk communication.

Detection of brain-directed autoantibodies in the serum of non-small cell lung cancer patients.

PubMed

Banjara, Manoj; Ghosh, Chaitali; Dadas, Aaron; Mazzone, Peter; Janigro, Damir

2017-01-01

Antibodies against brain proteins were identified in the plasma of cancer patients and are defined to cause paraneoplastic neurological syndromes. The profiles of brain-directed antibodies in non-small cell lung cancer (NSCLC) are largely unknown. Here, for the first time, we compared autoantibodies against brain proteins in NSCLC (n = 18) against those present in age-matched non-cancer control subjects (n = 18) with a similar life-style, habit, and medical history. Self-recognizing immunoglobulin (IgG) are primarily directed against cells in the cortex (P = 0.008), hippocampus (P = 0.003-0.05), and cerebellum (P = 0.02). More specifically, IgG targets were prominent in the pyramidal, Purkinje, and granule cell layers. Furthermore, autoimmune IgG signals were localized to neurons (81%), astrocytes (48%), and endothelial (29%) cells. While cancer sera yielded overall higher intensity signals, autoantigens of 100, 65, 45, 37, and 30 kDa molecular weights were the most represented. Additionally, a group of 100 kDa proteins seem more prevalent in female adenocarcinoma patients (4/5, 80%). In conclusion, our results revealed autoantigen specificity in NSCLC, which implicitly depends on patient's demographics and disease history. Patients at risk for lung cancer but with no active disease revealed that the immune profile in NSCLC is disease-dependent.

8-Hydroxyquinoline and hydroxamic acid inhibitors of botulinum neurotoxin BoNT/A.

PubMed

Dickerson, Tobin J; Smith, Garry R; Pelletier, Jeffrey C; Reitz, Allen B

2014-01-01

We describe here the state of the art of certain aspects concerning potential small molecule therapy directed toward botulism, by inhibition of the zinc-protease containing light chain (LC) of botulinum neurotoxin BoNT/A from the anaerobic bacillus Clostridium botulinum. Botulinum neurotoxins (BoNTs) are comprised of eight serologically-distinct proteins (A - H), several of which are further divided, such as BoNT/A which has five subtypes. The BoNTs are the most toxic substances known to mankind, causing a form of flaccid paralysis that can be rapid and is often lethal. BoNT/A is comprised of a ~100 kDa heavy chain (HC) attached via a single disulfide Cys-Cys bond to a ~50 kDa LC. The HC mediates transport to and uptake by presynaptic glutamatergic neurons, where the LC cleaves the protein SNAP-25 and thus prevents vesicular trafficking and release of acetylcholine. The Zn-endoprotease activity of the LC of BoNT/A is a target for the development of small molecule inhibitors of BoNT/A-mediated toxicity. A variety of BoNT/A LC inhibitors have been described to date and we focus here primarily on the Zn-binding 8-hydroxyquinoline structural type as well as some of the previously-described hydroxamic acids.

Immunoblot patterns of Taenia asiatica taeniasis.

PubMed

Jeon, Hyeong-Kyu; Eom, Keeseon S

2009-03-01

Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97 kDa and 21.5 kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and 21.5 kDa against T. asiatica infected sera. Antigens of adult Taenia saginata revealed 110-100, 66, 58-56, and 46 kDa immunoblot bands against T. asiatica infected sera. Antigens of adult Taenia solium also revealed 99-97, 68-66, and 46 kDa bands against T. asiatica infected sera. The immunoblot band of 21.5 kDa exhibited specificity to T. asiatica.

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

PubMed Central

Choi, Min-Ho; Ryu, Jin-Sook; Lee, Mejeong; Li, Shunyu; Chung, Byung-Suk; Chai, Jong-Yil; Sithithaworn, Paiboon; Tesana, Smarn

2003-01-01

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection. PMID:12972729

Immunoblot Patterns of Taenia asiatica Taeniasis

PubMed Central

Jeon, Hyeong-Kyu

2009-01-01

Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97 kDa and 21.5 kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and 21.5 kDa against T. asiatica infected sera. Antigens of adult Taenia saginata revealed 110-100, 66, 58-56, and 46 kDa immunoblot bands against T. asiatica infected sera. Antigens of adult Taenia solium also revealed 99-97, 68-66, and 46 kDa bands against T. asiatica infected sera. The immunoblot band of 21.5 kDa exhibited specificity to T. asiatica. PMID:19290097

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

PubMed

Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

2017-07-19

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

RNAi silenced Dd-grp94 (Dictyostelium discoideum glucose-regulated protein 94 kDa) cell lines in Dictyostelium exhibit marked reduction in growth rate and delay in development.

PubMed

Baviskar, Sandhya N; Shields, Malcolm S

2010-01-01

Glucose-regulated 94 kDa protein (Grp94) is a resident of the endoplasmic reticulum (ER) of multicellular eukaryotes. It is a constitutively expressed protein that is overexpressed in certain abnormal conditions of the cell such as depletion of glucose and calcium, and low oxygen and pH. The protein is also implicated in diseased conditions like cancer and Alzheimer's disease. In this study, the consequences of downregulation of Grp94 were investigated at both unicellular and multicellular stages of Dictyostelium discoideum. Previous studies have shown the expression of Dd-Grp94 (Dictyostelium discoideum glucose-regulated 94 kDa protein) in wild-type cells varies during development, and overexpression of Dd-Grp94 leads to abnormal cell shape and inhibition of development (i.e., formation of fruiting bodies). Grp94 is a known calcium binding protein and an efficient calcium buffer. Therefore, in the present study we hypothesized that downregulation of Dd-Grp94 protein would affect Dictyostelium cell structure, growth, and development. We found that Dd-grp94 RNAi recombinants exhibited reduced growth rate, cell size, and a subtle change in cell motility compared to the parental cells. The recombinants also exhibited a delay in development and small fruiting bodies. These results establish that Dd-grp94 plays a crucial role in determining normal cell structure, growth and differentiation.

Depleted uranium mobility across a weapons testing site: isotopic investigation of porewater, earthworms, and soils.

PubMed

Oliver, Ian W; Graham, Margaret C; MacKenzie, Angus B; Ellam, Robert M; Farmer, John G

2008-12-15

The mobility and bioavailability of depleted uranium (DU) in soils at a UK Ministry of Defence (UK MoD) weapons testing range were investigated. Soil and vegetation were collected near a test-firing position and at eight points along a transect line extending approximately 200 m down-slope, perpendicular to the firing line, toward a small stream. Earthworms and porewaters were subsequently separated from the soils and both total filtered porewater (<0.2 microm) and discrete size fractions (0.2 microm-100 kDa, 100-30 kDa, 30-3 kDa, and <3 kDa)obtainedvia centrifugal ultrafiltration were examined. Uranium concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) for soils and ICP-mass spectrometry (MS) for earthworms and porewaters, while 235U:238U atom ratios were determined by multicollector (MC)-ICP-MS. Comparison of the porewater and earthworm isotopic values with those of the soil solids indicated that DU released into the environment during weapons test-firing operations was more labile and more bioavailable than naturally occurring U in the soils at the testing range. Importantly, DU was shown to be present in soil porewater even at a distance of approximately 185 m from the test-firing position and, along the extent of the transect was apparently associated with organic colloids.

A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex)

NASA Technical Reports Server (NTRS)

Weitman, D.; Etlinger, J. D.

1992-01-01

Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

PubMed Central

Davey, Michelle L.; Hancock, Robert E. W.; Mutharia, Lucy M.

1998-01-01

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli. PMID:9435071

Redox changes accompanying storage protein mobilization in moist chilled and warm incubated walnut kernels prior to germination.

PubMed

Shahmoradi, Zeynab; Tamaskani, Fatemeh; Sadeghipour, Hamid Reza; Abdolzadeh, Ahmad

2013-01-01

Alterations in the redox state of storage proteins and the associated proteolytic processes were investigated in moist-chilled and warm-incubated walnut (Juglans regia L.) kernels prior to germination. The kernel total protein labeling with a thiol-specific fluorochrome i.e. monobromobimane (mBBr) revealed more reduction of 29-32 kDa putative glutelins, while in the soluble proteins, both putative glutelins and 41, 55 and 58 kDa globulins contained reduced disulfide bonds during mobilization. Thus, the in vivo more reduced disulfide bonds of storage proteins corresponds to greater solubility. After the in vitro reduction of walnut kernel proteins pre-treated by N-ethyl maleimide (NEM) with dithioerythrethiol (DTT) and bacterial thioredoxin, the 58 kDa putative globulin and a 6 kDa putative albumin were identified as disulfide proteins. Thioredoxin stimulated the reduction of the H(2)O(2)-oxidized 6 kDa polypeptide, but not the 58 kDa polypeptide by DTT. The solubility of 6 kDa putative albumin, 58 and 19-24 kDa putative globulins and glutelins, respectively, were increased by DTT. The in vitro specific mobilization of the 58 kDa polypeptide that occurred at pH 5.0 by the kernel endogenous protease was sensitive to the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and stimulated by DTT. The specific degradation of the 58 kDa polypeptide might be achieved through thioredoxin-mediated activation of a serine protease and/or reductive unfolding of its 58 kDa polypeptide substrate. As redox changes in storage proteins occurred equally in both moist chilled and warm incubated walnut kernels, the regulatory functions of thioredoxins in promoting seed germination may be due to other germination related processes. Copyright © 2012 Elsevier GmbH. All rights reserved.

Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris--importance of correct processing of the N-terminus.

PubMed

Zhang, Rundong; Kim, Tae-Kang; Qiao, Zhaun-Hong; Cai, Jian; Pierce, William M; Song, Zhao-Hui

2007-10-01

This study was conducted to optimize the expression of human CB2 cannabinoid receptors in methylotrophic yeast Pichia pastoris (P. pastoris). Two major species of expressed CB2 proteins were seen on Western blot, i.e., a 42 kDa band which matches the calculated molecular weight for tagged CB2, and a 52/55 kDa doublet. Treatment of membranes with N-glycosidase F or inclusion of tunicamycin in the culture medium during induction resulted in the disappearance of the 55 kDa, but not the 52 kDa band, suggesting that the 3 kDa extra in the 55 kDa band is due to N-glycosylation, but the 10 kDa extra in the 52 kDa band is not due to N-glycosylation. Anti-FLAG M1 antibody had a much higher preference for the 42 kDa band over the 52/55 kDa doublet, and a 10 kDa fragment recognized by anti-FLAG M2 antibody was generated by CNBr digestion of the 52/55 doublet. These data strongly support the hypothesis that the 10 kDa increase in molecular weight was due to unprocessed alpha-factor sequence. This conclusion was further validated by finding several peptide sequences for alpha-factor fragments at the N-terminal of the CB2 receptor using pepsin/chymotrypsin digestion and LC/MS/MS approaches. Importantly, unprocessed alpha-factor was found to be associated with poor ligand binding. In addition, controlling the level of CB2 protein expression was found to be critical for minimizing the presence of unprocessed alpha-factor sequence. The information gained from this study should aid the proper expression of not only CB2 receptor but also other members of the GPCR family in P. pastoris.

Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes.

PubMed

Mooney, R A; Bordwell, K L

1991-03-01

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism.

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase.

PubMed

Chayko, C A; Orgebin-Crist, M C; Skudlarek, M D; Tulsiani, D R

2000-09-01

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.

A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity.

PubMed

Wang, Junyong; Zeng, Yan; Xu, Shuai; Yang, Jiayun; Wang, Wanbing; Zhong, Bo; Ge, Jinying; Yin, Lei; Bu, Zhigao; Shu, Hong-Bing; Chen, Hualan; Lei, Cao-Qi; Zhu, Qiyun

2018-06-01

Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals. IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines. Copyright © 2018 American Society for Microbiology.

Structural Study of the C-Terminal Domain of Nonstructural Protein 1 from Japanese Encephalitis Virus.

PubMed

Poonsiri, Thanalai; Wright, Gareth S A; Diamond, Michael S; Turtle, Lance; Solomon, Tom; Antonyuk, Svetlana V

2018-04-01

Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal β-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates that flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the 22NS1 monoclonal antibody, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This report highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships. IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Nonstructural protein 1 (NS1) plays a role in viral replication, and, because it is secreted, it can exhibit a wide range of interactions with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the C-terminal β-ladder domain of NS1 between flaviviruses, including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics. Copyright © 2018 American Society for Microbiology.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

PubMed

Khasnatinov, Maxim A; Tuplin, Andrew; Gritsun, Dmitri J; Slovak, Mirko; Kazimirova, Maria; Lickova, Martina; Havlikova, Sabina; Klempa, Boris; Labuda, Milan; Gould, Ernest A; Gritsun, Tamara S

2016-01-01

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity

PubMed Central

Khasnatinov, Maxim A.; Tuplin, Andrew; Gritsun, Dmitri J.; Slovak, Mirko; Kazimirova, Maria; Lickova, Martina; Havlikova, Sabina; Klempa, Boris; Gould, Ernest A.

2016-01-01

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras. PMID:27341437

The effectiveness of the Pilates method: reducing the degree of non-structural scoliosis, and improving flexibility and pain in female college students.

PubMed

Alves de Araújo, Maria Erivânia; Bezerra da Silva, Elirez; Bragade Mello, Danielli; Cader, Samária Ali; Shiguemi Inoue Salgado, Afonso; Dantas, Estélio Henrique Martin

2012-04-01

To evaluate the effectiveness of Pilates with regard to the degree of scoliosis, flexibility and pain. The study included 31 female students divided into two groups: a control group (CG = 11), which had no therapeutic intervention, and an experimental group (EG = 20), which underwent Pilates-based therapy. We used radiological goniometry measurements to assess the degree of scoliosis, standard goniometry measurements to determine the degree of flexibility and the scale of perceived pain using the Borg CR 10 to quantify the level of pain. The independent t test of the Cobb angle (t = - 2.317, p = 0.028), range of motion of trunk flexion (t = 3.088, p = 0.004) and pain (t = -2.478, p = 0.019) showed significant differences between the groups, with best values in the Pilates group. The dependent t test detected a significant decrease in the Cobb angle (Δ% = 38%, t = 6.115, p = 0.0001), a significant increase in trunk flexion (Δ% = 80%, t = -7.977, p = 0.0001) and a significant reduction in pain (Δ% = 60%, t = 7.102, p = 0.0001) in the EG. No significant difference in Cobb angle (t = 0.430, p = 0.676), trunk flexion, (t = 0.938p = 0.371) or pain (t = 0.896, p = 0.391) was found for the CG. The Pilates group was better than control group. The Pilates method showed a reduction in the degree of non-structural scoliosis, increased flexibility and decreased pain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe.

PubMed

Shien, J-H; Wang, Y-S; Chen, C-H; Shieh, H K; Hu, C-C; Chang, P-C

2008-10-01

Live attenuated vaccines have been used for control of the disease caused by goose parvovirus (GPV), but the mechanism involved in attenuation of GPV remains elusive. This report presents the complete nucleotide sequences of two live attenuated strains of GPV (82-0321V and VG32/1) that were independently developed in Taiwan and Europe, together with the parental strain of 82-0321V and a field strain isolated in Taiwan in 2006. Sequence comparisons showed that 82-0321V and VG32/1 had multiple deletions and substitutions in the inverted terminal repeats region when compared with their parental strain or the field virus, but these changes did not affect the formation of the hairpin structure essential for viral replication. Moreover, 82-0321V and VG32/1 had five amino acid changes in the non-structural protein, but these changes were located at positions distant from known functional motifs in the non-structural protein. In contrast, 82-0321V had nine changes and VG32/1 had 11 changes in their capsid proteins (VP1), and the majority of these changes occurred at positions close to the putative receptor binding sites of VP1, as predicted using the structure of adeno-associated virus 2 as the model system. Taken together, the results suggest that changes in sequence near the receptor binding sites of VP1 might be responsible for attenuation of GPV. This is the first report of complete nucleotide sequences of GPV other than the virulent B strain, and suggests a possible mechanism for attenuation of GPV.

A thermostable trypsin inhibitor with antiproliferative activity from small pinto beans.

PubMed

Chan, Yau Sang; Zhang, Yanbo; Sze, Stephen Cho Wing; Ng, Tzi Bun

2014-08-01

Small pinto bean is a cultivar of Phaseolus vulgaris. It produces a 16-kDa trypsin inhibitor that could be purified using anion exchange and size chromatography. Q-Sepharose, Mono Q and Superdex 75 columns were employed for the isolation process. Small pinto bean trypsin inhibitor demonstrated moderate pH stability (pH 2-10) and marked heat stability, with its trypsin inhibitory activity largely retained after exposure to 100 °C for half an hour. The activity was abolished in the presence of dithiothreitol, in a dose-dependent manner, implying that disulfide bonds in small pinto bean trypsin inhibitor are crucial for the activity. The trypsin inhibitor showed a blocked N-terminus. The trypsin inhibitor only slightly inhibited the viability of breast cancer MCF7 and hepatoma HepG2 cells at 125 μM.

Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever

PubMed Central

Durbin, Anna P.; Vargas, Maria José; Wanionek, Kimberli; Hammond, Samantha N.; Gordon, Aubree; Rocha, Crisanta; Balmaseda, Angel; Harris, Eva

2008-01-01

In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14+, CD32+, CD86+, CD11c+). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis. PMID:18452966

A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae▿

PubMed Central

Woolford, Lucy; Rector, Annabel; Van Ranst, Marc; Ducki, Andrea; Bennett, Mark D.; Nicholls, Philip K.; Warren, Kristin S.; Swan, Ralph A.; Wilcox, Graham E.; O'Hara, Amanda J.

2007-01-01

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families. PMID:17898069

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

PubMed Central

Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

2016-01-01

ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR. PMID:27122577

Cloning and Characterizing Genes Involved in Monoterpene Induced Mammary Tumor Regression.

DTIC Science & Technology

1996-10-01

causes morphologic differentiation within 4 hours as characterized by neurite outgrowths (12). Monoterpenes inhibit enzymes in the mevalonate-lipid...metabolism pathway, including a selective inhibition of isoprenylation of 21-26 kDa small G proteins (13-15) and inhibition of ubiquinone ( CoQ ) and...Letters 269(2), 305-10 18 FOOTNOTES 1 The abbreviations used are: DMBA, 7,12-dimethylbenz[a]anthracene; NMU, N-methyl-N- nitrosourea; CoQ , ubiquinone

The molar hydrodynamic volume changes of factor VIIa due to GlycoPEGylation.

PubMed

Plesner, Bitten; Westh, Peter; Hvidt, Søren; Nielsen, Anders D

2011-06-01

The effects of GlycoPEGylation on the molar hydrodynamic volume of recombinant human rFVIIa were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10kDa PEG and a branched 40kDa PEG, respectively. Molar hydrodynamic volumes were determined by capillary viscometry and mass spectrometry. The intrinsic viscosities of rFVIIa, its two GlycoPEGylated compounds, and of linear 8kDa, 10kDa, 20kDa and branched 40kDa PEG polymers were determined. The measured intrinsic viscosity of rFVIIa is 6.0mL/g, while the intrinsic viscosities of 10kDa PEG-rFVIIa and 40kDa PEG-rFVIIa are 29.5mL/g and 79.0mL/g, respectively. The intrinsic viscosities of the linear PEG polymers are 20, 22.6 and 41.4mL/g for 8, 10, and 20kDa, respectively, and 61.1mL/g for the branched 40kDa PEG. From the results of the intrinsic viscosity and MALDI-TOF measurements it is evident, that the molar hydrodynamic volume of the conjugated protein is not just an addition of the molar hydrodynamic volume of the PEG and the protein. The molar hydrodynamic volume of the GlycoPEGylated protein is larger than the volume of its composites. These results suggest that both the linear and the branched PEG are not wrapped around the surface of rFVIIa but are chains that are significantly stretched out when attached to the protein. Copyright © 2011 Elsevier B.V. All rights reserved.

Phosphorylation of the rat hepatic polymeric IgA receptor.

PubMed Central

Larkin, J M; Sztul, E S; Palade, G E

1986-01-01

In vivo labeling with [35S]cysteine has identified three transmembrane forms of the rat hepatic polymeric IgA receptor: (i) a 105-kDa core glycosylated precursor; (ii) a terminally glycosylated 116-kDa intermediate; and (iii) a mature 120-kDa form. In the current study we show that the 120-kDa form is phosphorylated. After in vivo labeling with [32P]orthophosphate, all receptor forms were immunoprecipitated from hepatic total microsomes (TM) (with an antireceptor antiserum), separated by NaDodSO4/PAGE, and detected by autoradiography. The 120-kDa form was selectively phosphorylated, whereas the 116- and 105-kDa forms incorporated no detectable 32P. To determine the topology of the phosphorylation sites, hepatic TM isolated from rats labeled in vivo with either [35S]cysteine or [32P]orthophosphate were treated with trypsin. TM were solubilized and receptors were immunoprecipitated from lysates. With increasing trypsin concentrations, the [35S]cysteine-labeled receptor triplet was degraded to a trypsin-resistant doublet of approximately 95 and 85 kDa, indicating that approximately 20 kDa was removed from the receptor endodomain by trypsin. The same treatment removed all detectable 32P from labeled receptors. Furthermore, no 32P was detected in the 80-kDa biliary form of the receptor. Serine was identified as the only phosphorylated residue in acid hydrolysates of 32P-labeled immunoprecipitated receptor. These findings indicate that (i) the 120-kDa form is the only phosphorylated species of the receptor; and (ii) the phosphorylated residues are serine(s) located in the endodomain of the protein. Images PMID:3460069

Characterization of low molecular weight fragments from gamma irradiated κ-carrageenan used as plant growth promoter

NASA Astrophysics Data System (ADS)

Abad, Lucille V.; Aurigue, Fernando B.; Relleve, Lorna S.; Montefalcon, Djowel Recto V.; Lopez, Girlie Eunice P.

2016-01-01

Radiation degraded κ-carrageenan (1% solution at absorbed doses of 20 kGy and 30 kGy) were tested for its plant growth promoter (PGP) effect on pechay plants under hydroponics condition. Results revealed that higher PGP effects were found in κ-carrageenan irradiated at an absorbed dose of 30 kGy. Mw of irradiated κ-carrageenan as measured by GPC were determined to be 7362 Da and 6762 Da for 20 kGy and 30 kGy, respectively. Fractionation of the irradiated κ-carrageenan (30 kGy) was done to separate different Mw fractions using Mw cut-off filters of 1 kDa, 3 kDa, and 5 kDa. The PGP effect of the different retentates showed that biological activity in plants followed the order of 5 kDa>3 kDa>1 kDa using hydroponics condition but the reverse was observed in the order of 1 kDa>3 kDa>5 kDa when absorbed in plants by foliar spraying. GPC chromatogram indicated at least three (3) low molecular weight (LMW) fragments from radiation modified κ-carrageenan solution with an Mw<2000 Da. A fragment has also been identified with an Mw of as low as 160 Da which was produced under acidic (un-neutralized) condition. This may be attributed to the formation of 5-hydroxymethylfurfural (5-HMF).

Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

NASA Technical Reports Server (NTRS)

Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

1987-01-01

Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

Monoclonal Antibodies as Probes for the Detection of Porcine Blood-Derived Food Ingredients.

PubMed

Ofori, Jack A; Hsieh, Yun-Hwa P

2016-05-11

The lack of effective methods to monitor the use of porcine blood-derived food ingredients (PBFIs) is a concern for the billions of individuals who avoid consuming blood. We therefore sought to develop a panel of porcine blood-specific monoclonal antibodies (mAbs) for use as probes in immunoassays for the detection of PBFIs. Ten selected mAbs were identified that react with either a 60 or 90 kDa protein in the plasma fraction or a 12 kDa protein in the red blood cell fraction of porcine blood. Western blot analysis of commercially produced PBFIs revealed that these antigenic proteins are not affected by various manufacturing processes. The utility of these mAbs was demonstrated in a prototype sandwich ELISA developed for this study using mAbs 19C5-E10 and 16F9-C11. The new assay is porcine blood-specific and capable of detecting ≤0.03% (v/v) of PBFIs in cooked (100 °C for 15 min) ground meats or fish.

Biosynthesis and intracellular transport of the receptor for platelet-derived growth factor.

PubMed Central

Claesson-Welsh, L; Rönnstrand, L; Heldin, C H

1987-01-01

The biosynthesis of the receptor for platelet-derived growth factor (PDGF) was examined in metabolically labeled human foreskin fibroblasts. The receptor was synthesized as a 145-kDa precursor, which, when incubated with endo-beta-N-acetylglucosaminidase H (endo H), underwent a 15-kDa decrease in molecular mass. This indicates that the size of the core protein is about 130 kDa and that the 145-kDa form represents a receptor precursor carrying high-mannose N-linked oligosaccharide groups. Within 15 min after synthesis, the receptor was converted to a 165-kDa form. This form was entirely resistant to endo H treatment and probably represents a receptor molecule that has undergone further posttranslational modification, including O-linked glycosylation. Subsequently, within 30 min, a molecule of 170 kDa--i.e., the size of the mature receptor--appeared. A slightly larger molecule, of 175 kDa, which could be immunoprecipitated from PDGF-stimulated 32P-labeled cells, probably represents a receptor further modified by autophosphorylation. The 170-kDa molecule had an isoelectric point of about 4.5. Addition of PDGF increased the turnover rate of the 170-kDa PDGF receptor. Images PMID:2827155

Sensitivity analysis of discrete structural systems: A survey

NASA Technical Reports Server (NTRS)

Adelman, H. M.; Haftka, R. T.

1984-01-01

Methods for calculating sensitivity derivatives for discrete structural systems are surveyed, primarily covering literature published during the past two decades. Methods are described for calculating derivatives of static displacements and stresses, eigenvalues and eigenvectors, transient structural response, and derivatives of optimum structural designs with respect to problem parameters. The survey is focused on publications addressed to structural analysis, but also includes a number of methods developed in nonstructural fields such as electronics, controls, and physical chemistry which are directly applicable to structural problems. Most notable among the nonstructural-based methods are the adjoint variable technique from control theory, and the Green's function and FAST methods from physical chemistry.

Functional assignment to JEV proteins using SVM.

PubMed

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Functional assignment to JEV proteins using SVM

PubMed Central

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice

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

Wang, Diane R.; Wolfrum, Edward J.; Virk, Parminder

Rice plants ( Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, establishedmore » an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. Lastly, we find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice.« less

Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice

PubMed Central

Wang, Diane R.; Wolfrum, Edward J.; Virk, Parminder; Ismail, Abdelbagi; Greenberg, Anthony J.; McCouch, Susan R.

2016-01-01

Rice plants (Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, established an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. We find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice. PMID:27707775

Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice

DOE PAGES

Wang, Diane R.; Wolfrum, Edward J.; Virk, Parminder; ...

2016-10-05

Rice plants ( Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, establishedmore » an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. Lastly, we find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice.« less

Performance of masonry enclosure walls: lessons learned from recent earthquakes

NASA Astrophysics Data System (ADS)

Vicente, Romeu Silva; Rodrigues, Hugo; Varum, Humberto; Costa, Aníbal; Mendes da Silva, José António Raimundo

2012-03-01

This paper discusses the issue of performance requirements and construction criteria for masonry enclosure and infill walls. Vertical building enclosures in European countries are very often constituted by non-load-bearing masonry walls, using horizontally perforated clay bricks. These walls are generally supported and confined by a reinforced concrete frame structure of columns and beams/slabs. Since these walls are commonly considered to be nonstructural elements and their influence on the structural response is ignored, their consideration in the design of structures as well as their connection to the adjacent structural elements is frequently negligent or insufficiently detailed. As a consequence, nonstructural elements, as for wall enclosures, are relatively sensitive to drift and acceleration demands when buildings are subjected to seismic actions. Many international standards and technical documents stress the need for design acceptability criteria for nonstructural elements, however they do not specifically indicate how to prevent collapse and severe cracking, and how to enhance the overall stability in the case of moderate to high seismic loading. Furthermore, a review of appropriate measures to improve enclosure wall performance and both in-plane and out-of-plane integrity under seismic actions is addressed.

Human Rhinovirus 16 Causes Golgi Apparatus Fragmentation without Blocking Protein Secretion

PubMed Central

Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V.; Walton, Ross; Johnston, Sebastian L.

2014-01-01

ABSTRACT The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins. PMID:25100828

Turgor-responsive starch phosphorylation in Oryza sativa stems: A primary event of starch degradation associated with grain-filling ability.

PubMed

Wada, Hiroshi; Masumoto-Kubo, Chisato; Tsutsumi, Koichi; Nonami, Hiroshi; Tanaka, Fukuyo; Okada, Haruka; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nakashima, Taiken; Hakata, Makoto; Morita, Satoshi

2017-01-01

Grain filling ability is mainly affected by the translocation of carbohydrates generated from temporarily stored stem starch in most field crops including rice (Oryza sativa L.). The partitioning of non-structural stem carbohydrates has been recognized as an important trait for raising the yield ceiling, yet we still do not fully understand how carbohydrate partitioning occurs in the stems. In this study, two rice subspecies that exhibit different patterns of non-structural stem carbohydrates partitioning, a japonica-dominant cultivar, Momiroman, and an indica-dominant cultivar, Hokuriku 193, were used as the model system to study the relationship between turgor pressure and metabolic regulation of non-structural stem carbohydrates, by combining the water status measurement with gene expression analysis and a dynamic prefixed 13C tracer analysis using a mass spectrometer. Here, we report a clear varietal difference in turgor-associated starch phosphorylation occurred at the initiation of non-structural carbohydrate partitioning. The data indicated that starch degradation in Hokuriku 193 stems occurred at full-heading, 5 days earlier than in Momiroman, contributing to greater sink filling. Gene expression analysis revealed that expression pattern of the gene encoding α-glucan, water dikinase (GWD1) was similar between two varieties, and the maximum expression level in Hokuriku 193, reached at full heading (4 DAH), was greater than in Momiroman, leading to an earlier increase in a series of amylase-related gene expression in Hokuriku 193. In both varieties, peaks in turgor pressure preceded the increases in GWD1 expression, and changes in GWD1 expression was correlated with turgor pressure. Additionally, a threshold is likely to exist for GWD1 expression to facilitate starch degradation. Taken together, these results raise the possibility that turgor-associated starch phosphorylation in cells is responsible for the metabolism that leads to starch degradation. Because the two cultivars exhibited remarkable varietal differences in the pattern of non-structural carbohydrate partitioning, our findings propose that the observed difference in grain-filling ability originated from turgor-associated regulation of starch phosphorylation in stem parenchyma cells. Further understanding of the molecular mechanism of turgor-regulation may provide a new selection criterion for breaking the yield barriers in crop production.

Turgor-responsive starch phosphorylation in Oryza sativa stems: A primary event of starch degradation associated with grain-filling ability

PubMed Central

Tsutsumi, Koichi; Nonami, Hiroshi; Tanaka, Fukuyo; Okada, Haruka; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nakashima, Taiken; Hakata, Makoto; Morita, Satoshi

2017-01-01

Grain filling ability is mainly affected by the translocation of carbohydrates generated from temporarily stored stem starch in most field crops including rice (Oryza sativa L.). The partitioning of non-structural stem carbohydrates has been recognized as an important trait for raising the yield ceiling, yet we still do not fully understand how carbohydrate partitioning occurs in the stems. In this study, two rice subspecies that exhibit different patterns of non-structural stem carbohydrates partitioning, a japonica-dominant cultivar, Momiroman, and an indica-dominant cultivar, Hokuriku 193, were used as the model system to study the relationship between turgor pressure and metabolic regulation of non-structural stem carbohydrates, by combining the water status measurement with gene expression analysis and a dynamic prefixed 13C tracer analysis using a mass spectrometer. Here, we report a clear varietal difference in turgor-associated starch phosphorylation occurred at the initiation of non-structural carbohydrate partitioning. The data indicated that starch degradation in Hokuriku 193 stems occurred at full-heading, 5 days earlier than in Momiroman, contributing to greater sink filling. Gene expression analysis revealed that expression pattern of the gene encoding α-glucan, water dikinase (GWD1) was similar between two varieties, and the maximum expression level in Hokuriku 193, reached at full heading (4 DAH), was greater than in Momiroman, leading to an earlier increase in a series of amylase-related gene expression in Hokuriku 193. In both varieties, peaks in turgor pressure preceded the increases in GWD1 expression, and changes in GWD1 expression was correlated with turgor pressure. Additionally, a threshold is likely to exist for GWD1 expression to facilitate starch degradation. Taken together, these results raise the possibility that turgor-associated starch phosphorylation in cells is responsible for the metabolism that leads to starch degradation. Because the two cultivars exhibited remarkable varietal differences in the pattern of non-structural carbohydrate partitioning, our findings propose that the observed difference in grain-filling ability originated from turgor-associated regulation of starch phosphorylation in stem parenchyma cells. Further understanding of the molecular mechanism of turgor-regulation may provide a new selection criterion for breaking the yield barriers in crop production. PMID:28727805

The characteristics of extracellular polymeric substances and soluble microbial products in moving bed biofilm reactor-membrane bioreactor.

PubMed

Duan, Liang; Jiang, Wei; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W

2013-11-01

The characteristics of extracellular polymeric substances (EPS) and soluble microbial products (SMP) in conventional membrane bioreactor (MBR) and in moving bed biofilm reactor-membrane bioreactors (MBBR-MBR) were investigated in long-term (170 days) experiments. The results showed that all reactors had high removal efficiency of ammonium and COD, despite very different fouling conditions. The MBBR-MBR with media fill ratio of 26.7% had much lower total membrane resistance and no obvious fouling were detected during the whole operation. In contrast, MBR and MBBR-MBR with lower and higher media fill experienced more significant fouling. Low fouling at optimum fill ratio may be due to the higher percentage of small molecular size (<1 kDa) and lower percentage of large molecular size (>100 kDa) of EPS and SMP in the reactor. The composition of EPS and SMP affected fouling due to different O-H bonds in hydroxyl functional groups, and less polysaccharides and lipids. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.

PubMed

Ritterhoff, Tobias; Das, Hrishikesh; Hao, Yuqing; Sakin, Volkan; Flotho, Annette; Werner, Andreas; Melchior, Frauke

2016-01-01

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

Genetic recombination and Cryptosporidium hominis virulent subtype IbA10G2.

PubMed

Li, Na; Xiao, Lihua; Cama, Vitaliano A; Ortega, Ynes; Gilman, Robert H; Guo, Meijin; Feng, Yaoyu

2013-10-01

Little is known about the emergence and spread of virulent subtypes of Cryptosporidium hominis, the predominant species responsible for human cryptosporidiosis. We conducted sequence analyses of 32 genetic loci of 53 C. hominis specimens isolated from a longitudinally followed cohort of children living in a small community. We identified by linkage disequilibrium and recombination analyses only limited genetic recombination, which occurred exclusively within the 60-kDa glycoprotein gene subtype IbA10G2, a predominant subtype for outbreaks in industrialized nations and a virulent subtype in the study community. Intensive transmission of virulent subtype IbA10G2 in the study area might have resulted in genetic recombination with other subtypes. Moreover, we identified selection for IbA10G2 at a 129-kb region around the 60-kDa glycoprotein gene in chromosome 6. These findings improve our understanding of the origin and evolution of C. hominis subtypes and the spread of virulent subtypes.

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins.

PubMed

Peng, Ivory X; Shiea, Jentaie; Ogorzalek Loo, Rachel R; Loo, Joseph A

2007-01-01

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements. Copyright (c) 2007 John Wiley & Sons, Ltd.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

PubMed

Tabib-Salazar, Aline; Liu, Bing; Shadrin, Andrey; Burchell, Lynn; Wang, Zhexin; Wang, Zhihao; Goren, Moran G; Yosef, Ido; Qimron, Udi; Severinov, Konstantin; Matthews, Steve J; Wigneshweraraj, Sivaramesh

2017-07-27

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Community's Emergency Preparedness for Flood Hazards in Dire-dawa Town, Ethiopia: A Qualitative Study.

PubMed

Ejeta, Luche Tadesse

2018-02-21

Emergency preparedness at all levels (individuals and communities) is the corner stone of effective response to the increasing trends of global disasters due to man-made and natural hazards. It is determined by different factors, including (among others) past direct and indirect exposures to hazards. This study was carried out in Dire Dawa town, Ethiopia, which in the past experienced frequent flooding events, yet dearth of information exists about preparedness in the area.  The aim of the study was to assess the levels of emergency preparedness for flood hazards at households and communities levels. The study was conducted in a qualitative approach and was conducted in Dire Dawa town, which has been divided into nine administrative-units called Kebeles. Two focus group discussions were held in two of these units (Kebele-05 and 06), each focus group comprising twelve people (all above 18 years of age), and in total 24 people (13 females and 11 males) took part in the study. Open ended questions were used that could guide the discussions, and the discussions were audio-taped and transcribed. The results were translated from local language to English and qualitatively presented. The findings of focus group discussions showed that the local government in collaboration with the federal government built the flood protection dams in areas where flood hazards have been thought to be repeatedly wreaking havoc, specifically after the flood disaster of the year 2006. In addition, in Kebele-05, where one Non-Governmental Organization (NGO) was operating on flood hazards prevention and mitigation program, some non-structural emergency preparedness measures were undertaken by the communities. These non-structural measures (the major ones) entailed: establishment of committees recruited from residents and training them to raise awareness among communities on emergency preparedness; some residents made changes to their own houses (retrofitted) and put sandbags around their houses to temporarily protect the flooding; establishment of communication channels between communities to alarm each other in the event of flood disaster; and reforestation of the already deforested mountainous areas surrounding the town. However, concerns were raised by study participants about strengths of the constructed flood protection dams. Furthermore, the non-structural emergency preparedness measures identified by this study were not comprehensive; for example, residents were not trained in first aid, first aid kits were not provided, there was no linkage being established between communities and health facilities so as to provide emergency medical care to victims in the event of flood disaster. The findings of this study concur with some of the previous quantitative studies' results in that the past direct and indirect disaster experiences invoke preparedness intention and actual preparedness for flood hazards at individuals, communities and organizations levels. The only one quantitative and behavioral based study conducted thus far in Dire Dawa town reported the strong association of past flood disaster experience with household emergency preparedness. Among the residents there was a tendency to rely on the dams to be constructed with "good quality" and "higher strength" than making preparedness efforts on their own at their households. Structural measures such as building of dams, dikes, levees, and channel improvements could be means of mitigation measures; however, solely relying on these measures could have far reaching consequences. To mitigate flood hazards, dams were built and in addition, in Kebele-05 where an NGO was operating, some non-structural emergency preparedness measures were undertaken. In the course of construction of flood protection dams, ensuring communities ' involvement is needed ; and at the same time undertaking comprehensive non-structural emergency preparedness measures in all Kebeles is highly recommended. Emergency, Preparedness, Flood, Dire Dawa, Ethiopia.

Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

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

Appolaire, Alexandre; Girard, Eric; Colombo, Matteo

2014-11-01

The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme. The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexesmore » is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2–TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.« less

Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes.

PubMed Central

Mooney, R A; Bordwell, K L

1991-01-01

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1848760

A critical evaluation of an asymmetrical flow field-flow fractionation system for colloidal size characterization of natural organic matter.

PubMed

Zhou, Zhengzhen; Guo, Laodong

2015-06-19

Colloidal retention characteristics, recovery and size distribution of model macromolecules and natural dissolved organic matter (DOM) were systematically examined using an asymmetrical flow field-flow fractionation (AFlFFF) system under various membrane size cutoffs and carrier solutions. Polystyrene sulfonate (PSS) standards with known molecular weights (MW) were used to determine their permeation and recovery rates by membranes with different nominal MW cutoffs (NMWCO) within the AFlFFF system. Based on a ≥90% recovery rate for PSS standards by the AFlFFF system, the actual NMWCOs were determined to be 1.9 kDa for the 0.3 kDa membrane, 2.7 kDa for the 1 kDa membrane, and 33 kDa for the 10 kDa membrane, respectively. After membrane calibration, natural DOM samples were analyzed with the AFlFFF system to determine their colloidal size distribution and the influence from membrane NMWCOs and carrier solutions. Size partitioning of DOM samples showed a predominant colloidal size fraction in the <5 nm or <10 kDa size range, consistent with the size characteristics of humic substances as the main terrestrial DOM component. Recovery of DOM by the AFlFFF system, as determined by UV-absorbance at 254 nm, decreased significantly with increasing membrane NMWCO, from 45% by the 0.3 kDa membrane to 2-3% by the 10 kDa membrane. Since natural DOM is mostly composed of lower MW substances (<10 kDa) and the actual membrane cutoffs are normally larger than their manufacturer ratings, a 0.3 kDa membrane (with an actual NMWCO of 1.9 kDa) is highly recommended for colloidal size characterization of natural DOM. Among the three carrier solutions, borate buffer seemed to provide the highest recovery and optimal separation of DOM. Rigorous calibration with macromolecular standards and optimization of system conditions are a prerequisite for quantifying colloidal size distribution using the flow field-flow fractionation technique. In addition, the coupling of AFlFFF with fluorescence EEMs could provide new insights into DOM heterogeneity in different colloidal size fractions. Copyright © 2015 Elsevier B.V. All rights reserved.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

PubMed

Schmucker, B; Ballhausen, W G; Kressel, M

1997-01-01

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

Isoform composition and stoichiometry of the approx. 90-kDa heat shock protein associated with glucocorticoid receptors

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

Mendel, D.B.; Orti, E.

1988-05-15

The authors observed that the approx. 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approx. 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approx. 90-kDa heat shock protein. The observation that TSTA and the approx. 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested thatmore » the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the approx. 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approx. 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approx. 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with (/sup 35/S)methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two approx. 90-kDa non-steroid-binding subunits. The consistency with which a approx. 1:2 stoichiometric ratio of steroid binding to approx. 90-kDa protein is observed supports the view that the approx. 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes.« less

Diffusion of macromolecules through sclera.

PubMed

Miao, Heng; Wu, Bi-Dong; Tao, Yong; Li, Xiao-Xin

2013-02-01

To quantify the in vitro permeability coefficient over different topographical locations of porcine sclera to macromolecules with different molecular weight. Fresh equatorial and posterior superotemporal porcine sclera was mounted in a two-chamber diffusion apparatus, and its permeability to fluorescein isothiocyanate (FITC)-conjugated dextrans ranging in molecular weight from 40 kDa to 150 kDa was determined by fluorescence spectrophotometry. The sclera was processed as frozen sections and viewed with a fluorescence microscope. The thickness of the area and the thickness that macromolecules enriched in the surface of sclera were measured. The permeability coefficient (Pc) of porcine sclera to macromolecules was significantly higher (40 kDa, p = 0.028; 70 kDa, p = 0.033; 150 kDa, p = 0.007) in equatorial region than posterior, which could be attributed to the significant difference of thickness (p < 0.001, Kruskal-Wallis) between them. Moreover, linear regression indicated a significant negative relationship (40 kDa, p < 0.001; 70 kDa, p = 0.015; 150 kDa, p < 0.001) between scleral permeability coefficient and thickness. Also, Pc declined significantly with increasing molecular weight (MW, p < 0.001, Kruskal-Wallis). The area that the macromolecules enriched in the scleral surface was thicker for those with larger MW (p < 0.001, Kruskal-Wallis). The maximum MW and size for equatorial and posterior superotemporal scleral tissue were 185.01 KDa and 180.42 KDa, 9.92 nm and 9.67 nm, respectively. The permeability coefficient of porcine sclera has a significant negative relationship with scleral thickness and MW of macromolecules. Larger macromolecules are more likely to accumulate in scleral surface. The difference between topographical locations may have pharmacokinetic implications when considering transscleral diffusion of macromolecules. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

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

Butler, C.A.; Hoffman, P.S.

1990-05-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid permore » mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.« less

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

PubMed

MacDonald, Justin A; Sutherland, Cindy; Carlson, David A; Bhaidani, Sabreena; Al-Ghabkari, Abdulhameed; Swärd, Karl; Haystead, Timothy A J; Walsh, Michael P

2016-01-01

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Entericidin is required for a probiotic treatment (Enterobacter sp. strain C6-6) to protect trout from cold-water disease challenge.

PubMed

Schubiger, Carla B; Orfe, Lisa H; Sudheesh, Ponnerassery S; Cain, Kenneth D; Shah, Devendra H; Call, Douglas R

2015-01-01

Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analysis of colonization factor antigen I, an adhesin of enterotoxigenic Escherichia coli O78:H11: fimbrial morphology and location of the receptor-binding site.

PubMed Central

Bühler, T; Hoschützky, H; Jann, K

1991-01-01

Colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli was dissociated into one type of subunit (15 kDa). The dissociation was achieved either by heating CFA/I in sodium dodecyl sulfate at 100 degrees C or by heating it for 20 min in water. Heating in water to 100 degrees C yielded only in the 15-kDa subunit, but heating to 85 degree C yielded small amounts of oligomers in addition. The monomeric subunits obtained after heating in water are stable, as demonstrated by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heating prior to the electrophoretic run. These subunits inhibited CFA/I-induced hemagglutination, indicating that they had maintained their receptor-binding properties. When the hybridoma technique was used, two types of monoclonal anti-CFA/I antibodies were obtained. Antibodies obtained by immunization with the purified subunits were more reactive with subunits than with fimbriae, as shown by enzyme-linked immunosorbent assay. These antibodies strongly inhibited CFA/I-induced hemagglutination. When examined by immunoelectron microscopy, these antibodies seemed to label the fimbrial tips. A similar labeling pattern was obtained with gold particles modified with the receptor ganglioside GM2. Antibodies obtained by immunization with fimbriae reacted in enzyme-linked immunosorbent assays equally well with fimbriae and subunits. They inhibited CFA/I-induced hemagglutination only slightly. Immunoelectron microscopy revealed that these antibodies labeled the fimbriae densely and regularly over their entire lengths. In a coagglutination experiment with Staphylococcus aureus and monoclonal antibodies, the subunits retained their receptor-binding properties. From these results, we conclude that CFA/I fimbriae consist entirely of one type of adhesive subunit, of which only the one at the tip is accessible to the receptor. Images PMID:1682253

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors▿

PubMed Central

Hayes, M.; Stanton, C.; Slattery, H.; O'Sullivan, O.; Hill, C.; Fitzgerald, G. F.; Ross, R. P.

2007-01-01

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to αs1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract. PMID:17483275

Mass Spectrometric Identification of Mtb81, a Novel Serological Marker for Tuberculosis

PubMed Central

Hendrickson, Ronald C.; Douglass, John F.; Reynolds, Lisa D.; McNeill, Patricia D.; Carter, Darrick; Reed, Steven G.; Houghton, Raymond L.

2000-01-01

We have used serological proteome analysis in conjunction with tandem mass spectrometry to identify and sequence a novel protein, Mtb81, which may be useful for the diagnosis of tuberculosis (TB), especially for patients coinfected with human immunodeficiency virus (HIV). Recombinant Mtb81 was tested by an enzyme-linked immunosorbent assay to detect antibodies in 25 of 27 TB patients (92%) seropositive for HIV as well as in 38 of 67 individuals (57%) who were TB positive alone. No reactivity was observed in 11 of 11 individuals (100%) who were HIV seropositive alone. In addition, neither sera from purified protein derivative (PPD)-negative (0 of 29) nor sera from healthy (0 of 45) blood donors tested positive with Mtb81. Only 2 of 57 of PPD-positive individuals tested positive with Mtb81. Sera from individuals with smear-positive TB and seropositive for HIV but who had tested negative for TB in the 38-kDa antigen immunodiagnostic assay were also tested for reactivity against Mtb81, as were sera from individuals with lung cancer and pneumonia. Mtb81 reacted with 26 of 37 HIV+ TB+ sera (70%) in this group, compared to 2 of 37 (5%) that reacted with the 38-kDa antigen. Together, these results demonstrate that Mtb81 may be a promising complementary antigen for the serodiagnosis of TB. PMID:10835002

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

PubMed

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

PubMed Central

Millar, A H; Knorpp, C; Leaver, C J; Hill, S A

1998-01-01

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins. PMID:9729464

Synergistic effect of high and low molecular weight molecules in the foamability and foam stability of sparkling wines.

PubMed

Coelho, Elisabete; Reis, Ana; Domingues, M Rosário M; Rocha, Sílvia M; Coimbra, Manuel A

2011-04-13

The foam of sparkling wines is a key parameter of their quality. However, the compounds that are directly involved in foam formation and stabilization are not yet completely established. In this work, seven sparkling wines were produced in Bairrada appellation (Portugal) under different conditions and their foaming properties evaluated using a Mosalux-based device. Fractionation of the sparkling wines into four independent fractions, (1) high molecular weight material, with molecular weight higher than 12 kDa (HMW), (2) hydrophilic material with molecular weigh between 1 and 12 kDa (AqIMW), (3) hydrophobic material with molecular weigh between 1 and 12 kDa (MeIMW), and (4) hydrophobic material with a molecular weight lower than 1 kDa (MeLMW), allowed the observation that the wines presenting the lower foam stability were those that presented lower amounts of the MeLMW fraction. The fraction that presented the best foam stability was HMW. When HMW is combined with MeLMW fraction, the foam stability largely increased. This increase was even larger, approaching the foam stability of the sparkling wine, when HMW was combined with the less hydrophobic subfraction of MeLMW (fraction 3). Electrospray tandem mass spectrometry (ESI-MS/MS) of fraction 3 allowed the assignment of polyethylene glycol oligomers (n = 5-11) and diethylene glycol 8-hydroxytridecanoate glyceryl acetate. To observe if these molecules occur in sparkling wine foam, the MeLMW was recovered directly from the sparkling wine foam and was also analyzed by ESI-MS/MS. The presence of monoacylglycerols of palmitic and stearic acids, as well as four glycerylethylene glycol fatty acid derivatives, was observed. These surface active compounds are preferentially partitioned by the sparkling wine foam rather than the liquid phase, allowing the inference of their role as key components in the promotion and stabilization of sparkling wine foam.

Effects of endoplasmic reticulum stressors on maturation and signaling of hemizygous and heterozygous wild-type and mutant forms of KIT.

PubMed

Brahimi-Adouane, Sabrina; Bachet, Jean-Baptiste; Tabone-Eglinger, Séverine; Subra, Frédéric; Capron, Claude; Blay, Jean-Yves; Emile, Jean-François

2013-06-01

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild-type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation-dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra-cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra-cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Trace element fractionation and transport in boreal rivers and soil porewaters of permafrost-dominated basaltic terrain in Central Siberia

NASA Astrophysics Data System (ADS)

Pokrovsky, O. S.; Schott, J.; Dupré, B.

2006-07-01

The chemical status of ˜40 major and trace elements (TE) and organic carbon (OC) in pristine boreal rivers draining the basaltic plateau of Central Siberia (Putorana) and interstitial solutions of permafrost soils was investigated. Water samples were filtered in the field through progressively decreasing pore size (5 μm → 0.22 μm → 0.025 μm → 10 kDa → 1 kDa) using cascade frontal filtration technique. Most rivers and soil porewaters exhibit 2-5 times higher than the world average concentration of dissolved (i.e., <0.22 μm) iron (0.03-0.4 mg/L), aluminum (0.03-0.4 mg/L), OC (10-20 mg/L) and various trace elements that are usually considered as immobile in weathering processes (Ti, Zr, Ga, Y, REEs). Ultrafiltration revealed strong relationships between concentration of TE and that of colloidal Fe and Al. According to their partition during filtration and association with colloids, two groups of elements can be distinguished: (i) those weakly dependent on ultrafiltration and that are likely to be present as truly dissolved inorganic species (Li, Na, K, Si, Mn, Mo, Rb, Cs, As, Sb) or, partially (20-30%) associated with small size Fe- and Al-colloids (Ca, Mg, Sr, Ba) and to small (<1-10 kDa) organic complexes (Co, Ni, Cu, Zn), and (ii) elements strongly associated with colloidal iron and aluminum in all ultrafiltrates largely present in 1-100 kDa fraction (Ga, Y, REEs, Pb, V, Cr, Ti, Ge, Zr, Th, U). TE concentrations and partition coefficients did not show any detectable variations between different colloidal fractions for soil porewaters, suprapermafrost flow and surface streams. TE concentration measurements in river suspended particles demonstrated significant contribution (i.e., ⩾30%) of conventionally dissolved (<0.22 μm) forms for usually "immobile" elements such as divalent transition metals, Cd, Pb, V, Sn, Y, REEs, Zr, Hf, Th. The Al-normalized accumulation coefficients of TE in vegetation litter compared to basalts achieve 10-100 for B, Mn, Zn, As, Sr, Sn, Sb, and the larch litter degradation is able to provide the major contribution to the annual dissolved flux of most trace elements. It is hypothesized that the decomposition of plant litter in the topsoil horizon leads to Fe(III)-, Al-organic colloids formation and serves as an important source of elements in downward percolating fluids.

[Comparison of Dengue viral nonstructural protein 1 antigen testing kits].

PubMed

Wu, D; Zhao, L Z; Wu, Y H; Zhang, H; Zhang, M; Tan, Q Q; Zhou, H Q; Zhang, F C; He, J F

2018-02-06

Objective: To investigate the sensitivity and specificity of commercial nonstructural protein 1 (NS1) testing kits for Dengue fever diagnose, and provide the evidence for diagnostic criteria revision. Methods: 300 PCR or virus isolation positive blood samples for dengue virus were collected from sentinel hospitals for dengue surveillance in Guangzhou, Dongguang and Zhongshang from May 2015 to Nov. 2016. At the same time, 308 PCR negative samples for Dengue virus were collected as control group. The information of the sample was collected using questionnaires. These samples were tested using imported and domestic ELISA and the colloidal gold-labeled kits that were widely used for detecting dengue NS1. Sensitivity, specificity and coincidence were calculated and analyzed, and Z hongshan's result was regarded as the reslut of the third part. Results: The positive group includes 133 males and 167 females, average ages are 47.2±13.3, 179, 110 and 11 of them is Dengue Ⅰ, Ⅱ and Ⅲ respectively. The negative group includes 154 males and 154 females, average ages are (40.1±11.6) years old. The sensitivity of domestic ELISA Kits (94.5%) is less than imported (99.5%), and the result has statistical significance (χ(2)=8.59, P= 0.030), the specificity is 99.7% and 97.7% respectively; The sensitivity of imported and domestic the colloidal gold-labeled Kits is 97.5% and 96.5% respectively, both of specificities are 100%. The sensitivity and specificity of Dengue Ⅰ for NS1 test are more than 97.0%. The sensitivity of domestic ELISA and gold-labeled Kits is 90.0% and 95.0%, and the specificity is 96.8% and 100% respectively for Dengue Ⅱ test. The sensitivity of imported ELISA and gold-labeled Kits is 100% and 98.0%, and the specificity is 99.4% and 100% respectively for Dengue Ⅱ test. The result of the third party show the sensitivity and specificity of domestic ELISA and gold-labeled Kits are 90.0% and 98.0%, the differences has statistical significance (χ(2)=5.67, P= 0.020). Conclusion: NS1 testing can be used as early dengue fever diagnose for higher sensitivity and specificity.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

PubMed

Bruno, Raphael R; Neuhaus, Winfried; Roewer, Norbert; Wunder, Christian; Schick, Martin A

2014-09-01

Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2-21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-α. All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-α did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10-30 kDa) were significantly deleterious. For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

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

Valles, Steven M., E-mail: Steven.Valles@ars.usda.go; Hashimoto, Yoshifumi

2009-06-05

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number (FJ528584)), comprised of 10,386 nucleotides, and polyadenylated at the 3' terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5' proximal ORF (ORFmore » 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3' proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 +- 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.« less

Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3.

PubMed

Habraken, Y; Sung, P; Prakash, L; Prakash, S

1996-09-01

DNA-mismatch repair removes mismatches from the newly replicated DNA strand. In humans, mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1 and hPMS2 result in hereditary non-polyposis colorectal cancer (HNPCC) [1-8]. The hMSH2 (MSH for MutS homologue) protein forms a complex with a 160 kDa protein, and this heterodimer, hMutSalpha, has high affinity for a G/T mismatch [9,10]. Cell lines in which the 160 kDa subunit of hMutSalpha is mutated are specifically defective in the repair of base-base and single-nucleotide insertion/deletion mismatches [9,11]. Genetic studies in S. cerevisiae have suggested that MSH2 functions with either MSH3 or MSH6 in mismatch repair, and, in the absence of the latter two genes, MSH2 is inactive [12,13]. MSH6 encodes the yeast counterpart of the 160 kDa subunit of hMutSalpha [12,13]. As in humans, yeast MSH6 forms a complex with MSH2, and the MSH2-MSH6 heterodimer binds a G/T mismatch [14]. Here, we find that MSH2 and MSH3 form another stable heterodimer, and we purify this heterodimer to near homogeneity. We show that MSH2-MSH3 has low affinity for a G/T mismatch but binds to insertion/deletion mismatches with high specificity, unlike MSH2-MSH6.

Biopolymers as an Alternative to Petroleum-Based Polymers for Soil Modification; ESTCP ER-0920: Treatability Studies

DTIC Science & Technology

2012-08-01

carboxylic, amine, hydroxyl). Molasses produced more carboxylic acid groups than the corn syrup -based material and was composed of high ...molecular weight EPS units (as high as 800 KDa). Corn syrup -derived biopolymer, on the other hand, showed a greater number of small molecular weight...biopolymer by R. tropici: corn syrup , maltose, sorghum, and molasses. The maltose, being a very expensive carbon source, has been replaced by the sorghum

Biopolymers as an Alternative to Petroleum-Based Polymers for Soil Modification, ESTCP ER-0920: Treatability Studies

DTIC Science & Technology

2012-08-01

carboxylic, amine, hydroxyl). Molasses produced more carboxylic acid groups than the corn syrup -based material and was composed of high ...molecular weight EPS units (as high as 800 KDa). Corn syrup -derived biopolymer, on the other hand, showed a greater number of small molecular weight...biopolymer by R. tropici: corn syrup , maltose, sorghum, and molasses. The maltose, being a very expensive carbon source, has been replaced by the sorghum

The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

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

Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

1996-04-15

We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of differentmore » factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.« less

Mercuric chloride induces autoantibodies against U3 small nuclear ribonucleoprotein in susceptible mice.

PubMed Central

Reuter, R; Tessars, G; Vohr, H W; Gleichmann, E; Lührmann, R

1989-01-01

Autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. While animal models, which spontaneously develop abundant anti-nucleolar antibodies, have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. We have identified the nucleolar autoantigen against which the HgCl2-induced IgG autoantibodies from mice of strain B10.S are directed. It is a protein with an apparent molecular mass of 36 kDa and a pI value of approximately 8.6, which is associated with the nucleolar small nuclear RNA U3, and by these criteria must be identical with a polypeptide called fibrillarin. It is striking that scleroderma patients spontaneously produce autoantibodies against the same U3 ribonucleoprotein (RNP). The HgCl2-induced murine and the scleroderma-specific human anti-U3 RNP autoantibodies were indistinguishable in their reactivities toward fibrillarin. They further resemble each other insofar as both recognize epitopes on the 36-kDa protein, which have been highly conserved throughout evolution. Our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential anti-U3 RNP autoantibody production in the animal model and in scleroderma patients. Images PMID:2521387

Brushed block copolymer micelles with pH-sensitive pendant groups for controlled drug delivery.

PubMed

Lee, Hyun Jin; Bae, Younsoo

2013-08-01

To investigate the effects of small aliphatic pendent groups conjugated through an acid-sensitive linker to the core of brushed block copolymer micelles on particle properties. The brushed block copolymers were synthesized by conjugating five types of 2-alkanone (2-butanone, 2-hexanone, 2-octanone, 2-decanone, and 2-dodecanone) through an acid-labile hydrazone linker to poly(ethylene glycol)-poly(aspartate hydrazide) block copolymers. Only block copolymers with 2-hexanone and 2-octanone (PEG-HEX and PEG-OCT) formed micelles with a clinically relevant size (< 50 nm in diameter), low critical micelle concentration (CMC, < 20 μM), and drug entrapment yields (approximately 5 wt.%). Both micelles degraded in aqueous solutions in a pH-dependent manner, while the degradation was accelerated in an acidic condition (pH 5.0) in comparison to pH 7.4. Despite these similar properties, PEG-OCT micelles controlled the entrapment and pH-dependent release of a hydrophobic drug most efficiently, without altering particle size, shape, and stability. The molecular weight of PEG (12 kDa vs 5 kDa) induced no change in pH-controlled drug release rates of PEG-OCT micelles. Acid-labile small aliphatic pendant groups are useful to control the entrapment and release of a hydrophobic drug physically entrapped in the core of brushed block copolymer micelles.

Rice (Oryza sativa japonica) Albumin Suppresses the Elevation of Blood Glucose and Plasma Insulin Levels after Oral Glucose Loading.

PubMed

Ina, Shigenobu; Ninomiya, Kazumi; Mogi, Takashi; Hase, Ayumu; Ando, Toshiki; Matsukaze, Narumi; Ogihara, Jun; Akao, Makoto; Kumagai, Hitoshi; Kumagai, Hitomi

2016-06-22

The suppressive effect of rice albumin (RA) of 16 kDa on elevation of blood glucose level after oral loading of starch or glucose and its possible mechanism were examined. RA suppressed the increase in blood glucose levels in both the oral starch tolerance test and the oral glucose tolerance test. The blood glucose concentrations 15 min after the oral administration of starch were 144 ± 6 mg/dL for control group and 127 ± 4 mg/dL for RA 200 mg/kg BW group, while those after the oral administration of glucose were 157 ± 7 mg/dL for control group and 137 ± 4 mg/dL for RA 200 mg/kg BW group. However, in the intraperitoneal glucose tolerance test, no significant differences in blood glucose level were observed between RA and the control groups, indicating that RA suppresses the glucose absorption from the small intestine. However, RA did not inhibit the activity of mammalian α-amylase. RA was hydrolyzed to an indigestible high-molecular-weight peptide (HMP) of 14 kDa and low-molecular-weight peptides by pepsin and pancreatin. Furthermore, RA suppressed the glucose diffusion rate through a semipermeable membrane like dietary fibers in vitro. Therefore, the indigestible HMP may adsorb glucose and suppress its absorption from the small intestine.

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats.

PubMed

Salinas-Tobon, Maria Del Rosario; Navarrete-Leon, Anaid; Mendez-Loredo, Blanca Esther; Esquivel-Aguirre, Dalia; Martínez-Abrajan, Dulce Maria; Hernandez-Sanchez, Javier

2007-02-01

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.

Investigation of antioxidant potential of peptide fractions from the Tra Catfish by-product-derived hydrolysate using Alcalase® 2.4 L FG

NASA Astrophysics Data System (ADS)

Vo, Tam D. L.; Chung, Duy T. M.; Doan, Kien T.; Le, Duy T.; Trinh, Hung V.

2017-09-01

In this study, the antioxidant capacity of peptide fractions isolated from the Tra Catfish (Pangasius hypophthalmus) by-product-derived proteolysate using ultrafiltration centrifugal devices with 5 distinct molecular-weight cutoffs (MWCOs) of 1 kDa, 3 kDa, 5 kDa, 10 kDa, and 30 kDa was investigated. Firstly, the chemical composition of the Tra Catfish by-products was analyzed. The result showed that the Tra Catfish by-products contained 58.5% moisture, 33.9% crude protein, 50.1% crude lipid and 15.8% ash (on dry weight basis). Secondly, the effects of hydrolysis time, enzyme content on the antioxidant potential of the proteolysate were studied using DPPH• (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method (DPPH• SM) and FRAP (Ferric Reducing Antioxidant Potential) method. Alcalase® 2.4 L FG was used for hydrolysis. The result of antioxidant activity of the hydrolysate showed that the 50% DPPH• inhibition concentration (IC50) of the hydrolysate reached about 6775 µg/mL which was 1645-fold higher than that of vitamin C and 17-fold higher than that of BHT (ButylatedHydroxytoluene) with the degree of hydrolysis (DH) of the hydrolysate of 14.6% when hydrolysis time was 5 hours, enzyme/substrate (E/S) ratio was 30 U/g protein, hydrolysis temperature was 55°C, and pH was 7.5. The antioxidant potential of hydrolysate using FRAP method reached about 52.12 µM Trolox equivalent which was 53-fold and 18-fold lower than those of vitamin C and BHT, respectively, when the hydrolysis time was 5 h, enzyme/substrate ratio was 30 U/g protein, temperature was 500C, and pH level was 8. Next, the proteolysate was further fractionated using MWCOs of 1 kDa, 3 kDa, 5 kDa, 10 kDa, and 30 kDa and the peptide fractions were investigated for their antioxidant activity. The result showed that the <1 kDa fraction showed strongest antioxidant activity with the IC50 of 1313.31 ± 50.65 µg/mL and FRAP value of 906.90 ± 44.32 µM Trolox equivalent. The second strongest fraction was 1-3 kDa with the IC50 and FRAP value of 2897.85 ± 128.38 µg/mL and 517.7 ± 21.08 µM Trolox equivalent, respectively. The fractions of 3-5 kDa, 5-10 kDa were the third and fourth strongest, respectively. The 10-30 kDa fraction showed weakest antioxidant capacity with the IC50 of 8012.88 ± 280.68 µg/mL and FRAP value of 92.92 ± 0.61 µM Trolox equivalent. The antioxidant peptides derived from Tra Catfish by-products proteolysate showed the potential to be used as natural antioxidant components in pharmaceutical and food industry.

Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

PubMed

Chen, S; Blanck, G; Pollack, R E

1983-09-01

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation.

Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

PubMed Central

Chen, S; Blanck, G; Pollack, R E

1983-01-01

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation. Images PMID:6310588

Quantitative control of poly(ethylene oxide) surface antifouling and biodetection through azimuthally enhanced grating coupled-surface plasmon resonance sensing

NASA Astrophysics Data System (ADS)

Sonato, Agnese; Silvestri, Davide; Ruffato, Gianluca; Zacco, Gabriele; Romanato, Filippo; Morpurgo, Margherita

2013-12-01

Grating Coupled-Surface Plasmon reflectivity measurements carried out under azimuth and polarization control (GC-SPR φ ≠ 0°) were used to optimize the process of gold surface dressing with poly(ethylene oxide) (PEO) derivatives of different molecular weight, with the final goal to maximize the discrimination between specific and non-specific binding events occurring at the surface. The kinetics of surface deposition of thiol-ending PEOs (0.3, 2 and 5 kDa), introduced as antifouling layers, was monitored. Non-specific binding events upon immersion of the surfaces into buffers containing either 0.1% bovine serum albumin or 1% Goat Serum, were evaluated as a function of polymer size and density. A biorecognition event between avidin and biotin was then monitored in both buffers at selected low and high polymer surface densities and the contribution of analyte and fouling elements to the signal was precisely quantified. The 0.3 kDa PEO film was unable to protect the surface from non-specific interactions at any tested density. On the other hand, the 2 and 5 kDa polymers at their highest surface densities guaranteed full protection from non-specific interactions from both buffers. These densities were reached upon a long deposition time (24-30 h). The results pave the way toward the application of this platform for the detection of low concentration and small dimension analytes, for which both non-fouling and high instrumental sensitivity are fundamental requirements.

Induction of heat-shock response and alterations of protein phosphorylation by a novel topoisomerase II inhibitor, withangulatin A, in 9L rat brain tumor cells.

PubMed

Lee, W C; Lin, K Y; Chen, C M; Chen, Z T; Liu, H J; Lai, Y K

1991-10-01

Withangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat-shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat-shock protein 70 (HSP70) since both proteins migrated to the same position on two-dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre-treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 65 kDa protein which was also detected in the heat-shocked cells. Moreover, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent one of the earliest signals generated by both heat-shock and withangluatin A and may be involved in the upstream regulation of heat-shock response in cells.

Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex.

PubMed

Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet

2007-01-01

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.

Two cases of false-positive dengue non-structural protein 1 (NS1) antigen in patients with hematological malignancies and a review of the literature on the use of NS1 for the detection of Dengue infection.

PubMed

Chung, Shimin J; Krishnan, Prabha U; Leo, Yee Sin

2015-02-01

Early diagnosis of dengue has been made easier in recent years owing to the advancement in diagnostic technologies. The rapid non-structural protein 1 (NS1) test strip is widely used in many developed and developing regions at risk of dengue. Despite the relatively high specificity of this test, we recently encountered two cases of false-positive dengue NS1 antigen in patients with underlying hematological malignancies. We reviewed the literature for causes of false-positive dengue NS1. © The American Society of Tropical Medicine and Hygiene.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

PubMed

Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

2012-10-01

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

A novel PGC-1α isoform in brain localizes to mitochondria and associates with PINK1 and VDAC

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

Choi, Joungil, E-mail: jochoi@som.umaryland.edu; Veterans Affairs Medical Center, Baltimore, MD 21201; Batchu, Vera Venkatanaresh Kumar

2013-06-14

Highlights: •Novel 35 kDa PGC-1α localizes to mitochondrial inner membrane and matrix in brain. •Mitochondrial localization of 35 kDa PGC-1α depends on VDAC protein. •Mitochondrial localization of 35 kDa PGC-1α depends on membrane potential. •The 35 kDa PGC-1α associates and colocalizes with PINK in brain mitochondria. -- Abstract: Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) and PTEN-induced putative kinase 1 (PINK1) are powerful regulators of mitochondrial function. Here, we report that a previously unrecognized, novel 35 kDa PGC-1α isoform localizes to the mitochondrial inner membrane and matrix in brain as determined by protease protection and carbonate extraction assays, as well asmore » by immunoelectron microscopy. Immunoelectron microscopy and import experiments in vitro revealed that 35 kDa PGC-1α colocalizes and interacts with the voltage-dependent anion channel (VDAC), and that its import depends on VDAC. Valinomycin treatment which depolarizes the membrane potential, abolished mitochondrial localization of the 35 kDa PGC-1α. Using blue native-PAGE, co-immunoprecipitation, and immunoelectron microscopy analyses, we found that the 35 kDa PGC-1α binds and colocalizes with PINK1 in brain mitochondria. This is the first report regarding mitochondrial localization of a novel 35 kDa PGC-1α isoform and its association with PINK1, suggesting possible regulatory roles for mitochondrial function in the brain.« less

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed

Laferté, S; Loh, L C

1992-04-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours.

/sup 3/H)forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter

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

Shanahan, M.F.; Morris, D.P.; Edwards, B.M.

1987-05-05

Irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of themore » glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of (/sup 3/H)cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.« less

Surface component characterization as taxonomic tools for Phytomonas spp identification.

PubMed

Abreu Filho, B A; Dias Filho, B P; Vermelho, A B; Jankevicius, S I; Jankevicius, J V; dos Santos, R L

2001-02-01

The genus Phytomonas arbitrarily includes all protozoa of the family Trypanosomatidae isolated from plants, but its differentiation is a complex task. The phase separation technique using Triton X-114 was used to analyze hydrophobic and hydrophilic surface proteins in ten strains of Phytomonas isolated from various fruits. The iodination of surface proteins by the Iodo-Gen method was also used for Phytomonas isolates from tomatoes, corn and annatto, Herpetomonas samuelpessoai and Crithidia fasciculata. The distribution of protein-bound radioactivity in acrylamide gels was determined by autoradiograms and showed the presence of protein bands of 36-68 kDa in all strains of Phytomonas: there were two major bands at 88 kDa and 94 kDa, with minor bands at 36 kDa and 142 kDa in H. samuelpessoai; and there were three bands at 74, 86 and 94 kDa, with minor bands at 23 kDa and 105 kDa in C. fasciculata. The results demonstrated that samples of plant parasites can be clearly differentiated from H. samuelpessoai and C. fasciculata. These plant parasites were also submitted to polysaccharide analysis by gas-liquid chromatography of the corresponding alditol acetate. Arabinose, galactose, glucose and mannose, were the major monosaccharides found, while fucose, rhamnose and xylose were found in smaller amounts. The results of all these methods indicated that, after extension to a wider range of trypanosomatid strains, they may be useful in Phytomonas taxonomy.

Biofortification of soybean meal: immunological properties of the 27 kDa γ-zein.

PubMed

Krishnan, Hari B; Jang, Sungchan; Kim, Won-Seok; Kerley, Monty S; Oliver, Melvin J; Trick, Harold N

2011-02-23

Legumes, including soybeans ( Glycine max ), are deficient in sulfur-containing amino acids, which are required for the optimal growth of monogastric animals. This deficiency can be overcome by expressing heterologous proteins rich in sulfur-containing amino acids in soybean seeds. A maize 27 kDa γ-zein, a cysteine-rich protein, has been successfully expressed in several crops including soybean, barley, and alfalfa with the intent to biofortify these crops for animal feed. Previous work has shown that the maize 27 kDa zein can withstand digestion by pepsin and elicit an immunogenic response in young pigs. By use of sera from patients who tested positive by ImmunoCAP assay for elevated IgE to maize proteins, specific IgE binding to the 27 kDa γ-zein is demonstrated. Bioinformatic analysis using the full-length and 80 amino acid sliding window FASTA searches identified significant sequence homology of the 27 kDa γ-zein with several known allergens. Immunoblot analysis using human serum that cross-reacts with maize seed proteins also revealed specific IgE-binding to the 27 kDa γ-zein in soybean seed protein extracts containing the 27 kDa zein. This study demonstrates for the first time the allergenicity potential of the 27 kDa γ-zein and the potential that this protein has to limit livestock performance when used in soybeans that serve as a biofortified feed supplement.

Kinetics and magnitude of antibody responses against the conserved 47-kilodalton antigen and the variable 56-kilodalton antigen in scrub typhus patients.

PubMed

Chen, Hua-Wei; Zhang, Zhiwen; Huber, Erin; Mutumanje, Elissa; Chao, Chien-Chung; Ching, Wei-Mei

2011-06-01

Western blot analysis of Orientia tsutsugamushi whole-cell lysates with scrub typhus patient sera has identified at least five protein antigens of O. tsutsugamushi with molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

PubMed

Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

2016-10-01

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Robust phenotyping strategies for evaluation of stem non-structural carbohydrates (NSC) in rice.

PubMed

Wang, Diane R; Wolfrum, Edward J; Virk, Parminder; Ismail, Abdelbagi; Greenberg, Anthony J; McCouch, Susan R

2016-11-01

Rice plants (Oryza sativa) accumulate excess photoassimilates in the form of non-structural carbohydrates (NSCs) in their stems prior to heading that can later be mobilized to supplement photosynthate production during grain-filling. Despite longstanding interest in stem NSC for rice improvement, the dynamics of NSC accumulation, remobilization, and re-accumulation that have genetic potential for optimization have not been systematically investigated. Here we conducted three pilot experiments to lay the groundwork for large-scale diversity studies on rice stem NSC. We assessed the relationship of stem NSC components with 21 agronomic traits in large-scale, tropical yield trials using 33 breeder-nominated lines, established an appropriate experimental design for future genetic studies using a Bayesian framework to sample sub-datasets from highly replicated greenhouse data using 36 genetically diverse genotypes, and used 434 phenotypically divergent rice stem samples to develop two partial least-squares (PLS) models using near-infrared (NIR) spectra for accurate, rapid prediction of rice stem starch, sucrose, and total non-structural carbohydrates. We find evidence that stem reserves are most critical for short-duration varieties and suggest that pre-heading stem NSC is worthy of further experimentation for breeding early maturing rice. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

A quantitative flood risk analysis methodology for urban areas with integration of social research data

NASA Astrophysics Data System (ADS)

Escuder-Bueno, I.; Castillo-Rodríguez, J. T.; Zechner, S.; Jöbstl, C.; Perales-Momparler, S.; Petaccia, G.

2012-09-01

Risk analysis has become a top priority for authorities and stakeholders in many European countries, with the aim of reducing flooding risk, considering the population's needs and improving risk awareness. Within this context, two methodological pieces have been developed in the period 2009-2011 within the SUFRI project (Sustainable Strategies of Urban Flood Risk Management with non-structural measures to cope with the residual risk, 2nd ERA-Net CRUE Funding Initiative). First, the "SUFRI Methodology for pluvial and river flooding risk assessment in urban areas to inform decision-making" provides a comprehensive and quantitative tool for flood risk analysis. Second, the "Methodology for investigation of risk awareness of the population concerned" presents the basis to estimate current risk from a social perspective and identify tendencies in the way floods are understood by citizens. Outcomes of both methods are integrated in this paper with the aim of informing decision making on non-structural protection measures. The results of two case studies are shown to illustrate practical applications of this developed approach. The main advantage of applying the methodology herein presented consists in providing a quantitative estimation of flooding risk before and after investing in non-structural risk mitigation measures. It can be of great interest for decision makers as it provides rational and solid information.

The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

PubMed Central

Le Gall, Sylvie; Neuhof, Andrea; Rapoport, Tom

2004-01-01

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of ∼5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER. PMID:14617815

Biopolymers: Protein and Nucleic Acids

DTIC Science & Technology

1987-09-15

characterized a 45 kDa "ZSB, de!•ignted S’SB1, most extensively. SSB1 was isolated on the basis of preferential binding to sin ie-stranded versus double...stranded DNA and was subsequently shown also to bind to RNA (9). Although SSB1 seems to bind without sequen-e specificity, it is interesting to note...that it copurifies through several a inity steps with the yeast poly (A) binding protein (11, Sach3 and Jong, unpublished observations). SSB1 stimulates

Characterization of auxin-binding proteins from zucchini plasma membrane

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rice, M. S.; Lomax, T. L.

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases.

PubMed

Tsuji, Akihiko; Nishiyama, Nami; Ohshima, Miki; Maniwa, Saori; Kuwamura, Shuji; Shiraishi, Masataka; Yuasa, Keizo

2014-01-01

Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca(2+) or Cl(-). Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase-glucoamylase and sucrase-isomaltase complexes.

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases

PubMed Central

Tsuji, Akihiko; Nishiyama, Nami; Ohshima, Miki; Maniwa, Saori; Kuwamura, Shuji; Shiraishi, Masataka; Yuasa, Keizo

2014-01-01

Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes. PMID:25161866

Characterization of auxin-binding proteins from zucchini plasma membrane.

PubMed

Hicks, G R; Rice, M S; Lomax, T L

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects.

PubMed

Corbau, R; Duverger, V; Rommelaere, J; Nüesch, J P

2000-12-05

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation. Copyright 2000 Academic Press.

A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.

PubMed

Neumaier, M; Gaida, F J; Lewis, M R; Hefta, L J; Shively, L E; Raubitschek, A; Shively, J E

1999-01-01

We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.66 was expressed in high yield in a bioreactor and purified to homogeneity in a single step on an anti-idiotypic antibody affinity column. The molecular size of the antibody was 150 kDa as judged by gel filtration, SDS gel electrophoresis under non-reducing conditions, and MALDI-TOF/MS. The 150 kDa antibody had nearly identical kinetic (Kon = 1.53 x 10(6) M-1 s-1, .koff = 1.14 x 10(-5) s-1) and affinity constants (Kaff = 1.34 x 10(11) M-1) compared to the parent murine (Kaff = 1.25 x 10(11) M-1) and chimeric (Kaff = 1.16 x 10(11) M-1) antibodies when tested on biosensor chips. When delta SSchT84.66 was conjugated to the isothiocynato derivative of DTPA, radiolabeled with 111In, and injected into either normal or nude mice bearing tumor xenografts, it gave nearly identical biodistributions to chT84.66. delta SSchT84.66 and chT84.66 antibodies gave a maximum tumor uptake of 48 and 74% ID/g, and tumor to blood ratios of 5.3 and 6.2 at 48 h, respectively. We conclude that delta SSchT84.66 irreversibly associates into H2L2 dimers after concentration, that the dimers are stable under both the in vitro and in vivo conditions used in this study, and the properties of the antibody are virtually indistinguishable from the parent chT84.66 antibody.

Anti-inflammatory action of high molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways.

PubMed

Kim, Young-Sang; Ahn, Chang-Bum; Je, Jae-Young

2016-07-01

Anti-inflammatory Mytilus edulis hydrolysates (MEHs) were prepared by peptic hydrolysis and MEH was further fractionated into three fractions based on molecular weight, namely >5kDa, 1-5kDa, and <1kDa. The >5kDa peptide fraction exerted the highest nitric oxide (NO) inhibitory activity and inhibited prostaglandin E2 (PGE2) secretion in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pretreatment with the >5kDa peptide fraction markedly inhibited LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and gene expressions. Stimulation by LPS induced the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β), whereas co-treatment with the >5kDa peptide fraction suppressed pro-inflammatory cytokine production. The >5kDa peptide fraction inhibited the translocation of NF-κB (nuclear factor-kappa B) through the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation and also inhibited the MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

PubMed

Mc Donald, Jessica M; O'Malley, Tiernan T; Liu, Wen; Mably, Alexandra J; Brinkmalm, Gunnar; Portelius, Erik; Wittbold, William M; Frosch, Matthew P; Walsh, Dominic M

2015-11-01

Much knowledge about amyloid β (Aβ) aggregation and toxicity has been acquired using synthetic peptides and mouse models, whereas less is known about soluble Aβ in human brain. We analyzed aqueous extracts from multiple AD brains using an array of techniques. Brains can contain at least four different Aβ assembly forms including: (i) monomers, (ii) a ∼7 kDa Aβ species, and larger species (iii) from ∼30-150 kDa, and (iv) >160 kDa. High molecular weight species are by far the most prevalent and appear to be built from ∼7 kDa Aβ species. The ∼7 kDa Aβ species resist denaturation by chaotropic agents and have a higher Aβ42/Aβ40 ratio than monomers, and are unreactive with antibodies to Asp1 of Ab or APP residues N-terminal of Asp1. Further analysis of brain-derived ∼7 kDa Aβ species, the mechanism by which they assemble and the structures they form should reveal therapeutic and diagnostic opportunities. Copyright © 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Protein Subcellular Localization with Gaussian Kernel Discriminant Analysis and Its Kernel Parameter Selection.

PubMed

Wang, Shunfang; Nie, Bing; Yue, Kun; Fei, Yu; Li, Wenjia; Xu, Dongshu

2017-12-15

Kernel discriminant analysis (KDA) is a dimension reduction and classification algorithm based on nonlinear kernel trick, which can be novelly used to treat high-dimensional and complex biological data before undergoing classification processes such as protein subcellular localization. Kernel parameters make a great impact on the performance of the KDA model. Specifically, for KDA with the popular Gaussian kernel, to select the scale parameter is still a challenging problem. Thus, this paper introduces the KDA method and proposes a new method for Gaussian kernel parameter selection depending on the fact that the differences between reconstruction errors of edge normal samples and those of interior normal samples should be maximized for certain suitable kernel parameters. Experiments with various standard data sets of protein subcellular localization show that the overall accuracy of protein classification prediction with KDA is much higher than that without KDA. Meanwhile, the kernel parameter of KDA has a great impact on the efficiency, and the proposed method can produce an optimum parameter, which makes the new algorithm not only perform as effectively as the traditional ones, but also reduce the computational time and thus improve efficiency.

Limitation of Unloading in the Developing Grains Is a Possible Cause Responsible for Low Stem Non-structural Carbohydrate Translocation and Poor Grain Yield Formation in Rice through Verification of Recombinant Inbred Lines

PubMed Central

Li, Guohui; Pan, Junfeng; Cui, Kehui; Yuan, Musong; Hu, Qiuqian; Wang, Wencheng; Mohapatra, Pravat K.; Nie, Lixiao; Huang, Jianliang; Peng, Shaobing

2017-01-01

Remobilisation of non-structural carbohydrates (NSC) from leaves and stems and unloading into developing grains are essential for yield formation of rice. In present study, three recombinant inbred lines of rice, R91, R156 and R201 have been tested for source-flow-sink related attributes determining the nature of NSC accumulation and translocation at two nitrogen levels in the field. Compared to R91 and R156, R201 had lower grain filling percentage, harvest index, and grain yield. Meanwhile, R201 had significantly lower stem NSC translocation during grain filling stage. Grain filling percentage, harvest index, and grain yield showed the consistent trend with stem NSC translocation among the three lines. In comparison with R91 and R156, R201 had similarity in leaf area index, specific leaf weight, stem NSC concentration at heading, biomass, panicles m-2, spikelets per panicle, remobilization capability of assimilation in stems, sink capacity, sink activity, number and cross sectional area of small vascular bundles, greater number and cross sectional area of large vascular bundles, and higher SPAD, suggesting that source, flow, and sink were not the limiting factors for low stem NSC translocation and grain filling percentage of R201. However, R201 had significant higher stem and rachis NSC concentrations at maturity, which implied that unloading in the developing grains might result in low NSC translocation in R201. The results indicate that stem NSC translocation could be beneficial for enhancement of grain yield potential, and poor unloading into caryopsis may be the possible cause of low stem NSC translocation, poor grain filling and yield formation in R201. PMID:28848573

Differential Activation of Diverse Glutathione Transferases of Clonorchis sinensis in Response to the Host Bile and Oxidative Stressors

PubMed Central

Bae, Young-An; Ahn, Do-Whan; Lee, Eung-Goo; Kim, Seon-Hee; Cai, Guo-Bin; Kang, Insug; Sohn, Woon-Mok; Kong, Yoon

2013-01-01

Background Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. Methodology/Principal Findings We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa μ-class GST (Cs26μGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses. Conclusions/Significance Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26μGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions. PMID:23696907

Yolk proteins during ovary and egg development of mature female freshwater crayfish (Cherax quadricarinatus).

PubMed

Serrano-Pinto, Vania; Vazquez-Boucard, Celia; Villarreal-Colmenares, Humberto

2003-01-01

Vitellins from ovaries and eggs at different stages of development in freshwater crayfish (Cherax quadricarinatus) were examined by chromatography, PAGE and SDS-PAGE. With these methods, two forms of vitellin (Vt1 and Vt2) were observed in ovaries and eggs (stages I and V). In ovaries in secondary vitellogenesis, native molecular mass was 470 (Vt1) and 440 (Vt2) kDa. The electrophoretic pattern of the eggs proved to be more complex. The protein molecular mass depend on the development stage of the egg: stage I, 650 kDa (Vt1) and 440 kDa (Vt2); stage V, 390 kDa (Vt1) and 340 kDa (Vt2). The identified vitellins appear to be lipo-glycocarotenoprotein. A similar vitellin polypeptide composition was observed in the two forms of vitellin from ovaries and eggs in stage V. In ovaries the SDS-PAGE analysis showed four subunits with molecular weights of approximately 180, 120, 95 and 80 kDa (Vt1 and Vt2). The polypeptide composition in the two forms of vitellins in stage I and stage III eggs were different at 195, 190, 130 and 110 kDa (Vt1) and 116 and 107 kDa (Vt2). On the other hand, in stage V eggs, 110, 95, 87 and 75 kDa (Vt1 and Vt2) were identified. Two antibodies (Ab1 and Ab2) were prepared against the purified proteins of stage V eggs and their specificity was demonstrated by radial immunoprecipitation, and Western blotting analysis. Two forms of vitellins were also found in stage V eggs after chromatography on Sepharose CL-2B column and hydroxylapatite and polyacrylamide gel electrophoresis.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

PubMed

Shiomi, Kazuo; Sato, Yuichiro; Hamamoto, Shohei; Mita, Hajime; Shimakura, Kuniyoshi

2008-01-01

Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish. (c) 2008 S. Karger AG, Basel.

Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa.

PubMed

Tipton, Jeremiah D; Tran, John C; Catherman, Adam D; Ahlf, Dorothy R; Durbin, Kenneth R; Lee, Ji Eun; Kellie, John F; Kelleher, Neil L; Hendrickson, Christopher L; Marshall, Alan G

2012-03-06

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

Presence of Human T-Cell Responses to the Mycobacterium leprae 45-Kilodalton Antigen Reflects Infection with or Exposure to M. leprae

PubMed Central

Macfarlane, Anne; Mondragon-Gonzalez, Rafael; Vega-Lopez, Francisco; Wieles, Brigitte; de Pena, Josefina; Rodriguez, Obdulia; Suarez y de la Torre, Raul; de Vries, Rene R. P.; Ottenhoff, Tom H. M.; Dockrell, Hazel M.

2001-01-01

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-γ) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-γ secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-γ secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test. PMID:11329466

Toxins not neutralized by brown snake antivenom

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

Judge, Roopwant K.; Henry, Peter J.; Mirtschin, Peter

2006-06-01

The Australian snakes of the genus Pseudonaja (dugite, gwardar and common brown) account for the majority of snake bite related deaths in Australia. Without antivenom treatment, the risk of mortality is significant. There is an accumulating body of evidence to suggest that the efficacy of the antivenom is limited. The current study investigates the protein constituents recognized by the antivenom using 2-DE, immuno-blot techniques and rat tracheal organ bath assays. The 2-DE profiles for all three snake venoms were similar, with major species visualized at 78-132 kDa, 32-45 kDa and 6-15 kDa. Proteins characterized by LC-MS/MS revealed a coagulant toxinmore » ({approx}42 kDa) and coagulant peptide ({approx}6 kDa), as well as two PLA{sub 2} ({approx}14 kDa). Peptides isolated from {approx}78 kDa and 15-32 kDa protein components showed no similarity to known protein sequences. Protein recognition by the antivenom occurred predominantly for the higher molecular weight components with little recognition of 6-32 kDa MW species. The ability of antivenom to neutralize venom activity was also investigated using rat tracheal organ bath assays. The venoms of Pseudonaja affinis affinis and Pseudonaja nuchalis incited a sustained, significant contraction of the trachea. These contractions were attributed to PLA{sub 2} enzymatic activity as pre-treatment with the PLA{sub 2} inhibitor 4-BPB attenuated the venom-induced contractions. The venom of Pseudonaja textilis incited tracheal contractility through a non-PLA{sub 2} enzymatic activity. Neither activity was attenuated by the antivenom treatment. These results represent the first proteomic investigation of the venoms from the snakes of the genus Pseudonaja, revealing a possible limitation of the brown snake antivenom in binding to the low MW protein components.« less

Tight ceramic UF membrane as RO pre-treatment: the role of electrostatic interactions on phosphate rejection.

PubMed

Shang, Ran; Verliefde, Arne R D; Hu, Jingyi; Zeng, Zheyi; Lu, Jie; Kemperman, Antoine J B; Deng, Huiping; Nijmeijer, Kitty; Heijman, Sebastiaan G J; Rietveld, Luuk C

2014-01-01

Phosphate limitation has been reported as an effective approach to inhibit biofouling in reverse osmosis (RO) systems for water purification. The rejection of dissolved phosphate by negatively charged TiO2 tight ultrafiltration (UF) membranes (1 kDa and 3 kDa) was observed. These membranes can potentially be adopted as an effective process for RO pre-treatment in order to constrain biofouling by phosphate limitation. This paper focuses on electrostatic interactions during tight UF filtration. Despite the larger pore size, the 3 kDa ceramic membrane exhibited greater phosphate rejection than the 1 kDa membrane, because the 3 kDa membrane has a greater negative surface charge and thus greater electrostatic repulsion against phosphate. The increase of pH from 6 to 8.5 led to a substantial increase in phosphate rejection by both membranes due to increased electrostatic repulsion. At pH 8.5, the maximum phosphate rejections achieved by the 1 kDa and 3 kDa membrane were 75% and 86%, respectively. A Debye ratio (ratio of the Debye length to the pore radius) is introduced in order to evaluate double layer overlapping in tight UF membranes. Threshold Debye ratios were determined as 2 and 1 for the 1 kDa and 3 kDa membranes, respectively. A Debye ratio below the threshold Debye ratio leads to dramatically decreased phosphate rejection by tight UF membranes. The phosphate rejection by the tight UF, in combination with chemical phosphate removal by coagulation, might accomplish phosphate-limited conditions for biological growth and thus prevent biofouling in the RO systems. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo

PubMed Central

Choi, James J.; Wang, Shougang; Tung, Yao-Sheng; Morrison, Barclay; Konofagou, Elisa E.

2009-01-01

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agent's molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525 MHz, peak-rarefactional pressure in situ: 569 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared to 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa, into the brain parenchyma. This noninvasive and localized BBB opening technique could thus provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro, but whose in vivo translation has been hampered by their associated BBB impermeability. PMID:19900750

Amino acid composition and functional properties of giant red sea cucumber ( Parastichopus californicus) collagen hydrolysates

NASA Astrophysics Data System (ADS)

Liu, Zunying; Su, Yicheng; Zeng, Mingyong

2011-03-01

Giant red sea cucumber ( Parastichopus californicus) is an under-utilized species due to its high tendency to autolysis. The aim of this study was to evaluate the functional properties of collagen hydrolysates from this species. The degree of hydrolysis (DH), amino acid composition, SDS-PAGE, emulsion activity index (EAI), emulsion stability index (ESI), foam expansion (FE), and foam stability (FS) of hydrolysates were investigated. The effects of pH on the EAI, ESI FE and FS of hydrolysates were also investigated. The results indicated that the β and α 1 chains of the collagen were effectively hydrolyzed by trypsin at 50°c with an Enzyme/Substrate (E/S) ration of 1:20 (w:w). The DH of collagen was up to 17.3% after 3 h hydrolysis with trypsin. The hydrolysates had a molecular weight distribution of 1.1-17 kDa, and were abundant in glycine (Gly), proline (Pro), glutamic acid (Glu), alanine (Ala) and hydroxyproline (Hyp) residues. The hydrolysates were fractionated into three fractions (< 3 kDa, 3-10 kDa, and > 10 kDa), and the fraction of 3-10 kDa exhibited a higher EAI value than the fraction of > 10 kDa ( P<0.05). The fraction of > 10 kDa had higher FE and FS values than other fractions ( P<0.05). The pH had an important effect on the EAI, ESI, FE and FS. All the fractions showed undesirable emulsion and forming properties at pH 4.0. Under pH 7.0 and pH 10.0, the 3-10 kDa fraction showed higher EAI value and the fraction of > 10 kDa showed higher FE value, respectively. They are hoped to be utilized as functional ingredients in food and nutraceutical industries.

Ion Mobility Separation of Variant Histone Tails Extending to the “Middle-down” Range

PubMed Central

Shvartsburg, Alexandre A.; Zheng, Yupeng; Smith, Richard D.; Kelleher, Neil L.

2012-01-01

Differential ion mobility spectrometry (FAIMS) can baseline-resolve multiple variants of post-translationally modified peptides extending to the 3 - 4 kDa range, which differ in the localization of a PTM as small as acetylation. Essentially orthogonal separations for different charge states expand the total peak capacity in proportion to the number of observed states that increases for longer polypeptides. This might enable resolving localization variants for yet larger peptides and even intact proteins. PMID:22559289

Inhibition of the Androgen Receptor Amino-Terminal Domain by a Small Molecule as Treatment for Castrate-Resistant Prostate Cancer

DTIC Science & Technology

2013-10-01

IDPs have flexibility, thereby providing the plasticity to enable interactions with multiple partners where high-specificity and low-affinity...block protein-protein interactions is a rapidly evolving field, as the importance of these proteins in disease becomes established. The plasticity of...closest to the structure of EPI-002 — did not bind an abundance of other cellular proteins (Figure 3D , top). Only 3 bands between 200 and 75 kDa were

Burkholderia Mallei tssM Encodes a Secreted Deubiquitinase that is Expressed Inside Infected RAW 264.7 Murine Macrophages

DTIC Science & Technology

2008-10-13

Furthermore, the encoded protein of this gene is only 30 kDa. A potential GTG start codon at position 625 also encodes a protein that is too small...horizontal bar and putative alternate translation initiation sites (ATG, GTG , and TTG) are indicated. The sizes and locations of the proteins encoded... gray line with rounded rectangles showing sequence features and motifs, including the Ala- and Pro-rich N-terminal region and the C-terminal Cys and

Molecular characterization of African orthobunyaviruses.

PubMed

Yandoko, E Nakouné; Gribaldo, S; Finance, C; Le Faou, A; Rihn, B H

2007-06-01

The genus Orthobunyavirus is composed of segmented, negative-sense RNA viruses that are responsible for mild to severe human diseases. To date, no molecular studies of bunyaviruses in the genus Orthobunyavirus from central Africa have been reported, and their classification relies on serological testing. Four new primer pairs for RT-PCR amplification and sequencing of the complete genomic small (S) RNA segments of 10 orthobunyaviruses isolated from the Central African Republic and pertaining to five different serogroups have been designed and evaluated. Phylogenetic analysis showed that these 10 viruses belong to the Bunyamwera serogroup. The S segment sequences differ from those of the Bunyamwera virus reference strain by 5-15 % at the nucleotide level, and both overlapping reading frames, encoding the nucleocapsid (N) and non-structural (NS) proteins, were evident in sequenced genomes. This study should improve diagnosis and surveillance of African bunyaviruses.

Antiviral active peptide from oyster

NASA Astrophysics Data System (ADS)

Zeng, Mingyong; Cui, Wenxuan; Zhao, Yuanhui; Liu, Zunying; Dong, Shiyuan; Guo, Yao

2008-08-01

An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster ( Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10 5 kDa, 5 1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10 5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466

Biodegradability enhancement and detoxification of cork processing wastewater molecular size fractions by ozone.

PubMed

Santos, Diana C; Silva, Lúcia; Albuquerque, António; Simões, Rogério; Gomes, Arlindo C

2013-11-01

Cork boiling wastewater pollutants were fractionated by sequential use of four ultrafiltration membranes and five fractions were obtained: four retentates (>100, 50-100, 20-50 and 10-20 kDa) and one permeate (<10 kDa); which were used to study the correlation of molecular size with biodegradability and toxicity before and after ozonation. The results show that molecular size is correlated with organic load and restrains biodegradability. The fraction with >100 kDa corresponds to 56% of the organic load and the one with <10 kDa only 8%. The biodegradability of fractions increased 182% with fractions molecular size reduction from >100 to <10 kDa and the chemical oxygen demand (COD) was from 3436 to 386 mg L(-1). For biodegradability enhancement the best outcome of ozonation was obtained with compounds having molecular size >20 kDa and range from 5% up to 175% for applied ozone doses to COD ratios between 0.15 and 0.38. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

PubMed

Wang, Mu; Sheng, Xiu-Zhen; Xing, Jing; Tang, Xiao-Qian; Zhan, Wen-Bin

2011-03-16

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

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

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

Damage-reducing measures to manage flood risks in a changing climate

NASA Astrophysics Data System (ADS)

Kreibich, Heidi; Bubeck, Philip; Van Vliet, Mathijs; De Moel, Hans

2014-05-01

Damage due to floods has increased during the last few decades, and further increases are expected in several regions due to climate change and a growing vulnerability. To address the projected increase in flood risk, a combination of structural and non-structural flood risk mitigation measures is considered as a promising adaptation strategy. Such a combination takes into account that flood defence systems may fail, and prepare for unexpected crisis situations via land-use planning, building construction, evacuation and disaster response. Non-structural flood risk mitigation measures like shielding with water shutters or sand bags, building fortification or safeguarding of hazardous substances are often voluntary: they demand self-dependent action by the population at risk (Bubeck et al. 2012; 2013). It is believed that these measures are especially effective in areas with frequent flood events and low flood water levels, but some types of measures showed a significant damage-reducing effect also during extreme flood events, such as the Elbe River flood in August 2002 in Germany (Kreibich et al. 2005; 2011). Despite the growing importance of damage-reducing measures, information is still scarce about factors that motivate people to undertake such measures, the state of implementation of various non-structural measures in different countries and their damage reducing effects. Thus, we collected information and undertook an international review about this topic in the framework of the Dutch KfC project "Climate proof flood risk management". The contribution will present an overview about the available information on damage-reducing measures and draw conclusions for practical flood risk management in a changing climate. References: Bubeck, P., Botzen, W. J. W., Suu, L. T. T., Aerts, J. C. J. H. (2012): Do flood risk perceptions provide useful insights for flood risk management? Findings from central Vietnam. Journal of Flood Risk Management, 5, 4, 295-302 Bubeck, P., Botzen, W. J. W., Kreibich, H., Aerts, J. C. J. H. (2013) Detailed insights into the influence of flood-coping appraisals on mitigation behaviour. Global Environmental Change. DOI:10.1016/j.gloenvcha.2013.05.009. Kreibich, H., Thieken, A. H., Petrow, T., Müller, M., Merz, B. (2005): Flood loss reduction of private households due to building precautionary measures - Lessons Learned from the Elbe flood in August 2002. NHESS, 5, 1, 117-126. Kreibich, H., Christenberger, S., Schwarze, R. (2011) Economic motivation of households to undertake private precautionary measures against floods. NHESS, 11, 2, 309-321.

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus

PubMed Central

Zhu, Shaomei; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre

2016-01-01

ABSTRACT A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. IMPORTANCE HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines. PMID:27384651

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

PubMed

Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

2016-09-15

A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed Central

Laferté, S; Loh, L C

1992-01-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. PMID:1567368

Suppression of amyloid beta A11 antibody immunoreactivity by vitamin C: possible role of heparan sulfate oligosaccharides derived from glypican-1 by ascorbate-induced, nitric oxide (NO)-catalyzed degradation.

PubMed

Cheng, Fang; Cappai, Roberto; Ciccotosto, Giuseppe D; Svensson, Gabriel; Multhaup, Gerd; Fransson, Lars-Åke; Mani, Katrin

2011-08-05

Amyloid β (Aβ) is generated from the copper- and heparan sulfate (HS)-binding amyloid precursor protein (APP) by proteolytic processing. APP supports S-nitrosylation of the HS proteoglycan glypican-1 (Gpc-1). In the presence of ascorbate, there is NO-catalyzed release of anhydromannose (anMan)-containing oligosaccharides from Gpc-1-nitrosothiol. We investigated whether these oligosaccharides interact with Aβ during APP processing and plaque formation. anMan immunoreactivity was detected in amyloid plaques of Alzheimer (AD) and APP transgenic (Tg2576) mouse brains by immunofluorescence microscopy. APP/APP degradation products detected by antibodies to the C terminus of APP, but not Aβ oligomers detected by the anti-Aβ A11 antibody, colocalized with anMan immunoreactivity in Tg2576 fibroblasts. A 50-55-kDa anionic, sodium dodecyl sulfate-stable, anMan- and Aβ-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C terminus). anMan-containing HS oligo- and disaccharide preparations modulated or suppressed A11 immunoreactivity and oligomerization of Aβ42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by 3-β[2(diethylamino)ethoxy]androst-5-en-17-one (U18666A) and decreased when Gpc-1 autoprocessing was stimulated by ascorbate. Neither overexpression of Gpc-1 in Tg2576 fibroblasts nor addition of copper ion and NO donor to hippocampal slices from 3xTg-AD mice affected A11 immunoreactivity levels. However, A11 immunoreactivity was greatly suppressed by the subsequent addition of ascorbate. We speculate that temporary interaction between the Aβ domain and small, anMan-containing oligosaccharides may preclude formation of toxic Aβ oligomers. A portion of the oligosaccharides are co-secreted with the Aβ peptides and deposited in plaques. These results support the notion that an inadequate supply of vitamin C could contribute to late onset AD in humans.

Construction of a star-shaped copolymer as a vector for FGF receptor-mediated gene delivery in vitro and in vivo.

PubMed

Li, Da; Ping, Yuan; Xu, Fujian; Yu, Hai; Pan, Hongming; Huang, Hongliang; Wang, Qingqing; Tang, Guping; Li, Jun

2010-09-13

The success of cancer gene therapy highly relies on the gene delivery vector with high transfection activity and low toxicity. In the present study, eight-armed polyethylene glycol (EAP) and low molecular weight (LMW) polyethylenimine (PEI) were used as basic units to construct the architecture of a new star-shaped EAP-PEI copolymer (EAPP). MC11, a peptide capable of selectively binding fibroblast growth factor receptor (FGFR) on tumor cell membranes, was further conjugated to EAPP to produce the vector EAPP-MC11 (EAPPM) to enhance tumor targetability. This tumor-targeting vector EAPPM was observed to retard the plasmids mobility at a nitrogen/phosphorus (N/P) ratio of 3. The vector could efficiently condense plasmids within 300 nm nanoparticles with a positive zeta potential at the N/P ratio of 20 or above. While the cytotoxicity of EAPPM polyplexes was similar to that of LMW PEI, it was significantly lower than that of PEI (25 kDa) in HepG2 and PC3 cell lines. In vitro gene transfection with pDNA mediated by EAPPM showed that the transfection efficiency increased 15 times in HepG2 cells but remained at a similar level in PC3 cells in comparison with that of EAPP. By systemic injection of EAPPM/pDNA complexes into a HepG2-bearing mice model, luciferase expression detected in lung, liver, and tumor tissues demonstrated EAPPM could deliver in a targeted manner a reporter gene into tumor tissues, where the luciferase expression of EAPPM was 4 times higher than that of EAPP and even 23 times higher than that of PEI (25 kDa). Furthermore, it was found that the systemic delivery of EAPPM/pCSK-α-interferon complexes in vivo were much more effective in inhibiting tumor growth than EAPP or PEI (25 kDa). These results clearly show that EAPPM is an efficient and safe vector for FGFR-mediated targeted gene delivery both in vitro and in vivo. With low cytotoxicity and high targetability, EAPPM may have great potential as a delivery vector for future cancer gene therapy applications.

Production of Cry11A and Cry11Ba Toxins in Bacillus sphaericus Confers Toxicity towards Aedes aegypti and Resistant Culex Populations

PubMed Central

Servant, Pascale; Rosso, Marie-Laure; Hamon, Sylviane; Poncet, Sandrine; Delécluse, Armelle; Rapoport, Georges

1999-01-01

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297. PMID:10388698

Identification of proteinaceous material in the bone of the dinosaur Iguanodon.

PubMed

Embery, Graham; Milner, Angela C; Waddington, Rachel J; Hall, Rachel C; Langley, Martin S; Milan, Anna M

2003-01-01

This study has directed attention at the search for bone-related proteins in an extract of demineralized rib bone of the 120 mya Iguanodon. The inner compact bone was demineralized and the GuCl extract resolved into 11 fractions using anion exchange chromatography, which all contained silver-reactive proteins with various amino acid profiles. Two specific fractions, iv and xi, revealed characteristics typical of contemporary phosphoproteins and proteoglycans, respectively. Fraction iv, 43-57 kDa, contained a high ratio of aspartate and serine, although no phosphate was discernable. Fraction xi contained a band of 41-47 kDa and was rich in chondroitin sulphate and hyaluronan. In addition an early eluting fraction was immunoreactive with an antibody against osteocalcin. A cancellous bone fraction from the same bone sample was also analyzed using N-terminal sequencing and revealed potential similarities with cystatin. While we do not claim to have identified the presence of intact proteins, this study has value in demonstrating that extruded extracellular matrix is protected by its capacity to induce mineralization, which subsequently is important in conserving detectable protein products in ancient skeletal tissues.

Diffusive transfer to membranes as an effective interface between gel electrophoresis and mass spectrometry

NASA Astrophysics Data System (ADS)

Ogorzalek Loo, Rachel R.; Mitchell, Charles; Stevenson, Tracy I.; Loo, Joseph A.; Andrews, Philip C.

1997-12-01

Diffusive transfer was examined as a blotting method to transfer proteins from polyacrylamide gels to membranes for ultraviolet matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spectra have been obtained for 11 pmol of 66 kDa albumin loaded onto an IEF gel and subsequently blotted to polyethylene. Similarly, masses of intact carbonic anhydrase and hemoglobin were obtained from 14 and 20 pmol loadings. This methodology is also compatible with blotting high molecular weight proteins, as seen for 6 pmol of the 150 kDa monoclonal antibody anti-[beta]-galactosidase transferred to Goretex. Polypropylene, Teflon, Nafion and polyvinylidene difluoride (PVDF) also produced good spectra following diffusive transfer. Only analysis from PVDF required that the membrane be kept wet prior to application of matrix. Considerations in mass accuracy for analysis from large-area membranes with continuous extraction and delayed extraction were explored, as were remedies for surface charging. Vapor phase CNBr cleavage was applied to membrane-bound samples for peptide mapping.

Purification, characterization and immunomodulatory activity of polysaccharides from stem lettuce.

PubMed

Nie, Chenzhipeng; Zhu, Peilei; Ma, Shuping; Wang, Mingchun; Hu, Youdong

2018-05-15

Stem lettuce has a long history of cultivation in China and possesses high nutritional and medicinal value. In our previous studies, extraction optimization, characterization, and bioactivities of stem lettuce polysaccharides (SLP) were investigated. In this study, SLP were further separated into two purified polysaccharides, SLP-1 and SLP-2, by anion exchange chromatography followed by size exclusion chromatography. SLP-1, with a molecular weight of 90 KDa, was mainly composed of galacturonic acid, galactose and arabinose in a molar ratio of 17.6:41.7:33.9. SLP-2, with a molecular weight of 44 KDa, was mainly composed of mannose, galacturonic acid, galactose and arabinose in a molar ratio of 11.5:69.5:9.3:8.2. In addition, both purified polysaccharides contain sulphate radicals, have triple helical structures and can promote macrophage proliferation without cytotoxicity. SLP-2 was better able to stimulate phagocytic and nitric oxide production than SLP-1. The results suggest that polysaccharides from stem lettuce could be explored as immunomodulatory agents in the field of pharmaceuticals and functional foods. Copyright © 2018 Elsevier Ltd. All rights reserved.

Numerical modelling to assess maintenance strategy management options for a small tidal inlet

NASA Astrophysics Data System (ADS)

Shaeri, Saeed; Tomlinson, Rodger; Etemad-Shahidi, Amir; Strauss, Darrell

2017-03-01

Small tidal inlets are found to be more sensitive to anthropogenic alteration than their larger counterparts. Such alterations, although typically supported by technical design reports, sometimes require amendments or modification. One of the most suitable tools to conduct the necessary studies in this regard is numerical modelling, since the behaviour of the inlet system in response to proposed remedial actions, can easily be identified. In this paper, various alternative proposals are investigated to determine the most practical and viable option to mitigate the need for ongoing maintenance at a typical small, jettied tidal inlet. The main tool to investigate the alternatives is the hydro-sedimentological modelling of the inlet system, which was performed using the Delft3D software package. The proposed alternative entrance modifications were based upon structural alterations of the inlet system (such as a jetty extension or submerged weir) and non-structural scenarios (such as a change of the time of the dredging campaign or the deposition location of the dredged material). It was concluded that whilst a detailed study is inevitable in order to achieve a comprehensive design plan, based upon the results of this study the construction of a submerged weir at the entrance channel can satisfy the needs of most of the stakeholders, with justifiable costs over a longer period.

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes.

PubMed

Asem, K; Cornish-Bowden, A J; Cole, J A

1986-01-01

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.

Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

PubMed

Nam, Kihoon; Jung, Simhyun; Nam, Joung-Pyo; Kim, Sung Wan

2015-12-28

Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility. Copyright © 2015 Elsevier B.V. All rights reserved.

Nucleotide sequence and phylogenetic analysis of Cucurbit yellow stunting disorder virus RNA 2.

PubMed

Livieratos, Ioannis C; Coutts, Robert H A

2002-06-01

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly (Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5' to 3' direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses.

Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

PubMed

Albers, Shawn; Inthathirath, Fatima; Gill, Sandeep K; Winick-Ng, Warren; Jaworski, Ewa; Wong, Daisy Y L; Gros, Robert; Rylett, R Jane

2014-09-01

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aβ. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aβ production, with this potentiating the decline in function of cholinergic neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes.

PubMed

Swamy, Mahima; Kulathu, Yogesh; Ernst, Sandra; Reth, Michael; Schamel, Wolfgang W A

2006-04-15

SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.

The enzymatic hydrolysis of soy protein isolate by Corolase PP under high hydrostatic pressure and its effect on bioactivity and characteristics of hydrolysates.

PubMed

Guan, Haining; Diao, Xiaoqin; Jiang, Fan; Han, Jianchun; Kong, Baohua

2018-04-15

Enzymatic hydrolysis of soy protein isolate by Corolase PP under high hydrostatic pressure conditions was studied and the effects of hydrolysis on antioxidant and antihypertensive activities were investigated. As observed, high hydrostatic pressure (80-300MPa) enhanced the hydrolytic efficiency of Corolase PP and decreased the surface hydrophobicity of the hydrolysates. Hydrolysates obtained at 200MPa for 4h had higher bioactivities (reducing power, ABTS radical-scavenging and ACE inhibitory activities). The molecular weight (MW) determination indicated that hydrolysis at high hydrostatic pressure could increase the production of small peptides (<3kDa) and the amino acid sequences of these peptides with different inhibitory abilities, less than 3kDa, in hydrolysates were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS). These results indicated that high hydrostatic pressure combined with Corolase PP treatments could be used as a potential technology to produce bioactive peptides from soy protein isolate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alpha-crystallin homolog by exposure to nitric oxide donors.

PubMed

Garbe, T R; Hibler, N S; Deretic, V

1999-01-01

In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.

Developmental neurotoxicity elicited by prenatal or postnatal chlorpyrifos exposure: effects on neurospecific proteins indicate changing vulnerabilities.

PubMed

Garcia, Stephanie J; Seidler, Frederic J; Slotkin, Theodore A

2003-03-01

The developmental neurotoxicity of the organophosphate pesticide chlorpyrifos (CPF) is thought to involve both neurons and glia, thus producing a prolonged window of vulnerability. To characterize the cell types and brain regions involved in these effects, we administered CPF to developing rats and examined neuroprotein markers for oligodendrocytes (myelin basic protein, MBP), for neuronal cell bodies (neurofilament 68 kDa, NF68), and for developing axons (neurofilament 200 kDa, NF200). Prenatal CPF administration on gestational days (GDs) 17-20 elicited an immediate (GD21) enhancement of MBP and NF68; by postnatal day (PN) 30, however, there were deficits in all three biomarkers, with the effect restricted to females. Exposure in the early postnatal period, PN1-4, did not evoke significant short-term or long-term changes in the neuroproteins. However, with treatment on PN11-14, we found reductions in MBP in the immediate posttreatment period (PN15, PN20) throughout the brain, and deficiencies across all three proteins emerged by PN30. With this regimen, males were targeted preferentially. The sex-selective effects seen here for the GD17-20 and PN11-14 regimens match those reported earlier for subsequent behavioral performance. These results indicate a shift in the populations of neural cells targeted by CPF, dependent upon the period of exposure. Similarly, developmental differences in the sex selectivity of the biochemical mechanisms underlying neurotoxicant actions are likely to contribute to discrete behavioral outcomes.

Characterization of a Chitosanase from Jelly Fig (Ficus awkeotsang Makino) Latex and Its Application in the Production of Water-Soluble Low Molecular Weight Chitosans.

PubMed

Chang, Chen-Tien; Lin, Yen-Lu; Lu, Shu-Wei; Huang, Chun-Wei; Wang, Yu-Ting; Chung, Yun-Chin

2016-01-01

A chitosanase was purified from jelly fig latex by ammonium sulfate fractionation (50-80% saturation) and three successive column chromatography steps. The purified enzyme was almost homogeneous, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel activity staining. The molecular mass of the enzyme was 20.5 kDa. The isoelectric point (pI) was <3.5, as estimated by isoelectric focusing electrophoresis on PhastGel IEF 3-9. Using chitosan as the substrate, the optimal pH for the enzyme reaction was 4.5; the kinetic parameters Km and Vmax were 0.089 mg mL-1 and 0.69 μmol min-1 mg-1, respectively. The enzyme showed activity toward chitosan polymers which exhibited various degrees of deacetylation (21-94%). The enzyme hydrolyzed 70-84% deacetylated chitosan polymers most effectively. Substrate specificity analysis indicated that the enzyme catalyzed the hydrolysis of chitin and chitosan polymers and their derivatives. The products of the hydrolysis of chitosan polymer derivatives, ethylene glycol (EG) chitosan, carboxymethyl (CM) chitosan and aminoethyl (AE) chitosan, were low molecular weight chitosans (LMWCs); these products were referred to as EG-LMWC, CM-LMWC and AE-LMWC, respectively. The average molecular weights of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2 and 8.89 kDa, respectively. All of the LMWC products exhibited free radical scavenging activities toward ABTS•+, superoxide and peroxyl radicals.

Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin.

PubMed

Moraes, Claudia T P; Polatto, Juliana M; Rossato, Sarita S; Izquierdo, Mariana; Munhoz, Danielle D; Martins, Fernando H; Pimenta, Daniel C; Farfan, Mauricio J; Elias, Waldir P; Barbosa, Ângela S; Piazza, Roxane M F

2015-12-18

Enteropathogenic Escherichia coli (EPEC) is distinguished mainly by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC). The initial adherence to the intestinal mucosa is complex and mediated by adhesins other than bundle-forming pilus, which is not produced by aEPEC. Extracellular matrix (ECM) proteins of eukaryotic cells are commonly recognized by bacterial adhesins. Therefore, binding to ECM proteins may facilitate colonization, invasion and/or signaling by intestinal pathogens. Previous studies from our group demonstrated that aEPEC O26:H11 (strain BA2103) showed high binding activity to fibronectin, not shared by its counterpart, aEPEC O26:HNM. In the present study, using mass spectrometry after fibronectin-associated immunoprecipitation, two proteins, flagellin (50 kDa) and GroEL (52 kDa), were identified and BA2103 binding ability to fibronectin was inhibited in the presence of anti-H11 and anti-GroEL sera, but not by either naïve rabbit or other unrelated sera. It was also observed that the presence of purified flagellin inhibits adhesion of BA2103 to cellular fibronectin in a dose-dependent manner. Additionally, BA2103 GroEL is similar to the same protein of uropathogenic E. coli. Our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein in this process.

Heterogeneity of cellular proliferation within transitional cell carcinoma: correlation of protein kinase C alpha/betaI expression and activity.

PubMed

Aaltonen, Vesa; Koivunen, Jussi; Laato, Matti; Peltonen, Juha

2006-07-01

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-alpha and -betaI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-alpha in 13 out of 18 samples and -betaI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-alpha and/or -betaI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-alpha was located in the cytoplasm, whereas PKC-betaI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-alpha and -betaI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

Characterization of Antibodies for Grain-Specific Gluten Detection.

PubMed

Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

2016-03-01

Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation. © Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Dynamic Loading of Immature Epiphyseal Cartilage Pumps Nutrients out of Vascular Canals

PubMed Central

Albro, Michael B.; Banerjee, Rajan E.; Li, Roland; Oungoulian, Sevan R.; Chen, Bo; del Palomar, Amaya P.; Hung, Clark T.; Ateshian, Gerard A.

2011-01-01

The potential influence of mechanical loading on transvascular transport in vascularized soft tissues has not been explored extensively. This experimental investigation introduced and explored the hypothesis that dynamic mechanical loading can pump solutes out of blood vessels and into the surrounding tissue, leading to faster uptake and higher solute concentrations than could otherwise be achieved under unloaded conditions. Immature epiphyseal cartilage was used as a model tissue system, with fluorescein (332 Da), dextran (3, 10 and 70 kDa) and transferrin (80 kDa) as model solutes. Cartilage disks were either dynamically loaded (±10% compression over a 10% static offset strain, at 0.2 Hz) or maintained unloaded in solution for up to 20 hours. Results demonstrated statistically significant solute uptake in dynamically loaded (DL) explants relative to passive diffusion (PD) controls for all solutes except unbound fluorescein, as evidenced by the DL:PD concentration ratios after 20 hours (1.0 ± 0.2, 2.4 ± 1.1, 6.1 ± 3.3, 9.0 ± 4.0, and 5.5±1.6 for fluorescein, 3, 10, and 70 kDa dextran, and transferrin). Significant uptake enhancements were also observed within the first 30 seconds of loading. Termination of dynamic loading produced dissipation of enhanced solute uptake back to PD control values. Confocal images confirmed that solute uptake occurred from cartilage canals into their surrounding extracellular matrix. The incidence of this loading-induced transvascular solute pumping mechanism may significantly alter our understanding of the interaction of mechanical loading and tissue metabolism. PMID:21481875

An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera.

PubMed Central

Mahan, S M; Tebele, N; Mukwedeya, D; Semu, S; Nyathi, C B; Wassink, L A; Kelly, P J; Peter, T; Barbet, A F

1993-01-01

Heartwater, a major constraint to improved livestock production in Zimbabwe, threatens to invade areas which have been previously unaffected. To monitor its spread in Zimbabwe, an immunoblotting diagnostic assay based on the responses of animals to the immunodominant, conserved 32-kDa protein of Cowdria ruminantium was evaluated. In this assay, no false reactions were detected with sera known to be positive and negative, but sera from some cattle, sheep, and goats from heartwater-free areas of Zimbabwe reacted strongly with the 32-kDa protein, suggesting that either these animals had previous exposure to heartwater or they were false positives. To investigate the possibility of previous exposure to heartwater, 11 immunoblot-positive and 6 immunoblot-negative sheep from heartwater-free areas of Zimbabwe were compared regarding their susceptibilities to challenge with C. ruminantium. Prior to challenge, C. ruminantium could not be detected in any sheep by transmission to Amblyomma hebraeum ticks or by the polymerase chain reaction (PCR) conducted with plasma samples. All sheep were equally susceptible to the challenge, and infection was confirmed by brain biopsy, necropsy, PCR, and transmission of C. ruminantium to ticks. Our data suggest that the immunoblot-positive reactions of sera from heartwater-free areas were due not to previous C. ruminantium infection but rather to antigenic cross-reactivity between C. ruminantium and another agent(s) such as Ehrlichia species. In conclusion, the immunodominant 32-kDa protein is not antigenically specific to C. ruminantium and its use in serological diagnosis of heartwater requires reevaluation. Images PMID:8253974

Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells.

PubMed

Li, Yang; Basavappa, Megha; Lu, Jinfeng; Dong, Shuwei; Cronkite, D Alexander; Prior, John T; Reinecker, Hans-Christian; Hertzog, Paul; Han, Yanhong; Li, Wan-Xiang; Cheloufi, Sihem; Karginov, Fedor V; Ding, Shou-Wei; Jeffrey, Kate L

2016-12-05

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens 1 . However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago 2 , remains unknown 3 . Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence 4-7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs) 8,9 . Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice 10,11 . However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells 12-21 . Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

Ecological Risk Assessment of Land Use Change in the Poyang Lake Eco-economic Zone, China

PubMed Central

Xie, Hualin; Wang, Peng; Huang, Hongsheng

2013-01-01

Land use/land cover change has been attracting increasing attention in the field of global environmental change research because of its role in the social and ecological environment. To explore the ecological risk characteristics of land use change in the Poyang Lake Eco-economic Zone of China, an eco-risk index was established in this study by the combination of a landscape disturbance index with a landscape fragmentation index. Spatial distribution and gradient difference of land use eco-risk are analyzed by using the methods of spatial autocorrelation and semivariance. Results show that ecological risk in the study area has a positive correlation, and there is a decreasing trend with the increase of grain size both in 1995 and 2005. Because the area of high eco-risk value increased from 1995 to 2005, eco-environment quality declined slightly in the study area. There are distinct spatial changes in the concentrated areas with high land use eco-risk values from 1995 to 2005. The step length of spatial separation of land use eco-risk is comparatively long—58 km in 1995 and 11 km in 2005—respectively. There are still nonstructural factors affecting the quality of the regional ecological environment at some small-scales. Our research results can provide some useful information for land eco-management, eco-environmental harnessing and restoration. In the future, some measures should be put forward in the regions with high eco-risk value, which include strengthening land use management, avoiding unreasonable types of land use and reducing the degree of fragmentation and separation. PMID:23343986

Protein Buffering in Model Systems and in Whole Human Saliva

PubMed Central

Lamanda, Andreas; Cheaib, Zeinab; Turgut, Melek Dilek; Lussi, Adrian

2007-01-01

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and α-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva. PMID:17327922

Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor.

PubMed

Mitchell, Michael S; Rao, Venigalla B

2006-01-06

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.

In vivo Delivery of Fluoresceinated Dextrans to the Murine Growth Plate: Imaging of Three Vascular Routes by Multiphoton Microscopy

PubMed Central

Farnum, Cornelia; Lenox, Michelle; Zipfel, Warren; Horton, William; Williams, Rebecca

2008-01-01

Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and via a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature, and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4-5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da), or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa) vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few per cent of vascular concentration) 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. PMID:16342207

Papain cleavage of the 38,000-dalton fragment inhibits the binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate to lys-539 on the 60,000-dalton fragment in human band 3.

PubMed

Yamaguchi, Takeo; Kojima, Hideaki; Kawaguchi, Shiori; Shimada, Maiko; Aso, Haruka

2017-08-01

Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Direct role for the RNA polymerase domain of T7 primase in primer delivery

PubMed Central

Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C.

2010-01-01

Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys4 zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 phage lacking gene 4 (T7Δ4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Δ4 phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56- or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis. PMID:20439755

Maize 27 kDa gamma-zein is a potential allergen for early weaned pigs.

PubMed

Krishnan, Hari B; Kerley, Monty S; Allee, Gary L; Jang, Sungchan; Kim, Won-Seok; Fu, Chunjiang J

2010-06-23

Soybean and maize are extensively used in animal feed, primarily in poultry, swine, and cattle diets. Soybean meal can affect pig performance in the first few weeks following weaning and elicit specific antibodies in weaned piglets. Though maize is a major component of pig feed, it is not known if any of the maize proteins can elicit immunological response in young pigs. In this study, we have identified a prominent 27 kDa protein from maize as an immunodominant protein in young pigs. This protein, like some known allergens, exhibited resistance to pepsin digestion in vitro. Several lines of evidence identify the immunodominant 27 kDa protein as a gamma-zein, a maize seed storage protein. First, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of different solubility classes of maize seed proteins revealed the presence of an abundant 27 kDa protein in the prolamin (zein) fraction. Antibodies raised against the purified maize 27 kDa gamma-zein also reacted against the same protein recognized by the young pig serum. Additionally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptides generated by trypsin digestion of the immunodominant 27 kDa protein showed significant homology to the maize 27 kDa gamma-zein. Since eliminating the allergenic protein will have a great impact on the nutritive value of the maize meal and expand its use in the livestock industry, it will be highly desirable to develop maize cultivars completely lacking the 27 kDa allergenic protein.

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones*

PubMed Central

Zhou, Hai-Yan; Salih, Erdjan; Glimcher, Melvin J.

2010-01-01

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species. PMID:20833721

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

PubMed

Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

2016-07-01

Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR. Copyright © 2016 Kainulainen et al.

Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

PubMed

Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

2015-12-02

Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

PubMed Central

Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-01-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high throughput proteomic assays, we have previously identified Bruton’s tyrosine kinase (BTK) as a host protein that was uniquely up-regulated in the plasma membrane of HIV-1 infected T-cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant up-regulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells, however new BTK protein complexes were identified and distributed in both high molecular weight (~600 kDa) and a small molecular weight complex (~60–120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1 infected cells using siRNA resulted in selective death of infected, but not uninfected, cells. Using BTK specific antibody and small molecule inhibitors including LFM-A13 and a FDA approved compound, Ibrutinib (PCI – 32765), we have found that HIV-1 infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1 infected cells are sensitive to treatments targeting BTK expressed in infected cells. PMID:25672887

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia.

PubMed

Hershey, H P; Schwartz, L J; Gale, J P; Abell, L M

1999-07-01

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.

Isolation and characterization of an immunosuppressive protein from venom of the pupa-specific endoparasitoid Pteromalus puparum.

PubMed

Wu, Ma-li; Ye, Gong-yin; Zhu, Jia-ying; Chen, Xue-xin; Hu, Cui

2008-10-01

In hymenopteran parasitoids devoid of symbiotic viruses, venom proteins appear to play a major role in host immune suppression and host regulation. Not much is known about the active components of venom proteins in these parasitoids, especially those that have the functions involved in the suppression of host cellular immunity. Here, we report the isolation and characterization of a venom protein Vn.11 with 24.1 kDa in size from Pteromalus puparum, a pupa-specific endoparasitoid of Pieris rapae. The Vn.11 venom protein is isolated with the combination of ammonium sulfate precipitation and anion exchange chromatography, and its purity is verified using SDS-PAGE analysis. Like crude venom, the Vn.11 venom protein significantly inhibits the spreading behavior and encapsulation ability of host hemocytes in vitro. It is suggested that this protein is an actual component of P. puparum crude venom as host cellular-immune suppressive factor.

Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica.

PubMed

Guo, Mei; Lu, Fuping; Du, Lianxiang; Pu, Jun; Bai, Dongqing

2006-08-01

A cDNA encoding for laccase (Lcc1) was isolated from the ligninolytic fungus Trametes versicolor by reverse transcriptase polymerase chain reaction. The Lcc1 gene was subcloned into the Pichia methanolica expression vector pMETalphaA and transformed into the P. methanolica strains PMAD11 and PMAD16. The extracellular laccase activity of the PMAD11 recombinants was found to be 1.3-fold higher than that of the PMAD16 recombinants. The identity of the recombinant protein was further confirmed by immunodetection using the Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form. The effects of copper concentration, cultivation temperature, pH and methanol concentration in the BMMY on laccase expression were investigated. The laccase activity in the PMAD11 recombinant was up to 12.6 U ml(-1) by optimization.

Antimicrobial compounds of porcine mucosa

NASA Astrophysics Data System (ADS)

Kotenkova, E. A.; Lukinova, E. A.; Fedulova, L. V.

2017-09-01

The aim of the study was to investigate porcine oral cavity mucosa (OCM), nasal cavity mucosa (NCM), rectal mucosa (RM) and tongue mucosa (TM) as sources of antimicrobial compounds. Ultrafiltrates with MW >30 kDa, MW 5-30 kDa and MW <5 kDa were obtained. All ultrafiltrates had antimicrobial activity against Escherichia coli and Proteus vulgaris. NCM ultrafiltrates revealed the highest antibacterial activity in respect to negative control: for the fraction with MW >30 kDa, the zone of microbial growth inhibition was 7.5 mm, for the MW<5 kDa fraction, it was 7 mm, and for MW 5-30 kDa fraction, it was 4.5 mm. No significant differences were found in high molecular weight proteomic profile, while qualitative and quantitative differences were observed in the medium and low molecular weight areas, especially in OCM and NCM. HPLC showed 221 tissue-specific peptides in OCM, 156 in NCM, 225 in RM, but only 5 in TM. The results observed confirmed porcine mucous tissues as a good source of antimicrobial compounds, which could be an actual alternative for reduction of microbial spoilage of foods.

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

PubMed

Song, Hyun-Ouk

2016-02-01

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica.

PubMed

Indra, D; Ramalingam, K; Babu, Mary

2005-09-01

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.

Further insights into cortactin conformational regulation

PubMed Central

Evans, Jason V; Kelley, Laura C; Hayes, Karen E; Ammer, Amanda Gatesman; Martin, Karen H

2011-01-01

The actin regulatory protein cortactin is involved in multiple signaling pathways impinging on the cortical actin cytoskeleton. Cortactin is phosphorylated by ERK1/2 and Src family tyrosine kinases, resulting in neuronal Wiskott Aldrich Syndrome protein (N-WASp) activation and enhanced actin related protein (Arp)2/3-mediated actin nucleation. Cortactin migrates as an 80/85 kDa doublet when analyzed by SDS-PAGE. Phosphorylation by ERK1/2 is associated with conversion of the 80 kDa to the 85 kDa form, postulated to occur by inducing a conformational alteration that releases the carboxyl-terminal SH3 domain from autoinhibition. Our recent analysis of the 80–85 kDa cortactin “shift” in tumor cells indicates that while ERK1/2 phosphorylation is associated with the 85 kDa shift, this phosphorylation event is not required for the shift to occur, nor does ERK1/2 phosphorylation appreciably alter global cortactin confirmation. These data indicate that additional factors besides ERK1/2 phosphorylation contribute to generating and/or maintaining the activated 85 kDa cortactin form in stimulated cells. PMID:21866257

In vivo exposure to ozone produces an increase in a 72-kDa heat shock protein in guinea pigs.

PubMed

Su, W Y; Gordon, T

1997-09-01

Although several lines of evidence have suggested that oxidizing agents can induce heat shock proteins (HSPs) in vitro, little is known about the induction of HSPs during in vivo exposure to oxidants. Guinea pigs were exposed to ozone for 6 h and euthanized up to 72 h later. Proteins from lavage cells and lung tissue were characterized by immunoblotting with 72- and 73/72-kDa HSP monoclonal antibodies. Although 73-kDa HSP was expressed constituitively in lung tissue, it was not affected by ozone. In contrast, 72-kDa HSP was significantly increased in lavage cells and lung tissue of animals exposed to 0.4 and 0.66 parts/million of ozone. Both heat treatment and arsenite induced 72-kDa HSP in cultured alveolar macrophages. The increase in 72-kDa HSP in the lavage cell pellet peaked at 24 h after ozone, whereas the influx of polymorphonuclear leukocytes peaked at 4 h. Examination of the induction of HSPs by ozone may provide clues to the development of ozone tolerance in humans and animals.

Fibroblast growth factor regulates insulin-like growth factor-binding protein production by vascular smooth muscle cells.

PubMed

Ververis, J; Ku, L; Delafontaine, P

1994-02-01

Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

Assessment of safety, efficacy, and dosimetry of a novel 18-kDa translocator protein ligand, [11C]CB184, in healthy human volunteers.

PubMed

Sakata, Muneyuki; Ishibashi, Kenji; Imai, Masamichi; Wagatsuma, Kei; Ishii, Kenji; Hatano, Kentaro; Ishiwata, Kiichi; Toyohara, Jun

2017-12-01

N,N-di-n-propyl-2-[2-(4-[ 11 C]methoxyphenyl)-6,8-dichloroimidazol[1,2-a]pyridine-3-yl]acetamide ([ 11 C]CB184) is a novel selective radioligand for the 18-kD translocator protein (TSPO), which is upregulated in activated microglia in the brain, and may be useful in positron emission tomography (PET). We examined the safety, radiation dosimetry, and initial brain imaging with [ 11 C]CB184 in healthy human volunteers. Dynamic [ 11 C]CB184 PET scans (90 min) were performed in five healthy male subjects. During the scan, arterial blood was sampled at various time intervals, and the fraction of the parent compound in plasma was determined with high-performance liquid chromatography. No serious adverse events occurred in any of the subjects throughout the study period. [ 11 C]CB184 was metabolized in the periphery: 36.7% ± 5.7% of the radioactivity in plasma was detected as the unchanged form after 60 min. The total distribution volume (V T ) was estimated with a two-tissue compartment model. The V T of [ 11 C]CB184 was highest in the thalamus (5.1 ± 0.4), followed by the cerebellar cortex (4.4 ± 0.2), and others. Although regional differences were small, the observed [ 11 C]CB184 binding pattern was consistent with the TSPO distribution in the normal human brain. Radiation dosimetry was determined in three healthy male subjects using a serial whole-body PET scan acquired over 2 h after [ 11 C]CB184 injection. [ 11 C]CB184 PET demonstrated high uptake in the gallbladder at a later time (>60 min). In urine obtained approximately 100 min post-injection, 0.3% of the total injected radioactivity was recovered, indicating hepatobiliary excretion of radioactivity. The absorbed dose (μGy/MBq) was highest in the kidneys (21.0 ± 0.5) followed by the lungs (16.8 ± 2.7), spleen (16.6 ± 6.6), and pancreas (16.5 ± 2.2). The estimated effective dose for [ 11 C]CB184 was 5.9 ± 0.6 μSv/MBq. This initial evaluation indicated that [ 11 C]CB184 is feasible for imaging of TSPO in the brain.

Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

PubMed

Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

2015-04-01

Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

Embryogenesis-promoting factors in rat serum.

PubMed

Katoh, M; Kimura, R; Shoji, R

1998-06-15

Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

Peptide-based Antibodies against Glutathione-binding Domains Suppress Superoxide Production Mediated by Mitochondrial Complex I*

PubMed Central

Chen, Jingfeng; Chen, Chwen-Lih; Rawale, Sharad; Chen, Chun-An; Zweier, Jay L.; Kaumaya, Pravin T. P.; Chen, Yeong-Renn

2010-01-01

Complex I (NQR) is a critical site of superoxide () production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain (200GAGAYICGEETALIESIEGK219 of 51-kDa protein and 361VDSDTLCTEEVFPTAGAGTDLR382 of 75-kDa protein) as chimeric epitopes incorporating a “promiscuous” T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain (21SGDTTAPKKTSFGSLKDFDR40 of 51-kDa peptide and 100WNILTNSEKTKKAREGVMEFL120 of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated generation to a significant level. However, binding of Ab75 inhibited NQR-mediated generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction. PMID:19940158

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

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

Karttunen, Jenni; Mäntynen, Sari; Ihalainen, Teemu O.

2015-08-15

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of themore » P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.« less

Upscaling the Use of Mixed Recycled Aggregates in Non-Structural Low Cement Concrete

PubMed Central

López-Uceda, Antonio; Ayuso, Jesús; Jiménez, José Ramón; Agrela, Francisco; Barbudo, Auxiliadora; De Brito, Jorge

2016-01-01

This research aims to produce non-structural concrete with mixed recycled aggregates (MRA) in upscaled applications with low-cement content. Four slabs were executed with concrete made with different ratios of coarse MRA (0%, 20%, 40% and 100%), using the mix design, the mixing procedures and the facilities from a nearby concrete production plant. The analysis of the long-term compressive and splitting tensile strengths in concrete cores, extracted from the slabs, allowed the highlighting of the long-term high strength development potential of MRA incorporation. The study of cast specimens produced in situ under the same conditions as the slabs showed, firstly, that the use of MRA has a great influence on the properties related to durability, secondly, that the loss of compressive strength for total MRA incorporation relative to control concrete increases proportionally with the class strength, and, thirdly, that the mechanical properties (including Schmidt hammer results) from the concrete slabs showed no significant differences relative to the control concrete for coarse aggregates replacements up to 40%. Therefore, this upscaled experimental study supports the application of concrete with 100% coarse MRA incorporation and low cement content in non-structural civil works such as bike lanes, gutters, ground slabs, leveling surfaces, and subgrades for foundations. To the best of the authors’ knowledge, there have not been any upscaled applications of concrete with MRA and low cement content. PMID:28787892

Upscaling the Use of Mixed Recycled Aggregates in Non-Structural Low Cement Concrete.

PubMed

López-Uceda, Antonio; Ayuso, Jesús; Jiménez, José Ramón; Agrela, Francisco; Barbudo, Auxiliadora; De Brito, Jorge

2016-02-02

This research aims to produce non-structural concrete with mixed recycled aggregates (MRA) in upscaled applications with low-cement content. Four slabs were executed with concrete made with different ratios of coarse MRA (0%, 20%, 40% and 100%), using the mix design, the mixing procedures and the facilities from a nearby concrete production plant. The analysis of the long-term compressive and splitting tensile strengths in concrete cores, extracted from the slabs, allowed the highlighting of the long-term high strength development potential of MRA incorporation. The study of cast specimens produced in situ under the same conditions as the slabs showed, firstly, that the use of MRA has a great influence on the properties related to durability, secondly, that the loss of compressive strength for total MRA incorporation relative to control concrete increases proportionally with the class strength, and, thirdly, that the mechanical properties (including Schmidt hammer results) from the concrete slabs showed no significant differences relative to the control concrete for coarse aggregates replacements up to 40%. Therefore, this upscaled experimental study supports the application of concrete with 100% coarse MRA incorporation and low cement content in non-structural civil works such as bike lanes, gutters, ground slabs, leveling surfaces, and subgrades for foundations. To the best of the authors' knowledge, there have not been any upscaled applications of concrete with MRA and low cement content.

The influence of elevated CO2 on non-structural carbohydrate distribution and fructan accumulation in wheat canopies

NASA Technical Reports Server (NTRS)

Smart, D. R.; Chatterton, N. J.; Bugbee, B.

1994-01-01

We grew 2.4 m2 wheat canopies in a large growth chamber under high photosynthetic photon flux (1000 micromoles m-2 s-1) and using two CO2 concentrations, 360 and 1200 micromoles mol-1. Photosynthetically active radiation (400-700 nm) was attenuated slightly faster through canopies grown in 360 micromoles mol-1 than through canopies grown in 1200 micromoles mol-1, even though high-CO2 canopies attained larger leaf area indices. Tissue fractions were sampled from each 5-cm layer of the canopies. Leaf tissue sampled from the tops of canopies grown in 1200 micromoles mol-1 accumulated significantly more total non-structural carbohydrate, starch, fructan, sucrose, and glucose (p < 0.05) than for canopies grown in 360 micromoles mol-1. Non-structural carbohydrate did not significantly increase in the lower canopy layers of the elevated CO2 treatment. Elevated CO2 induced fructan synthesis in all leaf tissue fractions, but fructan formation was greatest in the uppermost leaf area. A moderate temperature reduction of 10 degrees C over 5 d increased starch, fructan and glucose levels in canopies grown in 1200 micromoles mol-1, but concentrations of sucrose and fructose decreased slightly or remained unchanged. Those results may correspond with the use of fructosyl-residues and release of glucose when sucrose is consumed in fructan synthesis.

Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

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

Perkins, J; Parida, S; Clavijo, A

2007-05-14

Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright,more » UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.« less

High Molecular Weight Isoforms of Growth Hormone In Cells of the Immune System

PubMed Central

Weigent, Douglas A.

2013-01-01

A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100 kDa, 65 kDa, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 kDa and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm whereas the lower molecular weight 65 kDa and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost three-fold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress. PMID:21741628

The C-terminal domain of Nrf1 negatively regulates the full-length CNC-bZIP factor and its shorter isoform LCR-F1/Nrf1β; both are also inhibited by the small dominant-negative Nrf1γ/δ isoforms that down-regulate ARE-battery gene expression.

PubMed

Zhang, Yiguo; Qiu, Lu; Li, Shaojun; Xiang, Yuancai; Chen, Jiayu; Ren, Yonggang

2014-01-01

The C-terminal domain (CTD, aa 686-741) of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1) shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L) CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein) and its shorter isoform LCR-F1/Nrf1β (55-kDa). Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112Nrf1 factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER) membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1β and Nrf2-C112Nrf1, results in an increase in their transcriptional ability to regulate antioxidant response element (ARE)-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1γ and 25-kDa Nrf1δ, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1β and Nrf2. Relative to Nrf1, LCR-F1/Nrf1β is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST) domain and acidic domain 2 (AD2). Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1β fused within two different chimaeric contexts to yield Gal4D:Nrf1β607 and Nrf1β:C270Nrf2, positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1β and down-regulation by Nrf1γ and Nrf1δ.

The analysis Arabidopsis thaliana overexpressing a 14kDa self-folding protein [abstract

USDA-ARS?s Scientific Manuscript database

A recent study in banana identified a 14kDa protein that has been hypothesized to function in regulating the nucleation and growth of the needle-shaped crystals of calcium oxalate that accumulate within the tissues of this plant. To gain further insight in to the functional role of this 14 kDa prote...

Physicochemical stability and in vitro bioaccessibility of ß-carotene nanoemulsions stabilized with whey protein-dextran conjugates

USDA-ARS?s Scientific Manuscript database

In this study, ß-carotene (BC)-loaded nanoemulsions encapsulated with native whey protein isolate (WPI) and WPI-dextran (DT, 5 kDa, 20 kDa, and 70 kDa) conjugates were prepared and the effects of glycosylation with various molecular weight DTs on the physicochemical property, lipolysis, and BC bioac...

Surface Layer Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (SL-MALDI-ToF-MS) Analysis of Polymer Blend Surface Composition

NASA Astrophysics Data System (ADS)

Hill, Jacob A.

The composition of a polymer blend is generally different at the surface than in the bulk and the gradient in composition with depth has important implications for surface properties. The determination of the surface composition presents various challenges which continue to prompt the development of new techniques for quantifying the composition. Here the technique of surface layer matrix-assisted laser desorption ionization time-of-flight mass spectrometry (SL-MALDI-ToF-MS) has been further developed to address four specific questions of polymer blend surface behavior within the general category of surface composition determination. The first question is how chain length disparity affects surface segregation in the case that the disparity is quite small. While such segregation is known for blends containing low molecular weight additives or systems with large polydispersity, it has not been reported for anionically polymerized polymers that are viewed, in practice, as monodisperse. For 6 kDa polystyrene the number average molecular weight (Mn) at the surface is ca. 300 Da (5%) lower than that in the bulk and for 7 kDa polymethyl methacrylate the shift is ca. 500 Da. The second question is how chain-end functionalization effects not the location of chain ends at the surface, but rather the prevalence at the surface of any part of a chain having an end functionalization. A key issue of such an approach is understanding precisely where the functionalities ultimately reside and how this functionalization shifts the balance of forces that determine the distribution of chains with depth. The surface of a blend of 6 kDa polystyrene and 6 kDa polystyrene functionalized with hydroxymethyl ends is not only depleted of the higher energy end groups, but is depleted of any segments belonging to the functionalized chains. This is demonstrated using SL-MALDI-ToF-MS, which detects entire chains that have any repeat unit at the outer surface, and requires no labelling. This study was extended to the surfaces of blends of 6 kDa polystyrene and 6 kDa polystyrene functionalized with hydroxyethyl ends. Blends of all compositions less than 90 wt. % functionalized chains showed depletion. Finally, the challenge of determining lateral variations in the surface composition has been addressed with the development of SL-MALDI-ToF-MS imaging (SL-MALDI-ToF-MSI). Key to developing imaging capability was improving the lateral uniformity of the matrix deposition. This uniformity was achieved using solvent free sublimation of matrix and salt onto the sample's surface. The capabilities of SL-MALDI-ToF-MSI were demonstrated by imaging the absence of material due to masking during material deposition, mechanical scribing or solvent perturbation at the surface of low molecular weight poly(methyl methacrylate) and polystyrene thin films. SL-MALDI-ToF MSI was made possible through the first uniform, solvent free simultaneous sublimation of matrix and salt onto the material's surface.

Highlighting the wide variability in arsenic speciation in wetlands: A new insight into the control of the behavior of arsenic

NASA Astrophysics Data System (ADS)

Guénet, Hélène; Davranche, Mélanie; Vantelon, Delphine; Bouhnik-Le Coz, Martine; Jardé, Emilie; Pierson-Wickmann, Anne-Catherine; Dorcet, Vincent; Jestin, Jacques

2017-04-01

Although the behavior of Arsenic (As) under reducing conditions in periods of high water levels in wetlands is well understood and documented, there is a lack of information under oxidizing conditions when the water level decreases. In this study, we were interested in the first stage of the oxidizing period, when oxidation products are still in suspension. A soil sample from the Naizin Kervidy wetland (France) was incubated in the laboratory to produce a reduced soil solution. The reduced solution was subsequently oxidized, filtered and ultrafiltered using decreasing pore size membranes (5 μm, 3 μm, 0.2 μm, 30 kDa and 5 kDa). The distribution of As and Fe was investigated in each size fraction of the oxidized solution and their speciations were studied using XAS, HPLC and SEC-ICP-MS. Organic matter was characterized using thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry (THM-GC-MS) and fluorescence spectroscopy. The majority of the As was present as As(V) but a small amount of As(III) still remained despite the advanced oxidized conditions. In the >0.2 μm fractions, the XAS analyses showed that As was associated, in the second shell, with Fe (As-Fe = 3.35 Å) as bidentate binuclear complexes and C (As-C = 2.90 Å), suggesting the integration of As in biological objects. In the <30 kDa fraction, As was directly bound to C (As-C = 1.96 Å) in the first shell indicating the presence of organic As species. In the second shell, an As-Fe distance of 3.35 Å was found showing that part of the As was still complexed with Fe. The 0.2 μm-30 kDa fraction was a transitional fraction in terms of the Fe species and OM composition. In this fraction, organic matter exhibited a more humic character (aromatic molecules) inducing an increasing cation binding capacity. As a consequence, in this fraction and in the smallest one, As, Fe and OM seemed to form ternary complexes in which the Fe or nano-oxides in the >30 kDa fraction and as monomer, or cluster in <30 kDa fraction acted as a bridge. In all of the fractions, a proportion of As(V) was present as organic methylated species. These organic species might be produced by several organisms (animal or plant) via a detoxification process. They seemed to be bound to the particulate and colloidal Fe/OM phases as well as integrated in the remains of the organisms. Mass calculations provided evidence that 90% of the As was contained in the >5 μm particulate fraction and thus was hardly mobile. This study showed that although wetlands have been identified as a potential source of As, a number of biological and geochemical trapping mechanisms also favor As stabilization in wetlands.

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

PubMed Central

Chen, Chwen-Lih; Zhang, Liwen; Yeh, Alexander; Chen, Chun-An; Green-Church, Kari B.; Zweier, Jay L.; Chen, Yeong-Renn

2008-01-01

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2·-) and O2·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2·- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool. PMID:17444656

18 CFR 415.2 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... part, except as otherwise required by the context: Project means the same word as defined by section 1... combination of structural and nonstructural additions, changes, or adjustments to properties and structures...

Abnormal uterine bleeding unrelated to structural uterine abnormalities: management in the perimenopausal period.

PubMed

Sabbioni, Lorenzo; Zanetti, Isabella; Orlandini, Cinzia; Petraglia, Felice; Luisi, Stefano

2017-02-01

Abnormal uterine bleeding (AUB) is one of the commonest health problems encountered by women and a frequent phenomenon during menopausal transition. The clinical management of AUB must follow a standardized classification system to obtain the better diagnostic pathway and the optimal therapy. The PALM-COEIN classification system has been approved by the International Federation of Gynecology and Obstetrics (FIGO); it recognizes structural causes of AUB, which can be measured visually with imaging techniques or histopathology, and non-structural entities such as coagulopathies, ovulatory dysfunctions, endometrial and iatrogenic causes and disorders not yet classified. In this review we aim to evaluate the management of nonstructural causes of AUB during the menopausal transition, when commonly women experience changes in menstrual bleeding patterns and unexpected bleedings which affect their quality of life.

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

PubMed Central

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

Multiple polypeptides immunologically related to beta-poly(L-malate) hydrolase (polymalatase) in the plasmodium of the slime mold Physarum polycephalum.

PubMed

Karl, M; Holler, E

1998-01-15

Plasmodia of Physarum polycephalum contain large amounts of the cell-type-specific polyanion beta-poly(L-malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68-kDa form giving rise to a 54-kDa form when deglycosylated, and the 97-kDa form giving rise to an 88-kDa polypeptide that was indistinguishable from an 88-kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two-dimensional non-denaturing in the first dimension and SDS-denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200-kDa 'precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular-mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of beta-poly(L-malate) from the nucleus to the culture medium.

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough*

PubMed Central

Yamaguchi, Yoshihide; Hayashi, Akiko; Campagnoni, Celia W.; Kimura, Akio; Inuzuka, Takashi; Baba, Hiroko

2012-01-01

Myelin protein zero (P0 or MPZ) is a major myelin protein (∼30 kDa) expressed in the peripheral nervous system (PNS) in terrestrial vertebrates. Several groups have detected a P0-related 36-kDa (or 35-kDa) protein that is expressed in the PNS as an antigen for the serum IgG of patients with neuropathy. The molecular structure and function of this 36-kDa protein are, however, still unknown. We hypothesized that the 36-kDa protein may be derived from P0 mRNA by stop codon readthrough. We found a highly conserved region after the regular stop codon in predicted sequences from the 3′-UTR of P0 in higher animals. MS of the 36-kDa protein revealed that both P0 peptides and peptides deduced from the P0 3′-UTR sequence were found among the tryptic fragments. In transfected cells and in an in vitro transcription/translation system, the 36-kDa molecule was also produced from the identical mRNA that produced P0. We designated this 36-kDa molecule as large myelin protein zero (L-MPZ), a novel isoform of P0 that contains an additional domain at the C terminus. In the PNS, L-MPZ was localized in compact myelin. In transfected cells, just like P0, L-MPZ was localized at cell-cell adhesion sites in the plasma membrane. These results suggest that L-MPZ produced by the stop codon readthrough mechanism is potentially involved in myelination. Since this is the first finding of stop codon readthrough in a common mammalian protein, detailed analysis of L-MPZ expression will help to understand the mechanism of stop codon readthrough in mammals. PMID:22457349

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

PubMed

Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

Formation of Hirano Bodies Induced by Expression of an Actin Cross-Linking Protein with a Gain-of-Function Mutation

PubMed Central

Maselli, Andrew; Furukawa, Ruth; Thomson, Susanne A. M.; Davis, Richard C.; Fechheimer, Marcus

2003-01-01

Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. PMID:12912897

Anti-brain antibodies are associated with more severe cognitive and behavioral profiles in Italian children with Autism Spectrum Disorder

PubMed Central

Piras, I.S.; Haapanen, L.; Napolioni, V.; Sacco, R.; Van de Water, J.; Persico, A.M.

2014-01-01

Circulating 45 and 62 kDa antibodies targeting the cerebellum were previously associated with Autism Spectrum Disorder (ASD), lower adaptive/cognitive function and aberrant behaviors. Moreover, 37, 39 and 73 kDa maternal antibodies (mAb) targeting the fetal brain were previously correlated with broad autism spectrum, irritability, abnormal brain enlargement and impaired expressive language. The present study aims towards clinically characterizing individuals with brain-targeted IgG and/or exposed to maternal antibrain antibodies in a large sample of Italian autistic children (N = 355), their unaffected siblings (N = 142) and mothers (N = 333). The presence of patient- and mother-produced anti-brain antibodies does not confer increased risk of autism within the same sibship. However, the 45 and 62 kDa antibodies are correlated with autism severity: the 45 kDa Ab is associated with cognitive impairment and lower scores at the Vineland Adaptive Behavior Scales, the 62 kDa Ab with motor stereotypies, while both correlate with larger head circumference (all P < 0.05). On the other hand, maternal 37, 39 and 73 kDa antibrain antibodies, either alone or in combination, are correlated with impaired verbal and non-verbal language development, neurodevelopmental delay and sleep/wake cycle disturbances in their autistic children (P < 0.05). Presence of the 62 kDa autoAb in the child is significantly associated with presence of the 39 and/or 73 kDa antibodies in his/her mother. Our results confirm and extend previous observations in an ethnically distinct sample, providing further evidence of a pathomorphic role for antibrain antibodies in autism while demonstrating their familial clustering. PMID:24389156

Anti-brain antibodies are associated with more severe cognitive and behavioral profiles in Italian children with Autism Spectrum Disorder.

PubMed

Piras, I S; Haapanen, L; Napolioni, V; Sacco, R; Van de Water, J; Persico, A M

2014-05-01

Circulating 45 and 62kDa antibodies targeting the cerebellum were previously associated with Autism Spectrum Disorder (ASD), lower adaptive/cognitive function and aberrant behaviors. Moreover, 37, 39 and 73kDa maternal antibodies (mAb) targeting the fetal brain were previously correlated with broad autism spectrum, irritability, abnormal brain enlargement and impaired expressive language. The present study aims towards clinically characterizing individuals with brain-targeted IgG and/or exposed to maternal antibrain antibodies in a large sample of Italian autistic children (N=355), their unaffected siblings (N=142) and mothers (N=333). The presence of patient- and mother-produced anti-brain antibodies does not confer increased risk of autism within the same sibship. However, the 45 and 62kDa antibodies are correlated with autism severity: the 45kDa Ab is associated with cognitive impairment and lower scores at the Vineland Adaptive Behavior Scales, the 62kDa Ab with motor stereotypies, while both correlate with larger head circumference (all P<0.05). On the other hand, maternal 37, 39 and 73kDa antibrain antibodies, either alone or in combination, are correlated with impaired verbal and non-verbal language development, neurodevelopmental delay and sleep/wake cycle disturbances in their autistic children (P<0.05). Presence of the 62kDa autoAb in the child is significantly associated with presence of the 39 and/or 73kDa antibodies in his/her mother. Our results confirm and extend previous observations in an ethnically distinct sample, providing further evidence of a pathomorphic role for anti-brain antibodies in autism while demonstrating their familial clustering. Copyright © 2014 Elsevier Inc. All rights reserved.

Mass spectrometric identification of Au68(SR)34 molecular gold nanoclusters with 34-electron shell closing.

PubMed

Dass, Amala

2009-08-26

The molecular formula Au(68)(SCH(2)CH(2)Ph)(34) has been assigned to the 14 kDa nanocluster using MALDI-TOF mass spectrometry. The 34-electron shell closing in a macroscopically obtained thiolated gold nanocluster is demonstrated. The Au(68) nanocluster is predicted to have a 49 atom Marks decahedral core with 19 inner core atoms and 30 outer atoms chelating with the staple motifs. The nanoclusters' predicted formulation is [Au](19+30) [Au(SR)(2)](11) [Au(2)(SR)(3)](4).