A novel M1 PAM VU0486846 exerts efficacy in cognition models without displaying agonist activity or cholinergic toxicity.

Science.gov (United States)

Rook, Jerri M; Bertron, Jeanette L; Cho, Hyekyung P; Garcia-Barrantes, Pedro M; Moran, Sean P; Maksymetz, James T; Nance, Kellie D; Dickerson, Jonathan W; Remke, Daniel H; Chang, Sichen; Harp, Joel; Blobaum, Anna L; Niswender, Colleen M; Jones, Carrie K; Stauffer, Shaun R; Conn, P Jeffrey; Lindsley, Craig W

2018-04-27

Selective activation of the M1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M1, leading to agonist actions in the prefrontal cortex (PFC) that impairs cognitive function, induces behavioral convulsions, and results in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M1-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427 and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [3H]-NMS binding at theorthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M1 PAM activity (EC50s > 100 nM) and pure-PAM activity in native tissues display robust pro-cognitive efficacy without AEs mediated by excessive activation of M1. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: synthesis and biological properties.

Science.gov (United States)

Thomann, Jean-Sébastien; Monneaux, Fanny; Creusat, Gaëlle; Spanedda, Maria Vittoria; Heurtault, Béatrice; Habermacher, Chloé; Schuber, Francis; Bourel-Bonnet, Line; Frisch, Benoît

2012-05-01

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/β ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

Science.gov (United States)

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

OpenAIRE

Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

Science.gov (United States)

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

2006-05-01

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

Comparative molecular dynamics study of neuromyelitis optica-immunoglobulin G binding to aquaporin-4 extracellular domains.

Science.gov (United States)

Alberga, Domenico; Trisciuzzi, Daniela; Lattanzi, Gianluca; Bennett, Jeffrey L; Verkman, Alan S; Mangiatordi, Giuseppe Felice; Nicolotti, Orazio

2017-08-01

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which most patients have serum autoantibodies (called NMO-IgG) that bind to astrocyte water channel aquaporin-4 (AQP4). A potential therapeutic strategy in NMO is to block the interaction of NMO-IgG with AQP4. Building on recent observation that some single-point and compound mutations of the AQP4 extracellular loop C prevent NMO-IgG binding, we carried out comparative Molecular Dynamics (MD) investigations on three AQP4 mutants, TP 137-138 AA, N 153 Q and V 150 G, whose 295-ns long trajectories were compared to that of wild type human AQP4. A robust conclusion of our modeling is that loop C mutations affect the conformation of neighboring extracellular loop A, thereby interfering with NMO-IgG binding. Analysis of individual mutations suggested specific hydrogen bonding and other molecular interactions involved in AQP4-IgG binding to AQP4. Copyright © 2017 Elsevier B.V. All rights reserved.

SD-PAMs: What, Where, When and How?

Energy Technology Data Exchange (ETDEWEB)

NONE

2007-07-01

This paper examines what ''sustainable development policies and measures'' (SD-PAMs) could be, and how they could be implemented and could fit into a post-2012 climate regime. This paper assumes that the option to implement SD-PAMs instead of quantified GHG emission commitments post-2012 is an option that would be likely to be only open to non-Annex I countries. There are several key, but unanswered, questions related to SD-PAMs. These include policy-related issues such as which countries could take on commitments to implement SD-PAMs (rather than quantified emission commitments)? Why would particular countries decide to take on such commitments? They also include questions related to how SD-PAMs could be implemented. For many other options for possible post-2012 GHG mitigation actions, including by non-Annex I countries, have also been proposed. However, this paper focuses solely on SD-PAMs.

Deciphering, Communicating, and Engineering the CRISPR PAM.

Science.gov (United States)

Leenay, Ryan T; Beisel, Chase L

2017-01-20

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their flanking CRISPR-associated (cas) genes make up RNA-guided, adaptive immune systems in prokaryotes whose effector proteins have become powerful tools for basic research and biotechnology. While the Cas effector proteins are remarkably diverse, they commonly rely on protospacer-adjacent motifs (PAMs) as the first step in target recognition. PAM sequences are known to vary considerably between systems and have proven to be difficult to predict, spurring the need for new tools to rapidly identify and communicate these sequences. Recent advances have also shown that Cas proteins can be engineered to alter PAM recognition, opening new opportunities to develop CRISPR-based tools with enhanced targeting capabilities. In this review, we discuss the properties of the CRISPR PAM and the emerging tools for determining, visualizing, and engineering PAM recognition. We also propose a standard means of orienting the PAM to simplify how its location and sequence are communicated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pro-2-PAM therapy for central and peripheral cholinesterases.

Science.gov (United States)

Demar, James C; Clarkson, Edward D; Ratcliffe, Ruthie H; Campbell, Amy J; Thangavelu, Sonia G; Herdman, Christine A; Leader, Haim; Schulz, Susan M; Marek, Elizabeth; Medynets, Marie A; Ku, Therese C; Evans, Sarah A; Khan, Farhat A; Owens, Roberta R; Nambiar, Madhusoodana P; Gordon, Richard K

2010-09-06

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for

Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

Science.gov (United States)

Leenay, Ryan T; Maksimchuk, Kenneth R; Slotkowski, Rebecca A; Agrawal, Roma N; Gomaa, Ahmed A; Briner, Alexandra E; Barrangou, Rodolphe; Beisel, Chase L

2016-04-07

CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. Copyright © 2016 Elsevier Inc. All rights reserved.

The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

Science.gov (United States)

Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes

2015-01-15

Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.

Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter

DEFF Research Database (Denmark)

Plenge, Per; Shi, Lei; Beuming, Thijs

2012-01-01

be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory...

Interference by pralidoxime (PAM) salts in clinical laboratory tests.

Science.gov (United States)

Nagase, Sumika; Kohguchi, Katsunori; Tohyama, Kaoru; Watanabe, Mikio; Iwatani, Yoshinori

2013-02-01

Drugs sometimes alter the results of clinical laboratory tests. We examined the effects of pralidoxime (PAM) salts, a medicine used to treat organophosphorus poisoning, on clinical laboratory test results for the first time. The effects of PAM salts on glucose (GLU) measurements were examined using a point-of-care testing (POCT) meter, four self-monitoring of blood glucose (SMBG) meters, and two biochemical autoanalyzers. The effects of PAM salts on other clinical tests were also evaluated. The addition of PAM iodide or potassium iodide, but not of PAM chloride or potassium chloride, to blood samples increased the GLU values measured by one POCT meter and 4 SMBG meters using the enzyme electrode (hydrogen peroxidase or oxygen electrode) method. On the other hand, PAM iodide or PAM chloride, but not KI or KCl, affected the values measured at 340 nm by an autoanalyzer using absorption spectrophotometry in 8 of 14 clinical laboratory tests. The absorption spectrum of PAM changed from 294 to 338 nm due to the reaction between PAM and the alkaline buffer, a component of the measuring reagents. PAM iodide increases the GLU values measured by the enzyme electrode method, and PAM salts affected the values measured at 340 nm by absorption spectrophotometry in many other clinical test items. Copyright © 2012 Elsevier B.V. All rights reserved.

The Proteolytically Stable Peptidomimetic Pam-(Lys-ßNSpe)6-NH2 Selectively Inhibits Human Neutrophil Activation via Formyl Peptide Receptor 2

DEFF Research Database (Denmark)

Skovbakke, Sarah Line; Heegaard, Peter M. H.; Larsen, Camilla J.

2015-01-01

of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release...... of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogues of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging...... flow cytometry in primary neutrophils and FPR-transfected cell lines we found that a fluorescently labelled analogue of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating...

Psoriatic arthritis mutilans (PAM) in the Nordic countries: demographics and disease status. The Nordic PAM study.

Science.gov (United States)

Gudbjornsson, B; Ejstrup, L; Gran, J T; Iversen, L; Lindqvist, U; Paimela, L; Ternowitz, T; Ståhle, M

2013-01-01

To determine the prevalence and clinical characteristics of psoriatic arthritis mutilans (PAM) in the Nordic countries. Patients with putative PAM aged ≥ 18 years were recruited. Fifty-nine patients were included after clinical examination. The prevalence of PAM in the adult Nordic population was estimated to be 3.69 per million inhabitants [95% confidence interval (CI) 2.75-4.63]. The female to male ratio was close to 1:1. The mean age of skin disease onset was 25 years and the mean age of onset of joint disease was 30 years. The onset of skin disease was 2 years earlier among female patients. At inclusion, the mean duration of arthritis was 27 ± 11 years for male patients and 33 ± 11 years for female patients. PAM was most frequently seen in the distal interphalangeal (DIP) joints of the toes, followed by the IP joint of the thumb and the DIP joint of the little finger on the left hand. Female and male patients had similar numbers of painful and swollen joints. Enthesitis was found in 19 patients (32%), while 38 patients (64%) had a history of dactylitis. Twenty-three of these 38 patients (61%) had a history of dactylitis in the same finger/toe as they had PAM. At the time of inclusion, 45% of the patients were found to have clear or almost clear skin. PAM in the Nordic countries has a low prevalence, with only three to five cases per million inhabitants. The majority of the patients present with mild skin disease.

Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes

Science.gov (United States)

Hiraki, Masahiko; Watanabe, Shokei; Chavas, Leonard M. G.; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Wakatsuki, Soichi; Fujihashi, Masahiro; Miki, Kunio; Baba, Seiki; Ueno, Go; Yamamoto, Masaki; Suzuki, Mamoru; Nakagawa, Atsushi; Watanabe, Nobuhisa; Tanaka, Isao

2010-06-01

To remotely control and automatically collect data in high-throughput X-ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL-5A, BL-17A, AR-NW12A and AR-NE3A. We developed and installed software that manages the flow of the automated X-ray experiments; sample exchanges, loop-centering and X-ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two-dimensional bar code reader. New beamlines, BL-1A at the Photon Factory and BL32XU at SPring-8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), will be installed at BL-1A and BL32XU, respectively. For the convenience of the users of both facilities, pins and cassettes for PAM and SPACE are developed as part of the TPRP.

Electrochemical examination of ability of dsDNA/PAM composites for storing and releasing of doxorubicin.

Science.gov (United States)

Zabost, Ewelina; Liwinska, Wioletta; Karbarz, Marcin; Kurek, Eliza; Lyp, Marek; Donten, Mikolaj; Stojek, Zbigniew

2016-06-01

Composites consisting of ss- and ds-DNA strands and polyacrylamide (PAM) hydrogel have been synthesized. DNA was entrapped non-covalently. The obtained DNA biomaterial exhibited a strong increase in guanine and adenine anodic currents when temperature reached the physiological level. This increase was related to the unique oligonucleotide structural changes in the composite. The structural alterations in the PAM lattices were employed for the release of the drug accumulated in the composite. Doxorubicin (Dox) was selected as the drug; it was accumulated by intercalation to dsDNA and was slowly released from the dsDNA/PAM system by using a minor temperature increase (up to 40÷45 °C) as it is routinely done in hyperthermia. The applied release temperature was either constant or oscillating. The binding strength, the rate of Dox release and the properties of the composite were examined using voltammetry, SEM and ICP-MS. Copyright © 2015 Elsevier B.V. All rights reserved.

PAM-1616, a selective peroxisome proliferator-activated receptor γ modulator with preserved anti-diabetic efficacy and reduced adverse effects.

Science.gov (United States)

Kim, Mi-Kyung; Chae, Yu Na; Choi, Song-hyen; Moon, Ho Sang; Son, Moon-Ho; Bae, Myung-Ho; Choi, Hyun-ho; Hur, Youn; Kim, Eunkyung; Park, Yoo Hoi; Park, Chan Sun; Kim, Jae Gyu; Lim, Joong In; Shin, Chang Yell

2011-01-15

Peroxisome proliferator-activated receptor (PPAR) γ is known to be a key regulator of insulin resistance. PAM-1616 is a novel, non-thiazolidinedione small molecule compound synthesized in Dong-A Research Center. In this study, we characterized the pharmacological and safety profiles of PAM-1616 as a selective PPARγ modulator. PAM-1616 selectively binds to human PPARγ (IC(50), 24.1±5.6 nM) and is a partial agonist for human PPARγ with an EC(50) of 83.6±43.7 nM and a maximal response of 24.9±7.1% relative to the full agonist, rosiglitazone. PAM-1616 was selective for human PPARγ than for human PPARα (EC(50), 2658±828 nM) without activating human PPARδ, which makes it a selective modulator of PPARγ. Treatment of high fat diet-induced obese C57BL/6J mice with PAM-1616 for 21 days improved HOMA-IR. Furthermore, PAM-1616 significantly improved hyperglycemia in db/db mice with little side effect when orally administered at a dose of 1 mg/kg/day for 28 days. Intriguingly, PAM-1616 was seen to increase the gene expression of inducible glucose transporter (GLUT4), while it partially induced that of a fatty acid carrier, aP2 in 3T3-L1 adipocytes, and it also showed partial recruitment of an adipogenic cofactor, TRAP220 as compared to rosiglitazone. PAM-1616 did not cause a significant increase in plasma volume of ICR mice when orally administered at a dose of 10 mg/kg/day for 9 days. PAM-1616 increased the expression of fluid retention-inducing genes such as serum/glucocorticoid-regulated kinase (SGK)-1 to a lesser extent as compared to rosiglitazone in human renal epithelial cells. These results suggest that PAM-1616 acts as a selective modulator of PPARγ with excellent antihyperglycemic property. The differential modulation of target gene by PAM-1616 might contribute to the improved side effect profiles. Copyright © 2010 Elsevier B.V. All rights reserved.

[Research progress on wind erosion control with polyacrylamide (PAM).

Science.gov (United States)

Li, Yuan Yuan; Wang, Zhan Li

2016-03-01

Soil wind erosion is one of the main reasons for soil degradation in the northwest region of China. Polyacrylamide (PAM), as an efficient soil amendment, has gained extensive attention in recent years since it is effective in improving the structure of surface soil due to its special physical and chemical properties. This paper introduced the physical and chemical properties of PAM, reviewed the effects of PAM on soil wind erosion amount and threshold wind velocity, as well as the effect differences of PAM in soil wind erosion control under conditions of various methods and doses. Its effect was proved by comparing with other materials in detail. Furthermore, we analyzed the mecha-nism of wind erosion control with PAM according to its influence on soil physical characteristics. Comprehensive analysis showed that, although some problems existed in wind erosion control with (PAM), PAM as a sand fixation agent, can not only enhance the capacity of the soil resis-tance to wind erosion, but also improve soil physical properties to form better soil conditions. Besides, we proposed that combination of PAM and plant growth would increase the survival rate of plants greatly, control soil wind erosion in wind-erosive areas, and improve the quality of the ecological environment construction. Thus, PAM has practically important significance and wide application prospect in controlling soil wind erosion.

Plasma-activated medium (PAM) kills human cancer-initiating cells.

Science.gov (United States)

Ikeda, Jun-Ichiro; Tanaka, Hiromasa; Ishikawa, Kenji; Sakakita, Hajime; Ikehara, Yuzuru; Hori, Masaru

2018-01-01

Medical non-thermal plasma (NTP) treatments for various types of cancers have been reported. Cells with tumorigenic potential (cancer-initiating cells; CICs) are few in number in many types of tumors. CICs efficiently eliminate anti-cancer chemicals and exhibit high-level aldehyde dehydrogenase (ALDH) activity. We previously examined the effects of direct irradiation via NTP on cancer cells; even though we targeted CICs expressing high levels of ALDH, such treatment affected both non-CICs and CICs. Recent studies have shown that plasma-activated medium (PAM) (culture medium irradiated by NTP) selectively induces apoptotic death of cancer but not normal cells. Therefore, we explored the anti-cancer effects of PAM on CICs among endometrioid carcinoma and gastric cancer cells. PAM reduced the viability of cells expressing both low and high levels of ALDH. Combined PAM/cisplatin appeared to kill cancer cells more efficiently than did PAM or cisplatin alone. In a mouse tumor xenograft model, PAM exerted an anti-cancer effect on CICs. Thus, our results suggest that PAM effectively kills both non-CICs and CICs, as does NTP. Therefore, PAM may be a useful new anti-cancer therapy, targeting various cancer cells including CICs. © 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

DEFF Research Database (Denmark)

Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa

2002-01-01

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...

Biological applications of an LCoS-based programmable array microscope (PAM)

Science.gov (United States)

Hagen, Guy M.; Caarls, Wouter; Thomas, Martin; Hill, Andrew; Lidke, Keith A.; Rieger, Bernd; Fritsch, Cornelia; van Geest, Bert; Jovin, Thomas M.; Arndt-Jovin, Donna J.

2007-02-01

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS) spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging (FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible. The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins such as DRONPA.

Augmenting the spectral efficiency of enhanced PAM-DMT-based optical wireless communications.

Science.gov (United States)

Islim, Mohamed Sufyan; Haas, Harald

2016-05-30

The energy efficiency of pulse-amplitude-modulated discrete multitone modulation (PAM-DMT) decreases as the modulation order of M-PAM modulation increases. Enhanced PAM-DMT (ePAM-DMT) was proposed as a solution to the reduced energy efficiency of PAM-DMT. This was achieved by allowing multiple streams of PAM-DMT to be superimposed and successively demodulated at the receiver side. In order to maintain a distortion-free unipolar ePAM-DMT system, the multiple time-domain PAM-DMT streams are required to be aligned. However, aligning the antisymmetry in ePAM-DMT is complex and results in efficiency losses. In this paper, a novel simplified method to apply the superposition modulation on M-PAM modulated discrete multitone (DMT) is introduced. Contrary to ePAM-DMT, the signal generation of the proposed system, termed augmented spectral efficiency discrete multitone (ASE-DMT), occurs in the frequency domain. This results in an improved spectral and energy efficiency. The analytical bit error rate (BER) performance bound of the proposed system is derived and compared with Monte-Carlo simulations. The system performance is shown to offer significant electrical and optical energy savings compared with ePAM-DMT and DC-biased optical orthogonal frequency division multiplexing (DCO-OFDM).

Software system for reducing PAM-2 data

Science.gov (United States)

Pepin, T. J.

1982-01-01

A software system for reducing PAM-II data was constructed. The data reduction process concatenates data tapes; determines ephemeris; and inverts full sun extinction data. Tests of this data reduction process show that PAM-II data can be compared with data from other, similar satellites.

Psoriatic arthritis mutilans (PAM) in the Nordic countries

DEFF Research Database (Denmark)

Gudbjornsson, B; Ejstrup, L; Gran, J T

2013-01-01

To determine the prevalence and clinical characteristics of psoriatic arthritis mutilans (PAM) in the Nordic countries.......To determine the prevalence and clinical characteristics of psoriatic arthritis mutilans (PAM) in the Nordic countries....

40 CFR 52.1080 - Photochemical Assessment Monitoring Stations (PAMS) Program.

Science.gov (United States)

2010-07-01

... Stations (PAMS) Program. 52.1080 Section 52.1080 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... § 52.1080 Photochemical Assessment Monitoring Stations (PAMS) Program. On March 24, 1994 Maryland's... Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as required by...

40 CFR 52.2426 - Photochemical Assessment Monitoring Stations (PAMS) Program.

Science.gov (United States)

2010-07-01

... Stations (PAMS) Program. 52.2426 Section 52.2426 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... § 52.2426 Photochemical Assessment Monitoring Stations (PAMS) Program. On November 23, 1994 Virginia's... Photochemical Assessment Monitoring Stations (PAMS) Program as a state implementation plan (SIP) revision, as...

A comparative study of proliferative activity and tumor stage of pregnancy-associated melanoma (PAM) and non-PAM in gestational age women.

Science.gov (United States)

Merkel, Emily A; Martini, Mary C; Amin, Sapna M; Yélamos, Oriol; Lee, Christina Y; Sholl, Lauren M; Rademaker, Alfred W; Guitart, Joan; Gerami, Pedram

2016-01-01

The influence of pregnancy on the development, progression, and prognosis of melanoma is controversial. We sought to compare clinical characteristics, histologic features, and proliferative activity in pregnancy-associated melanoma (PAM) and melanoma in nonpregnant women of reproductive age (non-PAM). In this retrospective cohort study, we reviewed medical records and pathology reports from women given a diagnosis of melanoma between 2006 and 2015. We also examined tumor proliferation rates using mitotic count and 2 immunohistochemical markers of proliferation, phosphohistone H3 and Ki-67. In 50 PAM and 122 non-PAM cases, a diagnosis of melanoma in situ was associated with PAM. Among invasive melanomas, there was no difference in proliferative activity between groups. Pregnancy status was also not associated with age at diagnosis, tumor site, Breslow depth, Clark level, ulceration, or overall stage. This was a retrospective study with a small sample size of mostly patients with early-stage melanoma. In our study of primarily early-stage melanoma, pregnancy did not have a significant impact on tumor proliferation. Particularly for patients given a diagnosis of stage I melanoma who are undergoing close surveillance, a history of PAM should not outweigh traditional factors, such as advanced maternal age, in planning future pregnancies. Copyright © 2015 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

Science.gov (United States)

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

40 CFR 52.2035 - Photochemical Assessment Monitoring Stations (PAMS) Program.

Science.gov (United States)

2010-07-01

... Stations (PAMS) Program. 52.2035 Section 52.2035 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...) Pennsylvania § 52.2035 Photochemical Assessment Monitoring Stations (PAMS) Program. On September 23, 1994... (PAMS) Program as a state implementation plan (SIP) revision, as required by section 182(c)(1) of the...

Engineered Cpf1 variants with altered PAM specificities.

Science.gov (United States)

Gao, Linyi; Cox, David B T; Yan, Winston X; Manteiga, John C; Schneider, Martin W; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

2017-08-01

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

Bacterial binding to extracellular proteins - in vitro adhesion

DEFF Research Database (Denmark)

Schou, C.; Fiehn, N.-E.

1999-01-01

Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis......Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis...

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

OpenAIRE

Shrago, A W; Chassy, B M; Dobrogosz, W J

1986-01-01

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

Science.gov (United States)

Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

2015-01-01

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

Nanoparticle-Delivered 2-PAM for Rat Brain Protection against Paraoxon Central Toxicity.

Science.gov (United States)

Pashirova, Tatiana N; Zueva, Irina V; Petrov, Konstantin A; Babaev, Vasily M; Lukashenko, Svetlana S; Rizvanov, Ildar Kh; Souto, Eliana B; Nikolsky, Evgeny E; Zakharova, Lucia Ya; Masson, Patrick; Sinyashin, Oleg G

2017-05-24

Solid lipid nanoparticles (SLNs) are among the most promising nanocarriers to target the blood-brain barrier (BBB) for drug delivery to the central nervous system (CNS). Encapsulation of the acetylcholinesterase reactivator, pralidoxime chloride (2-PAM), in SLNs appears to be a suitable strategy for protection against poisoning by organophosphorus agents (OPs) and postexposure treatment. 2-PAM-loaded SLNs were developed for brain targeting and delivery via intravenous (iv) administration. 2-PAM-SLNs displayed a high 2-PAM encapsulation efficiency (∼90%) and loading capacity (maximum 30.8 ± 1%). Drug-loaded particles had a mean hydrodynamic diameter close to 100 nm and high negative zeta potential (-54 to -15 mV). These properties contribute to improve long-term stability of 2-PAM-SLNs when stored both at room temperature (22 °C) and at 4 °C, as well as to longer circulation time in the bloodstream compared to free 2-PAM. Paraoxon-poisoned rats (2 × LD 50 ) were treated with 2-PAM-loaded SLNs at a dose of 2-PAM of 5 mg/kg. 2-PAM-SLNs reactivated 15% of brain AChE activity. Our results confirm the potential use of SLNs loaded with positively charged oximes as a medical countermeasure both for protection against OPs poisoning and for postexposure treatment.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

DEFF Research Database (Denmark)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch

2015-01-01

) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced...... to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4...

Investigation of defects on PAMS microspheres fabricated with microencapsulation method

International Nuclear Information System (INIS)

Chen Sufen; Li Bo; Liu Yiyang; Zhang Zhanwen; Qi Xiaobo

2012-01-01

Poly-(α-methylstyrene) (PAMS) microspheres were fabricated with W1/O/W2 double emulsion microencapsulation method, and the effects of polyvinylalcohol (PVA) and CaCl 2 weight concentrations and the O/W2 phase ratio on the percentages of defected PAMS microspheres were studied. The weight concentrations of PVA and CaCl 2 and the O/W2 phase ratio in the fabrication process of PAMS microspheres were optimized. The results show that, for the three parameters being 1.0%, 1.5%, and 0.01, respectively, the percentage of the defect-free PAMS microspheres without vacuoles in the shell wall can be up to 60%. (authors)

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.

Science.gov (United States)

Hu, Johnny H; Miller, Shannon M; Geurts, Maarten H; Tang, Weixin; Chen, Liwei; Sun, Ning; Zeina, Christina M; Gao, Xue; Rees, Holly A; Lin, Zhi; Liu, David R

2018-04-05

A key limitation of the use of the CRISPR-Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

PHBV/PAM scaffolds with local oriented structure through UV polymerization for tissue engineering.

Science.gov (United States)

Ke, Yu; Wu, Gang; Wang, Yingjun

2014-01-01

Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100  μm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance.

DNA binding properties of the small cascade subunit Csa5.

Directory of Open Access Journals (Sweden)

Michael Daume

Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

Jonah, Pam Keziah

African Journals Online (AJOL)

Jonah, Pam Keziah. Vol 9, No 2 (2012) - Articles A Pragmatic Analysis and Interpretation of Verbal Offers by Vendors of Consumer Products in Jos Metropolis Abstract. ISSN: 1813-2227. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL · AJOL's ...

Photochemical Assessment Monitoring Stations (PAMS)

Data.gov (United States)

U.S. Environmental Protection Agency — Photochemical Assessment Monitoring Stations (PAMS). This file provides information on the numbers and distribution (latitude/longitude) of air monitoring sites...

Understanding AL-PAM assisted oil sands tailings treatment

Energy Technology Data Exchange (ETDEWEB)

Guo, L.; Maham, Y.; Masliyah, J.; Xu, Z. [Alberta Univ., Edmonton, AB (Canada). Dept. of Chemical and Materials Engineering

2010-07-01

Technologies currently used to treat oil sands tailings include the composite tailings (CT) process and the thickened tailings (TT) or paste technology process. This PowerPoint presentation discussed a flocculation and filtration method used to produce stackable tailings deposits. Magnafloc 1011 and AL-PAM additions were used as part of the filtration technique to produce very dry filter cakes. The effect of AL-PAM molecular weight and aluminum content on tailings treatments was investigated as well as the effect of tailings characteristics on the performance of flocculant-assisted tailings filtration processes. The AL-PAM molecular structure was studied. An experimental study was conducted to determine the effect of various polymer additions on fresh tailings from an oil sands plant. The study showed that the optimum dosage for settling and filtration was lower for higher molecular weight AL-PAM. A higher aluminum content was beneficial for settling. Increases in the aluminum content did not improve filtration rates, but reduced optimal dosages. Step-by-step details of the supernatant filtration process were provided, as well as photographs of the laboratory study. tabs., figs.

A study on contraction of pneumatic artificial muscle (PAM) for load-lifting

Science.gov (United States)

Najmuddin, W. S. W. A.; Mustaffa, M. T.

2017-10-01

Pneumatic Artificial Muscles (PAMs) have been known for its wide application in various aspects of industrial automation and robotic equipments. Many advantages in terms of high power-to-volume ratio, high power-to-weight ratio, stick-slip-free operation and high degree of safety offer by PAM compare to traditional actuators. However, behind this benefits lie a limitation of significant compatibility of PAM mechanism which have to be considered so as to fully understand how the PAM works during load-lifting. In this study, the mesh suitability experiment and the effect of force load on PAM contraction experiment have been carried out. PAM is constructed and compatibility of bladder and the braided mesh to produce uniform expansion is investigated. Moreover, the first experimental result of finding compatibility is used to verify the contraction value under various loads.

A Design V and V Process of Hardwired PAMS for KJRR

International Nuclear Information System (INIS)

Jang, Gwisook; Suh, Yongsuk; Kim, Youngki

2013-01-01

A hardwired PAMS (Post Accident Monitoring System) of KJRR (KIJANG Research Reactor) provides the necessary information for operators to monitor and take actions after an accident condition. The hardwired PAMS are designed according to HSI (Human System Interface) guidelines. However the HSI guidelines are too high level requirements and do not reflect the detailed characteristics of commercial products. Thus, the system designers have a difficulty in the following how the system requirements are reflected in the actual and detailed requirements for purchase. Thus, a design V and V (Verification and Validation) of the hardwired PAMS should be specially managed and can be traceable through the entire development life cycle. Thus, this paper proposes a tracking matrix method for the design V and V of hardwired PAMS for KJRR. The PAMS of KJRR consists of hardwired indicators. There are many differences between the system requirements and procurement specifications for hardwired devices. The existing design V and V method for hardwired indicators is not clear. Thus, this paper proposed a tracking matrix for the design V and V of the hardwired PAMS

[Sewage sludge conditioning by bioleaching-dual PAC and PAM addition].

Science.gov (United States)

Liu, Chang-Geng; Zhang, Pan-Yue; Zeng, Guang-Ming; Liu, Yong-Gang

2010-09-01

Bioleaching-dual polyaluminum chloride (PAC) and polyacrylamide (PAM) addition was used to condition sewage sludge. The results showed that FeSO4 x 7H2O addition improved the bioleaching rate obviously with a fixed sulfur power dosage of 3 g x L(-1); when the FeSO4 x 7H2O dosage was 8 g x L(-1), the bioleaching lasted 1.5 d to decrease the sludge pH below 2. Bioleaching improved the sludge dewaterability significantly with a specific resistance to filtration (SRF) reduction of 77.52% from 6.45 x 10(10)s2 x g(-10 to 1.45 x 10(10)s2 x g(-1), but the bioleached sludge was still difficult to be dewatered. After adjusting the bioleached sludge pH to 6, PAC and PAM were used to enhance conditioning of the bioleached sludge. The results indicated that the optimal dosage was 200 mg x L(-1) for PAC or 50 mg x L(-1) for PAM when single chemical was used. When PAC and PAM were dually used, the optimal dosages of PAC and PAM were 100 mg x L(-1) and 25 mg x L(-1), respectively; SRF and moisture of sludge cake reduced to 2.02 x 10(8) s2 x g(-1) and 74.81%, respectively, showing good dewaterability of the treated sludge. Compared with the single use of PAC and PAM, the dual use of PAC and PAM showed the advantages of lower cost and better conditioning effect.

Quaternary Benzyltriethylammonium Ion Binding to the Na,K-ATPase: a Tool to Investigate Extracellular K+ Binding Reactions†

Science.gov (United States)

Peluffo, R. Daniel; González-Lebrero, Rodolfo M.; Kaufman, Sergio B.; Kortagere, Sandhya; Orban, Branly; Rossi, Rolando C.; Berlin, Joshua R.

2009-01-01

This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (VM)-dependent inhibition and tested the prediction that such a VM-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K+ activation of Na,K-ATPase activity and steady-state 86Rb+ occlusion. The initial rate of 86Rb+ occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb+, a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a VM-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K+-K+ exchange by the Na,K-ATPase, step changes in VM elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K+ congener, Tl+. pNBTEA- and Tl+-dependent transient currents both displayed saturation of charge moved at extreme negative and positive VM, equivalence of charge moved during and after step changes in VM, and similar apparent valence. The rate constant (ktot) for Tl+-dependent transient current asymptotically approached a minimum value at positive VM. In contrast, ktot for pNBTEA-dependent transient current was a “U”-shaped function of VM with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly-accessible sites, one of them located at K+ binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme. PMID:19621894

Psychometric properties of the hebrew translation of the patient activation measure (PAM-13).

Science.gov (United States)

Magnezi, Racheli; Glasser, Saralee

2014-01-01

"Patient activation" reflects involvement in managing ones health. This cross-sectional study assessed the psychometric properties of the Hebrew translation (PAM-H) of the PAM-13. A nationally representative sample of 203 Hebrew-speaking Israeli adults answered the PAM-H, PHQ-9 depression scale, SF-12, and Self-efficacy Scale via telephone. Mean PAM-H scores were 70.7±15.4. Rasch analysis indicated that the PAM-H is a good measure of activation. There were no differences in PAM-H scores based on gender, age or education. Subjects with chronic disease scored lower than those without. Scores correlated with the Self-efficacy Scale (0.47), Total SF-12 (0.39) and PHQ-9 (-0.35, PPAM-H score of those who scored below 10 (72.1±14.8) on the PHQ-9 (not depressed) compared to those scoring ≥10 (i.e. probable depression) (59.2±15.8; t 3.75; P = 0.001). The PAM-H psychometric properties indicate its usefulness with the Hebrew-speaking Israeli population. PAM-H can be useful for assessing programs aimed at effecting changes in patient compliance, health behaviors, etc. Researchers in Israel should use a single translation of the PAM-13 so that findings can be compared, increasing understanding of patient activation.

GE Hitachi's parts asset management solutions (PAMS)

International Nuclear Information System (INIS)

Varno, M.; Lancaster, K.; Detamore, B.; Dinh, H.; Danielsson, J.; Vitaback, F.

2014-01-01

To ensure operational excellence and improve plant reliability, GE Hitachi Nuclear Energy (GEH) has partnered with its customers to develop PARTS Asset Management Solutions (PAMS). PAMS is a comprehensive set of services that enable plants to proactively manage inventory, obtain after-market replacement parts quickly, and resolve obsolescence issues, ensuring operation of equipment is maintained. PAMS includes: - Electronics Exchange Service Program (EESP) - On-demand exchange service program for GE electronics parts that guarantees rapid shipment of parts, results in cost savings and eliminates all obsolescence risk. - Engineering Services (ES) - Service that finds replacement electronics parts for customers encountering obsolescence. - System Assessment Report (SAR) - Comprehensive, customised report for customer systems that provides all information needed to minimise part availability risk. - Inventory Management (IM) - Visual application based on OEM drawings and decades of industry operational experience that enables customers to proactively identify obsolescence and inventory management issues. - Material Solutions (MS) - Fixed price and lead time model that allows the customer to set-up blanket contracts or purchase orders for obtaining needed parts with a focus on reduced overall cycle time. This paper will provide an overview of PAMS and showcase a case study of the PAMS Digitized deployment for an existing GEH customer. (authors)

Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

Science.gov (United States)

Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

2016-03-17

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. Copyright © 2016 Elsevier Inc. All rights reserved.

Bacillus subtilis generates a major specific deletion in pAM beta 1.

OpenAIRE

van der Lelie, D; Venema, G

1987-01-01

pAM beta 1, a 26.5-kilobase plasmid originally isolated from Streptococcus faecalis, was conjugally transferred from Streptococcus lactis to Bacillus subtilis. No conjugal transfer of pAM beta 1 from B. subtilis to S. lactis was observed. In addition, pAM beta 1 which had been reintroduced in S. lactis after cycling through B. subtilis had lost its conjugal transferability to Streptococcus cremoris, although under the same conditions noncycled pAM beta 1 was transferred at high efficiency. Re...

Improvement of biodegradability of oil wastewater contained PAM by pretreatment with Fenton oxidation

International Nuclear Information System (INIS)

Bao, M.; Wang, N.

2008-01-01

The use of polymer flooding in enhanced oil recovery operations has resulted in higher levels of polyacrylamide (PAM) found in oil wastewater. PAM is harmful to the environment, particularly the monomer acrylamide that is generated from PAM degradation. In this study, PAM derived from oil wastewater was pretreated by Fenton oxidation. This oxidation method is based on the use of a mixture of H 2 O 2 and iron salts which produce hydroxyl radicals in acidic conditions. The method offers a cost-effective source of hydroxyl radicals, using easy-to-handle reagents. The purpose of this study was to transform PAM to biodegradable intermediums. The optimal conditions for the Fenton reactions were also determined and described. Under optimal conditions, the removal ratios of PAM and chemical oxygen demand (COD) were 83.8 and 77 per cent respectively. It was concluded that Fenton's oxidation is an effective treatment to improved the biodegradability of PAM. 14 refs., 1 tab., 7 figs

Signaling from the secretory granule to the nucleus: Uhmk1 and PAM.

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Francone, Victor P; Ifrim, Marius F; Rajagopal, Chitra; Leddy, Christopher J; Wang, Yanping; Carson, John H; Mains, Richard E; Eipper, Betty A

2010-08-01

Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Science.gov (United States)

Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

2015-07-23

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

Binding of a cementum attachment protein to extracellular matrix components and to dental surfaces

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Pitaru, S; Hekmati, H [Department of Oral Biology, Goldschleger School of Dental Medicine, Tel Aviv University (Israel); Savion, N [Goldschleger Eye Institute, Sackler School of Medicine, Tel Aviv University (Israel); Olsen, S; Narayanan, S A [Department of Pathology, School of Medicine, University of Washington, Seattle, Washington (United States)

1992-01-01

Cementum proteins (CP) have been shown to mediate cell attachment. Among these, a 55 kDa protein was isolated. The purpose of the present study was to assess the capacity of CP to bind to non-demineralized and demineralized root surfaces and to support cell attachment to dentin. CP were prepared by sequential extraction of bovine cementum with 25 mM EDTA, 0.5 M acetic acid followed by 4 M guanidine HCl. The latter was subjected to ion exchange chromatography on a DEAE-3SW column and eluted stepwise with a 0-0.5 M NaCl gradient. CP were labelled with [sup 125]I and the capacity of [sup 125]I-CP to bind to mineralized and partially demineralized dentin, synthetic hydroxyapatite, collagen, fibronectin and fibrillar collagen-fibronectin cimplex was assessed. It was found that CP bind specifically to mineralized dentin and synthetic hydroxyapatite but not to demineralized dentin. The specific binding was 60% of the total binding. SDS-PAGE analysis of the proteins bound to dentin indicated that the main bound protein had a molecular weight of 55 kDa. CP exhibited high affinity for fibronectin (k[sub D] = 1.56 x 10[sup -10] M) and fibronectincollagen complex, but their binding to either molecular or fibrillar collagen was negligible. It is suggested that CP may play an important role in the attachment of cells of the periodontium to cementum extracellular matrix during homeostasis and regeneration. (au).

Performance Comparison of 112-Gb/s DMT, Nyquist PAM4, and Partial-Response PAM4 for Future 5G Ethernet-Based Fronthaul Architecture

Science.gov (United States)

Eiselt, Nicklas; Muench, Daniel; Dochhan, Annika; Griesser, Helmut; Eiselt, Michael; Olmos, Juan Jose Vegas; Monroy, Idelfonso Tafur; Elbers, Joerg-Peter

2018-05-01

For a future 5G Ethernet-based fronthaul architecture, 100G trunk lines of a transmission distance up to 10 km standard single mode fiber (SSMF) in combination with cheap grey optics to daisy chain cell site network interfaces are a promising cost- and power-efficient solution. For such a scenario, different intensity modulation and direct detect (IMDD) Formats at a data rate of 112 Gb/s, namely Nyquist four-level pulse amplitude modulation (PAM4), discrete multi-tone Transmission (DMT) and partial-response (PR) PAM4 are experimentally investigated, using a low-cost electro-absorption modulated laser (EML), a 25G driver and current state-of-the-art high Speed 84 GS/s CMOS digital-to-analog converter (DAC) and analog-to-digital converter (ADC) test chips. Each modulation Format is optimized independently for the desired scenario and their digital signal processing (DSP) requirements are investigated. The performance of Nyquist PAM4 and PR PAM4 depend very much on the efficiency of pre- and post-equalization. We show the necessity for at least 11 FFE-taps for pre-emphasis and up to 41 FFE coefficients at the receiver side. In addition, PR PAM4 requires an MLSE with four states to decode the signal back to a PAM4 signal. On the contrary, bit- and power-loading (BL, PL) is crucial for DMT and an FFT length of at least 512 is necessary. With optimized parameters, all Modulation formats result in a very similar performances, demonstrating a transmission distance of up to 10 km over SSMF with bit error rates (BERs) below a FEC threshold of 4.4E-3, allowing error free transmission.

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

OpenAIRE

1982-01-01

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular ...

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1.

Science.gov (United States)

Yamano, Takashi; Zetsche, Bernd; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2017-08-17

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases. Copyright © 2017 Elsevier Inc. All rights reserved.

Pam2 lipopeptides systemically increase myeloid-derived suppressor cells through TLR2 signaling

Energy Technology Data Exchange (ETDEWEB)

Maruyama, Akira; Shime, Hiroaki, E-mail: shime@med.hokudai.ac.jp; Takeda, Yohei; Azuma, Masahiro; Matsumoto, Misako; Seya, Tsukasa, E-mail: seya-tu@pop.med.hokudai.ac.jp

2015-02-13

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that exhibit potent immunosuppressive activity. They are increased in tumor-bearing hosts and contribute to tumor development. Toll-like receptors (TLRs) on MDSCs may modulate the tumor-supporting properties of MDSCs through pattern-recognition. Pam2 lipopeptides represented by Pam2CSK4 serve as a TLR2 agonist to exert anti-tumor function by dendritic cell (DC)-priming that leads to NK cell activation and cytotoxic T cell proliferation. On the other hand, TLR2 enhances tumor cell progression/invasion by activating tumor-infiltrating macrophages. How MDSCs respond to TLR2 agonists has not yet been determined. In this study, we found intravenous administration of Pam2CSK4 systemically up-regulated the frequency of MDSCs in EG7 tumor-bearing mice. The frequency of tumor-infiltrating MDSCs was accordingly increased in response to Pam2CSK4. MDSCs were not increased by Pam2CSK4 stimuli in TLR2 knockout (KO) mice. Adoptive transfer experiments using CFSE-labeled MDSCs revealed that the TLR2-positive MDSCs survived long in tumor-bearing mice in response to Pam2CSK4 treatment. Since the increased MDSC population sustained immune-suppressive properties, our study suggests that Pam2CSK4-triggered TLR2 activation enhances the MDSC potential and suppress antitumor immune response in tumor microenvironment. - Highlights: • Pam2CSK4 administration induces systemic accumulation of CD11b{sup +}Gr1{sup +} MDSCs. • TLR2 is essential for Pam2CSK4-induced accumulation of CD11b{sup +}Gr1{sup +} MDSCs. • Pam2CSK4 supports survival of CD11b{sup +}Gr1{sup +} MDSCs in vivo.

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

Science.gov (United States)

Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

2014-09-25

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

Directory of Open Access Journals (Sweden)

Gerd Krause

Full Text Available The hormone thyrotropin (TSH and its receptor (TSHR are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR and lutropin/choriogonadotropin (LHR and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One major reason for these open questions is the lack of any structural information about the extracellular segment of TSHR that connects the N-terminal leucine-rich repeat domain (LRRD with the transmembrane helix (TMH 1, the hinge region. It has been shown experimentally that this segment is important for fine tuning of signaling and ligand interactions. A new crystal structure containing most of the extracellular hFSHR region in complex with hFSH has recently been published. Now, we have applied these new structural insights to the homologous TSHR and have generated a structural model of the TSHR LRRD/hinge-region/TSH complex. This structural model is combined and evaluated with experimental data including hormone binding (bTSH, hTSH, thyrostimulin, super-agonistic effects, antibody interactions and signaling regulation. These studies and consideration of significant and non-significant amino acids have led to a new description of mechanisms at the TSHR, including ligand-induced displacements of specific hinge region fragments. This event triggers conformational changes at a convergent center of the LRRD and the hinge region, activating an "intramolecular agonistic unit" close to the transmembrane domain.

Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

Science.gov (United States)

Krause, Gerd; Kreuchwig, Annika; Kleinau, Gunnar

2012-01-01

The hormone thyrotropin (TSH) and its receptor (TSHR) are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR) and lutropin/choriogonadotropin (LHR) and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One major reason for these open questions is the lack of any structural information about the extracellular segment of TSHR that connects the N-terminal leucine-rich repeat domain (LRRD) with the transmembrane helix (TMH) 1, the hinge region. It has been shown experimentally that this segment is important for fine tuning of signaling and ligand interactions. A new crystal structure containing most of the extracellular hFSHR region in complex with hFSH has recently been published. Now, we have applied these new structural insights to the homologous TSHR and have generated a structural model of the TSHR LRRD/hinge-region/TSH complex. This structural model is combined and evaluated with experimental data including hormone binding (bTSH, hTSH, thyrostimulin), super-agonistic effects, antibody interactions and signaling regulation. These studies and consideration of significant and non-significant amino acids have led to a new description of mechanisms at the TSHR, including ligand-induced displacements of specific hinge region fragments. This event triggers conformational changes at a convergent center of the LRRD and the hinge region, activating an "intramolecular agonistic unit" close to the transmembrane domain.

Preparing for chemical terrorism: a study of the stability of expired pralidoxime (2-PAM).

Science.gov (United States)

Hoffman, Robert S; Mercurio-Zappala, Maria; Bouchard, Nicole; Ravikumar, Padinjarekuttu; Goldfrank, Lewis

2012-03-01

Oximes such as pralidoxime (2-PAM) are essential antidotes for life-threatening organophosphate poisoning. Unfortunately, oximes are expensive, have limited use, and have short shelf lives. As such, maintaining large stockpiles in preparation for terrorist activity is not always possible. We have demonstrated that atropine is stable well beyond its labeled shelf life and that recently expired 2-PAM was clinically efficacious in a series of poisoned patients. Because 2-PAM is often dosed empirically, clinical improvement does not guarantee pharmacological stability. We therefore chose to analyze the chemical stability of expired 2-PAM. Samples of lyophylized 2-PAM were maintained according to the manufacturer's recommendations for 20 years beyond the published shelf life. We studied 2-PAM contained in a MARK I autoinjector that was stored properly for 3 years beyond its expiration date. An Agilent LC/MSD 1100 with diode-array detector and an Agilent Sorbax SB-C-18, 4.6 × 150-mm, 5-μm column were used with the following solvent systems: water with 0.01% trifluoroacetic acid and methanol with 0.01% trifluoroacetic acid. Fresh reagent grade 2-PAM was used as a standard. Results were repeated for consistency. Lyophylized 2-PAM was a white powder that was clear and colorless in solution. Liquid chromatography was identical to the standard and resulted in 2 isolated peaks with identical mass spectra, suggesting that they are stereoisomers. The autoinjector discharged a clear, yellowish solution. In addition to the 2 peaks identified for lyophylized 2-PAM, a small third peak was identified with a mass spectra corresponding to the reported N -methyl pyridinium carboxaldehyde degradation product. When properly stored, lyophylized 2-PAM appears to be chemically stable well beyond its expiration date. Although the relative amount of degradation product found in solubilized (autoinjector) 2-PAM was small, it is unclear whether this may be toxic and therefore is of concern

Preparation and characterization of BC/PAM-AgNPs nanocomposites for antibacterial applications.

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Yang, Guang; Wang, Caixia; Hong, Feng; Yang, Xuexia; Cao, Zhangjun

2015-01-22

In this work, a bacterial cellulose/polyacrylamide (BC/PAM) double network composite was prepared to act as the template for in situ synthesis of silver nanoparticles (AgNPs). Effects of reaction conditions of the BC/PAM composite were investigated on its microstructure, mechanical properties and thermal stabilities. Both the BC/PAM composite and pure BC were utilized to prepare the corresponding silver impregnated nanocomposites, i.e., BC/PAM-AgNPs and BC-AgNPs, by an environmental friendly method, UV irradiation. The influences of the templates were investigated on the AgNPs formation and the antibacterial activities of the nanocomposites by both the zone of inhibition and dynamic shake flask methods. It was shown that the BC/PAM composite displayed a denser microstructure and higher thermal stabilities than pure BC. The BC/PAM-AgNPs nanocomposite exhibited a bigger particle size and lower mass content of AgNPs than the BC-AgNPs one. For the antibacterial test, two nanocomposites exhibited a close antibacterial effect, with a high log reduction above 3 and killing ratio above 99.9%, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1.

Science.gov (United States)

Nishimasu, Hiroshi; Yamano, Takashi; Gao, Linyi; Zhang, Feng; Ishitani, Ryuichiro; Nureki, Osamu

2017-07-06

The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

Science.gov (United States)

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Elevation of Transfection Efficiency by Conjugation of Poly(amindoamine)-diethylenetriamine (PAM-DET) with Dexamethasone

International Nuclear Information System (INIS)

Jeong, Yunseong; Park, Jihye; Jin, Geunwoo; Park, Jongsang

2012-01-01

We successfully conjugated hydrophobic group, dexamethasone onto the surface of PAM-DET to synthesize PAM-DET-DX to form polyplexes with enhanced stability against ionic strength. We evaluated its stability by measuring the size of its polyplexes; the conjugated PAM-DET polyplex showed decreased growth compared to the PAM-DET polyplex in an environment with increased ionic strength, which implies that the conjugated PAM-DET has enhanced stability against increased ionic strength. Furthermore, conjugation of hydrophobic group caused a slight increase in the transfection efficiency without inducing toxicity. Of course, it isn't a neglectable factor that nuclear localization effect of DX can drive the advanced transfection efficiency of PAM-DET-DX polyplex. It means that the hydrophobic moieties which have some other positive properties in transfection are good candidates that can be introduced to non-viral polymeric gene delivery carrier. This strongly indicates that the introduction of hydrophobic moiety on PAM-DET is a good method to enhance polyplex stability against ionic strength without diminishing its advantageous properties, such as high transfection efficiency and low cytotoxicity

Wind tunnel experimental study on the effect of PAM on soil wind erosion control.

Science.gov (United States)

He, Ji-Jun; Cai, Qiang-Guo; Tang, Ze-Jun

2008-10-01

In recent years, high-molecular-weight anionic polyacrylamide (PAM) have been widely tested on a variety of soils, primarily in water erosion control. However, little information is available regarding the effectiveness of PAM on preventing soil loss from wind erosion. The research adopted room wind tunnel experiment, two kinds of soils were used which were from the agro-pastoral area of Inner Mongolia, the northwest of China, the clay content of soils were 22.0 and 13.7%, respectively. For these tests, all the treatments were performed under the condition of wind velocity of 14 m s(-1) and a blown angle of 8.75%, according to the actual situation of experimented area. The study results indicated that using PAM on the soil surface could enhance the capability of avoiding the wind erosion, at the same time, the effect of controlling wind soil erosion with 4 g m(-2) PAM was better than 2 g m(-2) PAM's. Economically, the 2 g m(-2) PAM used in soil surface can control wind erosion effectively in this region. The prophase PAM accumulated in soil could not improve the capability of avoiding the wind erosion, owing to the degradation of PAM in the soil and the continual tillage year after year. The texture of soil is a main factor influencing the capability of soil avoiding wind erosion. Soil with higher clay content has the higher capability of preventing soil from wind erosion than one with the opposite one under the together action of PAM and water.

Pam (Peptidylglycine α-amidating monooxygenase) heterozygosity alters brain copper handling with region specificity

Science.gov (United States)

Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

2013-01-01

Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in postsynaptic vesicular fractions. Cu followed a similar pattern, with ~20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) is selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not the hippocampus in PAM+/− mice, along with GABAB receptor mRNA levels. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. PMID:24032518

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.

Science.gov (United States)

Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Nguyen, Nhu T; Topkar, Ved V; Zheng, Zongli; Joung, J Keith

2015-12-01

CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.

Elucidation of relaxin-3 binding interactions in the extracellular loops of RXFP3

Directory of Open Access Journals (Sweden)

Ross eBathgate

2013-02-01

Full Text Available Relaxin-3 is a highly conserved neuropeptide in vertebrate species and binds to the Class A G protein-coupled receptor RXFP3. Relaxin-3 is involved in a wide range of behaviours, including feeding, stress responses, arousal and cognitive processes and therefore targeting of RXFP3 may be relevant for a range of neurological diseases. Structural knowledge of RXFP3 and its interaction with relaxin-3 would both increase our understanding of ligand recognition in GPCRs that respond to protein ligands and enable acceleration of the design of drug leads. In this study we have used comparative sequence analysis, molecular modelling and receptor mutagenesis to investigate the binding site of the native ligand human relaxin-3 (H3 relaxin on the human RXFP3 receptor. Previous structure function studies have demonstrated that arginine residues in the H3 relaxin B-chain are critical for binding interactions with the receptor extracellular loops and/or N-terminal domain. Hence we have concentrated on determining the ligand interacting sites in these domains and have focussed on glutamic (E and aspartic acid (D residues in these regions that may form electrostatic interactions with these critical arginine residues. Conserved D/E residues identified from vertebrate species multiple sequence alignments were mutated to Ala in human RXFP3 to test the effect of loss of amino acid side chain on receptor binding using both Eu-labelled relaxin-3 agonist. Finally data from mutagenesis experiments have been used in ligand docking simulations to a homology model of human RXFP3 based on the peptide-bound CXCR4 structure. These studies have resulted in a model of the relaxin-3 interaction with RXFP3 which will inform further interrogation of the agonist binding site.

Community-level microalgal toxicity assessment by multiwavelength-excitation PAM fluorometry

International Nuclear Information System (INIS)

Schmitt-Jansen, Mechthild; Altenburger, Rolf

2008-01-01

In ecotoxicological studies involving community-level investigations, rapid and multiparametric fluorescence-based methods may provide substantial advantages over traditional methods used for structural and functional community analysis. Therefore, multiwavelength-excitation pulse-amplitude modulated (PAM) fluorometry was applied in this study to assess long-term changes in periphyton community structure, short-term effects on periphyton functioning (photosynthesis) and pollution induced community tolerance (PICT). For inter-calibration, periphyton structure was evaluated by chemotaxonomic analysis of accessory pigments and a four-wavelength-excitation PAM fluorometer. Short-term effects of herbicides were evaluated by fluorescence quenching analysis and 14 C-incorporation as a proxy of primary production. Subsequently, the method was applied to assess structural and functional changes in periphyton communities after isoproturon exposure for 14 and 26 days, respectively. Results showed good correlation of the PAM fluorescence-based measurements with traditional methods for biofilms in the initial colonisation phase for structural and functional parameters. However, for biofilms older than 9 weeks PAM fluorescence may underestimate biomass. Multiwavelength-excitation PAM fluorometry showed good correlation with marker pigment concentrations indicating that this method provides a reliable estimate of the community structure. PAM fluorometry was able to quantify changes of biomass and follow relative shifts in class composition of biofilms under exposure of isoproturon. Short-term tests based on the quantification of the inhibition of the effective quantum yield revealed a concentration-dependent increase of PICT. The observation of two succession phases of the biofilms after 14 and 26 days of growth, respectively, revealed that sensitivity of biofilms decreased with increasing age and biomass, respectively, but PICT remained a characteristic parameter of exposed

Community-level microalgal toxicity assessment by multiwavelength-excitation PAM fluorometry

Energy Technology Data Exchange (ETDEWEB)

Schmitt-Jansen, Mechthild [UFZ-Helmholtz Centre for Environmental Research, Department of Bioanalytical Ecotoxicology, Permoserstr. 15, 04318 Leipzig (Germany)], E-mail: Mechthild.Schmitt@ufz.de; Altenburger, Rolf [UFZ-Helmholtz Centre for Environmental Research, Department of Bioanalytical Ecotoxicology, Permoserstr. 15, 04318 Leipzig (Germany)

2008-01-20

In ecotoxicological studies involving community-level investigations, rapid and multiparametric fluorescence-based methods may provide substantial advantages over traditional methods used for structural and functional community analysis. Therefore, multiwavelength-excitation pulse-amplitude modulated (PAM) fluorometry was applied in this study to assess long-term changes in periphyton community structure, short-term effects on periphyton functioning (photosynthesis) and pollution induced community tolerance (PICT). For inter-calibration, periphyton structure was evaluated by chemotaxonomic analysis of accessory pigments and a four-wavelength-excitation PAM fluorometer. Short-term effects of herbicides were evaluated by fluorescence quenching analysis and {sup 14}C-incorporation as a proxy of primary production. Subsequently, the method was applied to assess structural and functional changes in periphyton communities after isoproturon exposure for 14 and 26 days, respectively. Results showed good correlation of the PAM fluorescence-based measurements with traditional methods for biofilms in the initial colonisation phase for structural and functional parameters. However, for biofilms older than 9 weeks PAM fluorescence may underestimate biomass. Multiwavelength-excitation PAM fluorometry showed good correlation with marker pigment concentrations indicating that this method provides a reliable estimate of the community structure. PAM fluorometry was able to quantify changes of biomass and follow relative shifts in class composition of biofilms under exposure of isoproturon. Short-term tests based on the quantification of the inhibition of the effective quantum yield revealed a concentration-dependent increase of PICT. The observation of two succession phases of the biofilms after 14 and 26 days of growth, respectively, revealed that sensitivity of biofilms decreased with increasing age and biomass, respectively, but PICT remained a characteristic parameter of exposed

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation.

Science.gov (United States)

Li, Ming; Wang, Rui; Xiang, Hua

2014-06-01

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring 'self' DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Ground processing of the McDonnell Douglas Payload Assist Module (PAM)

Science.gov (United States)

Bryan, C. E.; Maclean, D. A.

1985-01-01

The payload assist module (PAM) ground processing operations which have evolved since they were started in 1982 are described. The objective of the changes was to reduce the prelaunch testing of the airborne support equipment to increase the throughput of PAM systems while not compromising the reliability of the system when functioned on-orbit. The changes that resulted from the initial cargo element ground processing, the on-orbit performance of the systems, plus the postflight refurbishment and recertification of the airborne support equipment resulted in significant reductions in labor expenditures and work shifts required to prepare a PAM system for flight.

Resolving the contribution of the uncoupled phycobilisomes to cyanobacterial pulse-amplitude modulated (PAM) fluorometry signals.

Science.gov (United States)

Acuña, Alonso M; Snellenburg, Joris J; Gwizdala, Michal; Kirilovsky, Diana; van Grondelle, Rienk; van Stokkum, Ivo H M

2016-01-01

Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method allows determination of the quantum yields of maximal (F(M)) and minimal fluorescence (F(0)), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F(0) and F(M) are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PB(free)), PB-PSII complexes, and free PSI. The free PSII contribution was negligible. The PB(free) contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PB(free) and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.

PAM: A Program for Adolescent Mothers.

Science.gov (United States)

Robichaux, Faye B.; And Others

1989-01-01

Describes the Program for Adolescent Mothers (PAM) established to provide opportunities for teen mothers in Louisiana to increase their self-esteem, become productive citizens, and become aware of the physical and emotional development of children. (JOW)

10-Gbps duobinary-4-PAM for High-Performance Access Networks

DEFF Research Database (Denmark)

Suhr, Lau Frejstrup; Vegas Olmos, Juan José; Tafur Monroy, Idelfonso

2014-01-01

This paper reports on an experimental demonstration of a seven level duobinary-4-PAM signal operating at 10.16 Gbps, implemented analogically and featuring direct detection at the receiver side.......This paper reports on an experimental demonstration of a seven level duobinary-4-PAM signal operating at 10.16 Gbps, implemented analogically and featuring direct detection at the receiver side....

Extracellular and intracellular steroid binding proteins

International Nuclear Information System (INIS)

Wagner, R.K.

1978-01-01

Steroid hormone binding proteins can be measured, after the removal of endogenous steroids, as specific complexes with radio-labelled hormones. In this study all the requirements for a quantitative determination of steroid hormone binding proteins are defined. For different methods, agargel electrophoresis, density gradient centrifugation, equilibrium dialysis and polyacrylamide electrophoresis have been evaluated. Agar electrophoresis at low temperature was found to be the simplest and most useful procedure. With this method the dissociation rates of high affinity complexes can be assessed and absolute binding protein concentrations can be determined. The dissociation rates of the oestradiol-oestrogen receptor complex and the R-5020-progestin receptor complex are low (1-2% per h run time.) In contrast, that of complexes between androgen receptor and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one (DHT), progestin receptor and progesterone, corticosteroid binding globulin (CBG) and cortisol or progesterone and sex hormone binding globulin (SHBG) and DHT were hign (16-27% per h run time). Target tissue extracts (cytosols) contain, besides soluble tissue proteins, large amounts of plasma proteins. The extent of this plasma contamination can be determined by measuring the albumin concentration in cytosols by immunodiffusion. In cytosols of 4 different human target tissues the albumin content varied from 20-30% corresponding to an even higher whole plasma concentration. Steroid binding plasma proteins, such as CBG and SHBG are constituents of this containment. (author)

Effect of trace amounts of polyacrylamide (PAM) on long-term performance of activated sludge

International Nuclear Information System (INIS)

Luo, Yuan-ling; Yang, Zhao-hui; Xu, Zheng-yong; Zhou, Ling-jun; Zeng, Guang-ming; Huang, Jing; Xiao, Yong; Wang, Li-ke

2011-01-01

This study aims at evaluating the impacts of PAM addition on activated sludge performance. Four lab-scale sequencing batch reactors (SBRs), each with a working volume of 3 L, were investigated with different PAM concentrations. Experiments were conducted with varying organic loading rate and the sludge volume index (SVI), particle size, zeta potential, specific oxygen uptake rate (SOUR), mixed liquor suspended solids (MLSS), COD and ammonium removal efficiency were monitored over a 105-day period. The results showed that all of the PAM addition not only improved the removal efficiencies of COD and ammonium, but also exhibited some advantages on sludge performance. It was found that the sludge performance of settling property, flocculation and microbial activity increased with increasing concentration of PAM. However, high level of PAM (1 mg/L) led to the formation of large amounts of loose-structure flocs, which eliminated dissolved oxygen transfer and caused the sludge disintegration, resulting in bad settleability and lower microbial activity. In this way, when the dosage of PAM was 0.1 mg/L, the sludge had the best settling property and activity.

Concurrent validity of the PAM accelerometer relative to the MTI Actigraph using oxygen consumption as a reference.

Science.gov (United States)

Slootmaker, S M; Chin A Paw, M J M; Schuit, A J; van Mechelen, W; Koppes, L L J

2009-02-01

The purpose of this study was to examine the concurrent validity of the Personal Activity Monitor (PAM) accelerometer relative to the Actigraph accelerometer using oxygen consumption as a reference, and to assess the test-retest reliability of the PAM. Thirty-two fit, normal weight adults (aged 21-54) performed two activities, treadmill walking and stair walking, while wearing the PAM, the Actigraph and the Cosmed K4b(2). Correlation coefficients and agreement in absolute energy expenditure (EE) levels between PAM, Actigraph and Cosmed were calculated. The test-retest reliability was examined among 296 PAM's using a laboratory shaker. Intraclass correlation coefficients (ICC) and coefficient of variation (CV) were determined. Correlations for treadmill walking and stair walking, respectively, were r(2)=0.95 and r(2)=0.65 for PAM with Actigraph, r(2)=0.82 and r(2)=0.93 for PAM with VO(2) and r(2)=0.64 and 0.74 for Actigraph with VO(2). Both the PAM and Actigraph underestimated EE during treadmill and stair walking by a substantial amount. The test-retest reliability of the PAM was high [ICC=0.80; 95% confidence interval (CI) (0.28;0.92) and intra-CV=1.5%]. The PAM and Actigraph accelerometer are comparable in assessing bodily movement during treadmill and stair walking. The PAM is a valid device to rank subjects in EE and can be useful in collecting objective data to monitor habitual physical activity.

Investigation of PAM-4 for extending reach in data center interconnect applications

DEFF Research Database (Denmark)

Vegas Olmos, Juan José; Teipen, Brian; Eiselt, Nicklas

2015-01-01

Optical four-level pulse amplitude modulation (PAM-4) is being widely studied for various short-reach optical interfaces, motivated by the need to keep cost structure low, and to increase link capacity despite various constraints in component bandwidth. When considering PAM-4 in applications...... with reach significantly greater than 10km, such as in extended data center interconnects which require optical amplification, impairments such as chromatic dispersion, optical filtering, and ASE must be controlled. We investigate and report on requirements of PAM-4 for extended-reach, data center...

Power penalties for multi-level PAM modulation formats at arbitrary bit error rates

Science.gov (United States)

Kaliteevskiy, Nikolay A.; Wood, William A.; Downie, John D.; Hurley, Jason; Sterlingov, Petr

2016-03-01

There is considerable interest in combining multi-level pulsed amplitude modulation formats (PAM-L) and forward error correction (FEC) in next-generation, short-range optical communications links for increased capacity. In this paper we derive new formulas for the optical power penalties due to modulation format complexity relative to PAM-2 and due to inter-symbol interference (ISI). We show that these penalties depend on the required system bit-error rate (BER) and that the conventional formulas overestimate link penalties. Our corrections to the standard formulas are very small at conventional BER levels (typically 1×10-12) but become significant at the higher BER levels enabled by FEC technology, especially for signal distortions due to ISI. The standard formula for format complexity, P = 10log(L-1), is shown to overestimate the actual penalty for PAM-4 and PAM-8 by approximately 0.1 and 0.25 dB respectively at 1×10-3 BER. Then we extend the well-known PAM-2 ISI penalty estimation formula from the IEEE 802.3 standard 10G link modeling spreadsheet to the large BER case and generalize it for arbitrary PAM-L formats. To demonstrate and verify the BER dependence of the ISI penalty, a set of PAM-2 experiments and Monte-Carlo modeling simulations are reported. The experimental results and simulations confirm that the conventional formulas can significantly overestimate ISI penalties at relatively high BER levels. In the experiments, overestimates up to 2 dB are observed at 1×10-3 BER.

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

International Nuclear Information System (INIS)

O’Day, Danton H.; Huber, Robert J.; Suarez, Andres

2012-01-01

Highlights: ► Extracellular calmodulin is present throughout growth and development in Dictyostelium. ► Extracellular calmodulin localizes within the ECM during development. ► Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. ► Extracellular calmodulin exists in eukaryotic microbes. ► Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca 2+ /CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

Experimental demonstration of 56 Gbit/s PAM-4 over 15 km and 84 Gbit/s PAM-4 over 1 km SSMF at 1525 nm using a 25G VCSEL

DEFF Research Database (Denmark)

Eiselt, Nicklas; Griesser, Helmut; Wei, Jinlong

2016-01-01

Record 28-GBd PAM-4 transmission over 15 km SSMF and 42-GBd PAM-4 over 1 km SSMF using a low-power 25G VCSEL are demonstrated at 1525 nm without optical dispersion compensation and only simple transceiver DSPs....

Psychometric assessment of the patient activation measure short form (PAM-13) in rural settings.

Science.gov (United States)

Hung, Man; Carter, Marjorie; Hayden, Candace; Dzierzon, Rhonda; Morales, Jose; Snow, Laverne; Butler, Jorie; Bateman, Kim; Samore, Matthew

2013-04-01

The patient activation measure short form (PAM-13) assesses patients' self-reported health management skills, knowledge, confidence, and motivation. We used item response theory to evaluate the psychometric properties of the PAM-13 utilized in rural settings. A Rasch partial credit model analysis was conducted on the PAM-13 instrument using a sample of 812 rural patients recruited by providers and our research staff. Specially, we examined dimensionality, item fit, and quality of measures, category response curves, and item differential functioning. Convergent and divergent validities were also examined. The PAM-13 instrument has excellent convergent and divergent validities. It is fairly unidimensional, and all items fit the Rasch model well. It has relatively high person and item reliability indices. Majority of the items were free of item differential functioning. There were, however, some issues with ceiling effects. Additionally, there was a lack of responses for category one across all items. Patient activation measure short form (PAM-13) performs well in some areas, but not all. In general, more items need to be added to cover the upper end of the trait. The four response categories of PAM-13 should be collapsed into three.

Achievable Strength-Based Signal Detection in Quantity-Constrained PAM OOK Concentration-Encoded Molecular Communication.

Science.gov (United States)

Mahfuz, Mohammad Upal

2016-10-01

In this paper, the expressions of achievable strength-based detection probabilities of concentration-encoded molecular communication (CEMC) system have been derived based on finite pulsewidth (FP) pulse-amplitude modulated (PAM) on-off keying (OOK) modulation scheme and strength threshold. An FP-PAM system is characterized by its duty cycle α that indicates the fraction of the entire symbol duration the transmitter remains on and transmits the signal. Results show that the detection performance of an FP-PAM OOK CEMC system significantly depends on the statistical distribution parameters of diffusion-based propagation noise and intersymbol interference (ISI). Analytical detection performance of an FP-PAM OOK CEMC system under ISI scenario has been explained and compared based on receiver operating characteristics (ROC) for impulse (i.e., spike)-modulated (IM) and FP-PAM CEMC schemes. It is shown that the effects of diffusion noise and ISI on ROC can be explained separately based on their communication range-dependent statistics. With full duty cycle, an FP-PAM scheme provides significantly worse performance than an IM scheme. The paper also analyzes the performance of the system when duty cycle, transmission data rate, and quantity of molecules vary.

Effect of trace amounts of polyacrylamide (PAM) on long-term performance of activated sludge.

Science.gov (United States)

Luo, Yuan-ling; Yang, Zhao-hui; Xu, Zheng-yong; Zhou, Ling-jun; Zeng, Guang-ming; Huang, Jing; Xiao, Yong; Wang, Li-ke

2011-05-15

This study aims at evaluating the impacts of PAM addition on activated sludge performance. Four lab-scale sequencing batch reactors (SBRs), each with a working volume of 3L, were investigated with different PAM concentrations. Experiments were conducted with varying organic loading rate and the sludge volume index (SVI), particle size, zeta potential, specific oxygen uptake rate (SOUR), mixed liquor suspended solids (MLSS), COD and ammonium removal efficiency were monitored over a 105-day period. The results showed that all of the PAM addition not only improved the removal efficiencies of COD and ammonium, but also exhibited some advantages on sludge performance. It was found that the sludge performance of settling property, flocculation and microbial activity increased with increasing concentration of PAM. However, high level of PAM (1mg/L) led to the formation of large amounts of loose-structure flocs, which eliminated dissolved oxygen transfer and caused the sludge disintegration, resulting in bad settleability and lower microbial activity. In this way, when the dosage of PAM was 0.1mg/L, the sludge had the best settling property and activity. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder*

Science.gov (United States)

Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N.; Torda, Andrew E.

2014-01-01

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 μm) to aquo-cobalamin (H2OCbl+) but not to other cobalamins. Competition experiments with the H2OCbl+-coordinating ligand CN− and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl+ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl+. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins. PMID:25342754

Deletion of the calmodulin-binding domain of Grb7 impairs cell attachment to the extracellular matrix and migration

Energy Technology Data Exchange (ETDEWEB)

García-Palmero, Irene; Villalobo, Antonio, E-mail: antonio.villalobo@iib.uam.es

2013-06-28

Highlights: •Grb7 is a calmodulin (CaM)-binding protein. •Deleting the CaM-binding site impairs cell attachment and migration. •CaM antagonists inhibit Grb7-mediated cell migration. •We conclude that CaM controls Grb7-mediated cell migration. -- Abstract: The adaptor Grb7 is a calmodulin (CaM)-binding protein that participates in signaling pathways involved in cell migration, proliferation and the control of angiogenesis, and plays a significant role in tumor growth, its metastatic spread and tumor-associated neo-vasculature formation. In this report we show that deletion of the CaM-binding site of Grb7, located in the proximal region of its pleckstrin homology (PH) domain, impairs cell migration, cell attachment to the extracellular matrix, and the reorganization of the actin cytoskeleton occurring during this process. Moreover, we show that the cell-permeable CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) both retard the migration of cells expressing wild type Grb7, but not the migration of cells expressing the mutant protein lacking the CaM-binding site (Grb7Δ), underscoring the proactive role of CaM binding to Grb7 during this process.

USMA Study of the Residential Communities Initiative (RCI) Portfolio and Asset Management (PAM)

National Research Council Canada - National Science Library

Powell, Robert; Parnell, Gregory; Driscoll, Patrick J; Boylan, Gregory; Evans, Daniel; Underwood, Thaddeus; Moten, Margaret

2006-01-01

...) Portfolio and Asset Management (PAM) program and provide an independent assessment of the adequacy of the Army's RCI PAM program in achieving its intended objectives across all domains and all phases of the program...

Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.

Science.gov (United States)

Anders, Carolin; Bargsten, Katja; Jinek, Martin

2016-03-17

The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of deoxycholate conjugation on stability of pDNA/polyamidoamine-diethylentriamine (PAM-DET) polyplex against ionic strength.

Science.gov (United States)

Jeong, Yunseong; Jin, Geun-Woo; Choi, Eunjung; Jung, Ji Hyuk; Park, Jong-Sang

2011-11-28

Polyplexes formed from cationic polymer/pDNA have been known to be vulnerable to external ionic strength. To improve polyplex stability against ionic strength, we attempted the chemical conjugation of the hydrophobic deoxycholate (DC) moiety to the polyamidoamine-diethylenetriamine (PAM-DET) dendrimer. Dynamic light scattering studies showed that the tolerance of the resulting PAM-DET-DC against ionic strength is higher than that of PAM-DET. In addition, we confirmed that the stability of polyplex has a strong relationship with the degree of conjugation of the DC moiety to the PAM-DET dendrimer and the charge ratio of PAM-DET-DC. Furthermore, the transfection efficiency of the PAM-DET-DC polyplex is higher than that of PAM-DET but its cytotoxicity remains the same. Therefore, the chemical conjugation of DC is a safe and effective method for increasing the stability of supramolecules formed from electrostatic interaction. Copyright © 2011 Elsevier B.V. All rights reserved.

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding

DEFF Research Database (Denmark)

Barington, Line; Rummel, Pia C; Lückmann, Michael

2016-01-01

and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action...... in CCR8. We find that the 7 transmembrane (7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only...

Low-power DAC-less PAM-4 transmitter using a cascaded microring modulator.

Science.gov (United States)

Dubé-Demers, Raphaël; LaRochelle, Sophie; Shi, Wei

2016-11-15

Future super-computer interconnect systems and data centers request ultrahigh data rate links at low cost and power consumption, for which transmitters with a high level of integration and spectral efficient formats are key components. We report 60 Gb/s pulse-amplitude modulation (PAM-4) of an optical signal using a dual-microring silicon photonics circuit, making a low-power, digital-to-analog converter (DAC)-less PAM modulator. The power consumption is evaluated below 100 fJ/bit, including thermal adjustments. To the best of our knowledge, these results feature the lowest reported power consumption for PAM signaling in a DAC-less scheme for data rate beyond 40 Gb/s.

PAM-4 signal delivery in one radio-over-fiber system

Science.gov (United States)

Wang, Hada; Zhou, Wen; Yu, Jianjun

2017-10-01

We propose and experimentally demonstrate four-level pulse-amplitude-modulation (PAM-4) signal delivery in a radio-over-fiber system for the first time. Over 8-Gbit/s PAM-4 signals have been transmitted over 20-km single-mode fiber-28 and 1-m wireless distance. The signal after transmission is detected directly by an envelope detector at the receiver side. The maximal bit rate could be increased if the bandpass amplifier and envelope detector have more bandwidth.

A passive and active microwave-vector radiative transfer (PAM-VRT) model

International Nuclear Information System (INIS)

Yang, Jun; Min, Qilong

2015-01-01

A passive and active microwave vector radiative transfer (PAM-VRT) package has been developed. This fast and accurate forward microwave model, with flexible and versatile input and output components, self-consistently and realistically simulates measurements/radiation of passive and active microwave sensors. The core PAM-VRT, microwave radiative transfer model, consists of five modules: gas absorption (two line-by-line databases and four fast models); hydrometeor property of water droplets and ice (spherical and nonspherical) particles; surface emissivity (from Community Radiative Transfer Model (CRTM)); vector radiative transfer of successive order of scattering (VSOS); and passive and active microwave simulation. The PAM-VRT package has been validated against other existing models, demonstrating good accuracy. The PAM-VRT not only can be used to simulate or assimilate measurements of existing microwave sensors, but also can be used to simulate observation results at some new microwave sensors. - Highlights: • A novel microwave vector radiative transfer model is developed. • It can simulate passive and active microwave radiative transfer simultaneously. • It can be applied to simulate measurements for different types of viewing geometry. • The accuracy of this model has been validated against other existing models

A20 is critical for the induction of Pam3CSK4-tolerance in monocytic THP-1 cells.

Directory of Open Access Journals (Sweden)

Jinyue Hu

Full Text Available A20 functions to terminate Toll-like receptor (TLR-induced immune response, and play important roles in the induction of lipopolysacchride (LPS-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.

View of the deployed INSAT/PAM-D

Science.gov (United States)

1983-01-01

View of the deployed Indian National Satellite (INSAT) attached to Payload Assist Module (PAM) D drifts above the earth's surface. A faint shadow of the open payload bay can be seen in the top left of the frame.

Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

Directory of Open Access Journals (Sweden)

Natália Salazar

Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

Development of peat-oil (POM) and peat-alcohol (PAM) slurries as alternative fuels

Energy Technology Data Exchange (ETDEWEB)

Clemens, D F

1983-11-01

The preparation and evaluation of peat/No. 2 fuel oil mixtures (POM) and peat/methanol mixtures (PAM) is described. POM and PAM prepared using North Carolina peat and having varied peat loadings, peat moisture contents and peat particle sizes have been studied by measuring slurry sedimentation ratios and drain times from sedimentation tubes. The peat moisture content was particularly crucial in forming stable slurries. The effect of a variety of additives at 0.5-1.0 wt% on sedimentation ratios, drain times and viscosities was studied. Calorimetric studies of several PAM and POM slurries as well as preliminary combustion tests of POM slurries in a salamander burner are also reported.

BicPAMS: software for biological data analysis with pattern-based biclustering.

Science.gov (United States)

Henriques, Rui; Ferreira, Francisco L; Madeira, Sara C

2017-02-02

Biclustering has been largely applied for the unsupervised analysis of biological data, being recognised today as a key technique to discover putative modules in both expression data (subsets of genes correlated in subsets of conditions) and network data (groups of coherently interconnected biological entities). However, given its computational complexity, only recent breakthroughs on pattern-based biclustering enabled efficient searches without the restrictions that state-of-the-art biclustering algorithms place on the structure and homogeneity of biclusters. As a result, pattern-based biclustering provides the unprecedented opportunity to discover non-trivial yet meaningful biological modules with putative functions, whose coherency and tolerance to noise can be tuned and made problem-specific. To enable the effective use of pattern-based biclustering by the scientific community, we developed BicPAMS (Biclustering based on PAttern Mining Software), a software that: 1) makes available state-of-the-art pattern-based biclustering algorithms (BicPAM (Henriques and Madeira, Alg Mol Biol 9:27, 2014), BicNET (Henriques and Madeira, Alg Mol Biol 11:23, 2016), BicSPAM (Henriques and Madeira, BMC Bioinforma 15:130, 2014), BiC2PAM (Henriques and Madeira, Alg Mol Biol 11:1-30, 2016), BiP (Henriques and Madeira, IEEE/ACM Trans Comput Biol Bioinforma, 2015), DeBi (Serin and Vingron, AMB 6:1-12, 2011) and BiModule (Okada et al., IPSJ Trans Bioinf 48(SIG5):39-48, 2007)); 2) consistently integrates their dispersed contributions; 3) further explores additional accuracy and efficiency gains; and 4) makes available graphical and application programming interfaces. Results on both synthetic and real data confirm the relevance of BicPAMS for biological data analysis, highlighting its essential role for the discovery of putative modules with non-trivial yet biologically significant functions from expression and network data. BicPAMS is the first biclustering tool offering the

Series Pneumatic Artificial Muscles (sPAMs) and Application to a Soft Continuum Robot.

Science.gov (United States)

Greer, Joseph D; Morimoto, Tania K; Okamura, Allison M; Hawkes, Elliot W

2017-01-01

We describe a new series pneumatic artificial muscle (sPAM) and its application as an actuator for a soft continuum robot. The robot consists of three sPAMs arranged radially round a tubular pneumatic backbone. Analogous to tendons, the sPAMs exert a tension force on the robot's pneumatic backbone, causing bending that is approximately constant curvature. Unlike a traditional tendon driven continuum robot, the robot is entirely soft and contains no hard components, making it safer for human interaction. Models of both the sPAM and soft continuum robot kinematics are presented and experimentally verified. We found a mean position accuracy of 5.5 cm for predicting the end-effector position of a 42 cm long robot with the kinematic model. Finally, closed-loop control is demonstrated using an eye-in-hand visual servo control law which provides a simple interface for operation by a human. The soft continuum robot with closed-loop control was found to have a step-response rise time and settling time of less than two seconds.

Conjugal transfer of plasmid pAM beta 1 in Lactobacillus reuteri and between lactobacilli and Enterococcus faecalis.

OpenAIRE

Tannock, G W

1987-01-01

The broad-host-range plasmid pAM beta 1 (erythromycin resistance) was transferred conjugally from Streptococcus lactis to Lactobacillus reuteri, L. murinus, and L. fermentum. Transfer of pAM beta 1 between two L. reuteri strains occurred, and lactobacillus transconjugants could act as donors of pAM beta 1 in crosses with Enterococcus faecalis JH2-2.

Ordered ZnO/AZO/PAM nanowire arrays prepared by seed-layer-assisted electrochemical deposition

International Nuclear Information System (INIS)

Shen, Yu-Min; Pan, Chih-Huang; Wang, Sheng-Chang; Huang, Jow-Lay

2011-01-01

An Al-doped ZnO (AZO) seed layer is prepared on the back side of a porous alumina membrane (PAM) substrate by spin coating followed by annealing in a vacuum at 400 °C. Zinc oxide in ordered arrays mediated by a high aspect ratio and an ordered pore array of AZO/PAM is synthesized. The ZnO nanowire array is prepared via a 3-electrode electrochemical deposition process using ZnSO 4 and H 2 O 2 solutions at a potential of − 1 V (versus saturated calomel electrode) and temperatures of 65 and 80 °C. The microstructure and chemical composition of the AZO seed layer and ZnO/AZO/PAM nanowire arrays are characterized by field emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM), and energy-dispersive X-ray spectroscopy (EDS). Results indicate that the ZnO/AZO/PAM nanowire arrays were assembled in the nanochannel of the porous alumina template with diameters of 110–140 nm. The crystallinity of the ZnO nanowires depends on the AZO seed layer during the annealing process. The nucleation and growth process of ZnO/AZO/PAM nanowires are interpreted by the seed-layer-assisted growth mechanism.

Accelerated Biofluid Filling in Complex Microfluidic Networks by Vacuum-Pressure Accelerated Movement (V-PAM).

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Yu, Zeta Tak For; Cheung, Mei Ki; Liu, Shirley Xiaosu; Fu, Jianping

2016-09-01

Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micropumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Here, a technology has been developed termed Vacuum-Pressure Accelerated Movement (V-PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead-end channels and closed chambers. Operation of the V-PAM entails a pressurized fluid loading into microfluidic channels where gas confined inside can rapidly be dissipated through permeation through a thin, gas-permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. Effects of different structural and operational parameters of the V-PAM for promoting fluid filling in microfluidic environments have been studied systematically. This work further demonstrates the applicability of V-PAM for rapid filling of temperature-sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V-PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs-on-chips, and biomimetic studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of sample exchange robot PAM-HC for beamline BL-1A at the photon factory

Energy Technology Data Exchange (ETDEWEB)

Hiraki, Masahiko, E-mail: masahiko.hiraki@kek.jp [Mechanical Engineering Center, Applied Research Laboratory, KEK (High Energy Accelerator Research Organization), 1-1 Oho, Tsukuba, Ibaraki 305-0801 Japan (Japan); Department of Accelerator Science, SOKENDAI (the Graduate University for Advanced Studies), 1-1 Oho, Tsukuba, Ibaraki 305-0801 Japan (Japan); Matsugaki, Naohiro; Yamada, Yusuke; Senda, Toshiya [Structural Biology research Center, Institute of Materials Structure Science, KEK (Japan); Department of Materials Structure Science, SOKENDAI (Japan)

2016-07-27

A macromolecular crystallography beamline, BL-1A, has been built at the Photon Factory (PF) for low energy experiments and has been operational since 2010. We have installed a sample exchange robot, PAM (PF Automated Mounting system), similar to other macromolecular crystallography beamlines. However, following the installation of a helium chamber to reduce the absorption of the diffraction signal by air, we developed a new sample exchange robot to replace PAM. The new robot, named PAM-HC (Helium Chamber), is designed with the goal of minimizing leakage of helium gas from the chamber. Here, the PAM-HC hardware and the flow of its movement are described. Furthermore, measurements of temperature changes during sample exchange are presented in this paper.

Development of sample exchange robot PAM-HC for beamline BL-1A at the photon factory

International Nuclear Information System (INIS)

Hiraki, Masahiko; Matsugaki, Naohiro; Yamada, Yusuke; Senda, Toshiya

2016-01-01

A macromolecular crystallography beamline, BL-1A, has been built at the Photon Factory (PF) for low energy experiments and has been operational since 2010. We have installed a sample exchange robot, PAM (PF Automated Mounting system), similar to other macromolecular crystallography beamlines. However, following the installation of a helium chamber to reduce the absorption of the diffraction signal by air, we developed a new sample exchange robot to replace PAM. The new robot, named PAM-HC (Helium Chamber), is designed with the goal of minimizing leakage of helium gas from the chamber. Here, the PAM-HC hardware and the flow of its movement are described. Furthermore, measurements of temperature changes during sample exchange are presented in this paper.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

Science.gov (United States)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-01-01

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

The PAM-1 aminopeptidase regulates centrosome positioning to ensure anterior-posterior axis specification in one-cell C. elegans embryos.

Science.gov (United States)

Fortin, Samantha M; Marshall, Sara L; Jaeger, Eva C; Greene, Pauline E; Brady, Lauren K; Isaac, R Elwyn; Schrandt, Jennifer C; Brooks, Darren R; Lyczak, Rebecca

2010-08-15

In the one-cell Caenorhabditis elegans embryo, the anterior-posterior (A-P) axis is established when the sperm donated centrosome contacts the posterior cortex. While this contact appears to be essential for axis polarization, little is known about the mechanisms governing centrosome positioning during this process. pam-1 encodes a puromycin sensitive aminopeptidase that regulates centrosome positioning in the early embryo. Previously we showed that pam-1 mutants fail to polarize the A-P axis. Here we show that PAM-1 can be found in mature sperm and in cytoplasm throughout early embryogenesis where it concentrates around mitotic centrosomes and chromosomes. We provide further evidence that PAM-1 acts early in the polarization process by showing that PAR-1 and PAR-6 do not localize appropriately in pam-1 mutants. Additionally, we tested the hypothesis that PAM-1's role in polarity establishment is to ensure centrosome contact with the posterior cortex. We inactivated the microtubule motor dynein, DHC-1, in pam-1 mutants, in an attempt to prevent centrosome movement from the cortex and restore anterior-posterior polarity. When this was done, the aberrant centrosome movements of pam-1 mutants were not observed and anterior-posterior polarity was properly established, with proper localization of cortical and cytoplasmic determinants. We conclude that PAM-1's role in axis polarization is to prevent premature movement of the centrosome from the posterior cortex, ensuring proper axis establishment in the embryo. Copyright 2010 Elsevier Inc. All rights reserved.

The transporter SynPAM71 is located in the plasma membrane and thylakoids, and mediates manganese tolerance in Synechocystis PCC6803

DEFF Research Database (Denmark)

Gandini, Chiara; Schmidt, Sidsel Birkelund; Husted, Søren

2017-01-01

symptoms were observed in WT cells exposed to excess Mn. Moreover, CyanoP, which is involved in the early steps of PSII assembly, is massively upregulated in ΔSynPAM71. SynPAM71 was detected in both the plasma membrane and, to a lesser extent, the thylakoid membranes. Our results suggest that SynPAM71......Manganese (Mn) is an essential constituent of photosystem II (PSII) and therefore indispensable for oxygenic photosynthesis. Very little is known about how Mn is transported, delivered and retained in photosynthetic cells. Recently, the thylakoid-localized transporter PAM71 has been linked...... to chloroplast Mn homeostasis in Arabidopsis thaliana. Here, we characterize the function of its homolog in Synechocystis (SynPAM71). We used a loss-of-function line (ΔSynPAM71), wild-type (WT) cells exposed to Mn stress and strains expressing a tagged variant of SynPAM71 to characterize the role of SynPAM71...

Engineered Cpf1 variants with altered PAM specificities increase genome targeting range

Science.gov (United States)

Gao, Linyi; Cox, David B.T.; Yan, Winston X.; Manteiga, John C.; Schneider, Martin W.; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

2017-01-01

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1–7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 assay indicated that these variants retain high DNA targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately three-fold in human coding sequences to one cleavage site per ~11 bp. PMID:28581492

Binding of Hg by bacterial extracellular polysaccharide: a possible role in Hg tolerance.

Science.gov (United States)

Cruz, Kimberly; Guézennec, Jean; Barkay, Tamar

2017-07-01

Bacteria employ adaptive mechanisms of mercury (Hg) tolerance to survive in environments containing elevated Hg concentrations. The potential of extracellular polysaccharides (EPS) production by bacteria as a mechanism of Hg tolerance has not been previously investigated. The objectives of this study were to determine if bacterial EPS sorb Hg, and if so does sorption provide protection against Hg toxicity. Purified EPS with different chemical compositions produced by bacterial isolates from microbial mats in French Polynesian atolls and deep-sea hydrothermal vents were assessed for Hg sorption. The data showed that EPS sorbed up to 82% of Hg from solution, that this sorption was dependent on EPS composition, and that sorption was a saturable mechanism. Hg uptake capacities ranged from 0.005 to 0.454 mmol Hg/g for the different EPS. To determine if EPS production could alter bacterial Hg tolerance, Escherichia coli K-12 strains and their EPS defective mutants were tested by the disc inhibition assay. Mercury inhibited growth in a dose-dependent manner with wild-type strains having smaller (~1 mm), but statistically significant, zones of inhibition than various mutants and this difference was related to a 2-fold decline in the amount of EPS produced by the mutants relative to cell biomass. These experiments identified colanic acid and hexosamine as Hg-binding moieties in EPS. Together these data indicate that binding of Hg to EPS affords a low level of resistance to the producing bacteria.

The development of peat-oil (POM) and peat-alcohol (PAM) slurries as alternative fuels

Energy Technology Data Exchange (ETDEWEB)

Clemens, D F; Evans, G O; Harrell, P A; Whitehurst, B M

1983-11-01

The preparation and evaluation of peat/No. 2 fuel oil mixtures (POM) and peat/methanol mixtures (PAM) is described. POM and PAM prepared using North Carolina peat and having varied peat loadings, peat moisture contents, and peat particle sizes have been studied by measuring slurry sedimentation ratios and drain times from sedimentation tubes. The peat moisture content was particularly crucial in forming stable slurries. The effect of a variety of additives at 0.5-1.0 wt.% on sedimentation ratios, drain times, and viscosities was studied. Calorimetric studies of several PAM and POM slurries as well as preliminary combustion tests of POM slurries in a salamander burner are also reported.

Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae

Directory of Open Access Journals (Sweden)

Benjamin B. A. Raymond

2018-02-01

Full Text Available Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15 using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM, and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.

Science.gov (United States)

Raymond, Benjamin B A; Madhkoor, Ranya; Schleicher, Ina; Uphoff, Cord C; Turnbull, Lynne; Whitchurch, Cynthia B; Rohde, Manfred; Padula, Matthew P; Djordjevic, Steven P

2018-01-01

Mycoplasma hyopneumoniae , an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C.

Science.gov (United States)

Kumar, Dhivya; Mains, Richard E; Eipper, Betty A

2016-05-01

A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM in Chlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein. © 2016 Society for Endocrinology.

Review of UV spectroscopic, chromatographic, and electrophoretic methods for the cholinesterase reactivating antidote pralidoxime (2-PAM).

Science.gov (United States)

John, Harald; Blum, Marc-Michael

2012-01-01

Pralidoxime (2-PAM) belongs to the class of monopyridinium oximes with reactivating potency on cholinesterases inhibited by phosphylating organophosphorus compounds (OPC), for example, pesticides and nerve agents. 2-PAM represents an established antidote for the therapy of anticholinesterase poisoning since the late 1950s. Quite high therapeutic concentrations in human plasma (about 13 µg/ml) lead to concentrations in urine being about 100 times higher allowing the use of less sensitive analytical techniques that were used especially in the early years after 2-PAM was introduced. In this time (mid-1950s until the end of the 1970s) 2-PAM was most often analyzed by either paper chromatography or simple UV spectroscopic techniques omitting any sample separation step. These methods were displaced completely after the establishment of column liquid chromatography in the early 1980s. Since then, diverse techniques including cation exchange, size-exclusion, reversed-phase, and ligand-exchange chromatography have been introduced. Today, the most popular method for 2-PAM quantification is ion pair chromatography often combined with UV detection representing more than 50% of all column chromatographic procedures published. Furthermore, electrophoretic approaches by paper and capillary zone electrophoresis have been successfully used but are seldom applied. This review provides a commentary and exhaustive summary of analytical techniques applied to detect 2-PAM in pharmaceutical formulations and biological samples to characterize stability and pharmacokinetics as well as decomposition and biotransformation products. Separation techniques as well as diverse detectors are discussed in appropriate detail allowing comparison of individual preferences and limitations. In addition, novel data on mass spectrometric fragmentation of 2-PAM are provided. Copyright © 2011 John Wiley & Sons, Ltd.

[Effects of biochar and PAM application on saline soil hydraulic properties of coastal reclamation region].

Science.gov (United States)

Cao, Yu Tong; She, Dong Li

2017-11-01

Disc infiltration tests were carried out to study the soil infiltration characteristics under different rates of soil amendments application, and to investigate the effects of biochar and polyacrylamide (PAM) application on saline soil hydraulic properties, pore characteristics and contribution of each pore to soil water flow in coastal reclamation region. The results showed that soil satura-ted hydraulic conductivity increased by 46.4% when biochar was applied at 2% compared with the control, and decreased with increasing PAM application. The total effective soil porosity and r>100 μm pores were increased by 8.3% and 10.2% (PPAM application. Particularly, the total effective soil porosity decreased markedly when PAM was applied at 1‰ and the reduction was up to 88%. With the application of biochar and PAM, the contribution of r500 μm played a major role in determining water flows.

CYP51 is an essential drug target for the treatment of primary amoebic meningoencephalitis (PAM).

Science.gov (United States)

Debnath, Anjan; Calvet, Claudia M; Jennings, Gareth; Zhou, Wenxu; Aksenov, Alexander; Luth, Madeline R; Abagyan, Ruben; Nes, W David; McKerrow, James H; Podust, Larissa M

2017-12-01

Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered "rare" (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug 'repurposing'-a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially 'druggable' target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

Insights into the interactions among Surfactin, betaines, and PAM: surface tension, small-angle neutron scattering, and small-angle X-ray scattering study.

Science.gov (United States)

Xiao, Jingwen; Liu, Fang; Garamus, Vasil M; Almásy, László; Handge, Ulrich A; Willumeit, Regine; Mu, Bozhong; Zou, Aihua

2014-04-01

The interactions among neutral polymer polyacrylamide (PAM) and the biosurfactant Surfactin and four betaines, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SDDAB), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (STDAB), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SHDAB), and N-dodecyl-N,N-dimethyl-2-ammonio-acetate (C12BE), in phosphate buffer solution (PBS) have been studied by surface tension measurements, small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and rheological experiments. It has been confirmed that the length of alkyl chain is a key parameter of interaction between betaines and PAM. Differences in scattering contrast between X-ray and neutrons for surfactants and PAM molecules provide the opportunity to separately follow the changes of structure of PAM and surfactant aggregates. At concentrations of betaines higher than CMC (critical micelle concentration) and C2 (CMC of surfactant with the presence of polymer), spherical micelles are formed in betaines and betaines/PAM solutions. Transition from spherical to rod-like aggregates (micelles) has been observed in solutions of Surfactin and Surfactin/SDDAB (αSurfactin = 0.67 (molar fraction)) with addition of 0.8 wt % of PAM. The conformation change of PAM molecules only can be observed for Surfactin/SDDAB/PAM system. Viscosity values follow the structural changes suggested from scattering measurements i.e., gradually increases for mixtures PAM → Surfactin/PAM → Surfactin/SDDAB/PAM in PBS.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.

Science.gov (United States)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-06-05

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

International Nuclear Information System (INIS)

Bashkin, P.; Doctrow, S.; Klagsbrun, M.; Svahn, C.M.; Folkman, J.; Vlodavsky, I.

1989-01-01

Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125 I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 x 10 12 binding sites/mm 2 ECM with an apparent k D of 610 nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 μg/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 μg/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descement's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response

Kramers-Kronig PAM Transceiver and Two-Sided Polarization-Multiplexed Kramers-Kronig Transceiver

Science.gov (United States)

Antonelli, Cristian; Mecozzi, Antonio; Shtaif, Mark

2018-01-01

We propose two transceiver schemes based on Kramers Kronig (KK) detection. One targets low-cost high-throughput applications and uses PAM transmission in combination with direct detection and digital reconstruction of the optical phase. This scheme allows digital compensation of chromatic dispersion and provides a significant improvement in terms of spectral efficiency, compared to conventional PAM transmission. The second scheme targets high-channel-count coherent systems with the aim of simplifying the receiver complexity by reducing the optical components count.

The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together.

Science.gov (United States)

Bäck, Nils; Kanerva, Kristiina; Kurutihalli, Vishwanatha; Yanik, Andrew; Ikonen, Elina; Mains, Richard E; Eipper, Betty A

2017-08-01

Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. Copyright © 2017 Elsevier GmbH. All rights reserved.

Equilibrium-phase MR angiography: Comparison of unspecific extracellular and protein-binding gadolinium-based contrast media with respect to image quality.

Science.gov (United States)

Erb-Eigner, Katharina; Taupitz, Matthias; Asbach, Patrick

2016-01-01

The purpose of this study was to compare contrast and image quality of whole-body equilibrium-phase high-spatial-resolution MR angiography using a non-protein-binding unspecific extracellular gadolinium-based contrast medium with that of two contrast media with different protein-binding properties. 45 patients were examined using either 15 mL of gadobutrol (non-protein-binding, n = 15), 32 mL of gadobenate dimeglumine (weakly protein binding, n = 15) or 11 mL gadofosveset trisodium (protein binding, n = 15) followed by equilibrium-phase high-spatial-resolution MR-angiography of four consecutive anatomic regions. The time elapsed between the contrast injection and the beginning of the equilibrium-phase image acquisition in the respective region was measured and was up to 21 min. Signal intensity was measured in two vessels per region and in muscle tissue. Relative contrast (RC) values were calculated. Vessel contrast, artifacts and image quality were rated by two radiologists in consensus on a five-point scale. Compared with gadobutrol, gadofosveset trisodium revealed significantly higher RC values only when acquired later than 15 min after bolus injection. Otherwise, no significant differences between the three contrast media were found regarding vascular contrast and image quality. Equilibrium-phase high-spatial-resolution MR-angiography using a weakly protein-binding or even non-protein-binding contrast medium is equivalent to using a stronger protein-binding contrast medium when image acquisition is within the first 15 min after contrast injection, and allows depiction of the vasculature with high contrast and image quality. The protein-binding contrast medium was superior for imaging only later than 15 min after contrast medium injection. Copyright © 2015 John Wiley & Sons, Ltd.

Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

Science.gov (United States)

Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

2015-10-01

Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. Copyright © 2015 Elsevier GmbH. All rights reserved.

CYP51 is an essential drug target for the treatment of primary amoebic meningoencephalitis (PAM.

Directory of Open Access Journals (Sweden)

Anjan Debnath

2017-12-01

Full Text Available Primary Amoebic Meningoencephalitis (PAM is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered "rare" (but likely underreported the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug 'repurposing'-a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51 target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially 'druggable' target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

Pulmonary alveolar macrophages (PAM) engulf and regain elastin particles and do not respond to some stimuli of neutrophil (PMN) elastinolysis

International Nuclear Information System (INIS)

Tricomi, S.M.; Hyers, T.M.; Yu, S.Y.; Liao, J.J.

1986-01-01

Elastin degradation by PMN and by PAM differs in the proteinases produced and in the method of cellular attack on the substrate. To further characterize the elastinolytic mechanisms of these two cells, 14 C-labelled bovine ligament elastin was dried onto 24-well culture plates and live cells were placed on the substrate in culture medium. Incubation times were 4 hours for PMN and 20 hours for PAM. Elastinolytic activity was determined by counting 14 C-elastin peptides in the supernatant. By lidocaine release of PAM from the surface, 14 C-elastin retained by the cell was measured. Studies on rabbit PAM showed that 40% of dpm remain associated with the cell at 20 hours. Transmission electron microscopy of human PAM confirmed that PAM can engulf and retain elastin particles at 4 and 24 hours of incubation when in close contact with the substrate. Of the number of dpm released by PMN in 4 hours, PAM in 20 hours released only 23% of that number into supernatant and retained 17% closely associated with the cell after lidocaine treatment. Platelet factor 4, a protein released by platelets upon aggregation which stimulates activity of PMN elastase on elastin, was shown to enhance elastinolysis by whole PMN by 57% at 10 μg/ml in this assay. Platelet factor 4 did not enhance elastinolysis by PAM at concentrations up to 100 μg/ml

Integrins and extracellular matrix in mechanotransduction

Directory of Open Access Journals (Sweden)

Ramage L

2011-12-01

Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

Science.gov (United States)

Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

2014-10-01

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

High Speed PAM -8 Optical Interconnects with Digital Equalization based on Neural Network

DEFF Research Database (Denmark)

Gaiarin, Simone; Pang, Xiaodan; Ozolins, Oskars

2016-01-01

We experimentally evaluate a high-speed optical interconnection link with neural network equalization. Enhanced equalization performances are shown comparing to standard linear FFE for an EML-based 32 GBd PAM-8 signal after 4-km SMF transmission.......We experimentally evaluate a high-speed optical interconnection link with neural network equalization. Enhanced equalization performances are shown comparing to standard linear FFE for an EML-based 32 GBd PAM-8 signal after 4-km SMF transmission....

The effect of polyacrylamide (PAM) applications on infiltration, runoff ...

African Journals Online (AJOL)

. Anionic polyacrylamide (PAM) application to soils is an effective soil conservation practice for reducing runoff and soil losses caused by erosion. It also increases the infiltration rate of soils. The objective of this study was conducted to ...

OFDM and PAM comparison using a high baudrate low resolution IM/DD interface for 400G Ethernet access.

Science.gov (United States)

André, Nuno Sequeira; Louchet, Hadrien; Filsinger, Volker; Hansen, Erik; Richter, André

2016-05-30

We compare OFDM and PAM for 400G Ethernet based on a 3-bit high baudrate IM/DD interface at 1550nm. We demonstrate 27Gb/s and 32Gb/s transmission over 10km SSMF using OFDM and PAM respectively. We show that capacity can be improved through adaptation/equalization to achieve 42Gb/s and 64Gb/s for OFDM and PAM respectively. Experimental results are used to create realistic simulations to extrapolate the performance of both modulation formats under varied conditions. For the considered interface we found that PAM has the best performance, OFDM is impaired by quantization noise. When the resolution limitation is relaxed, OFDM shows better performance.

Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

Science.gov (United States)

Hayes, Robert P; Xiao, Yibei; Ding, Fran; van Erp, Paul B G; Rajashankar, Kanagalaghatta; Bailey, Scott; Wiedenheft, Blake; Ke, Ailong

2016-02-25

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

Directory of Open Access Journals (Sweden)

Graham M West

Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

Surveillance for malaria outbreak on malaria-eliminating islands in Tafea Province, Vanuatu after Tropical Cyclone Pam in 2015.

Science.gov (United States)

Chan, C W; Iata, H; Yaviong, J; Kalkoa, M; Yamar, S; Taleo, G; Isozumi, R; Fukui, M; Aoyama, F; Pomer, A; Dancause, K N; Kaneko, A

2017-01-01

The risk of malaria outbreak surfaced in Vanuatu after Tropical Cyclone (TC) Pam in March 2015. In June and July 2015 we conducted malariometric surveys on the islands of Tanna, Aneityum, and Erromango in Tafea Province, where malaria elimination had been targeted, to determine if malaria incidence had increased after TC Pam. No Plasmodium infection was detected by microscopy and PCR in 3009 survey participants. Only 6·3% (190/3007) of participants had fever. Spleen rates in children aged ⩽12 years from Aneityum and Tanna were low, at 3·6% (14/387) and 5·3% (27/510), respectively. Overall bed net use was high at 72·8% (2175/2986); however, a significantly higher (P Pam. The path towards malaria elimination in Tafea Province was not adversely affected by TC Pam.

Experimental Comparison of 56 Gbit/s PAM-4 and DMT for Data Center Interconnect Applications

DEFF Research Database (Denmark)

Eiselt, Nicklas; Dochhan, Annika; Griesser, Helmut

2016-01-01

Four-level pulse amplitude modulation (PAM-4) and discrete multi-tone transmission (DMT) in combination with intensity modulation and direct-detection are two promising approaches for a low-power and low-cost solution for the next generation of data center interconnect applications. We experiment......Four-level pulse amplitude modulation (PAM-4) and discrete multi-tone transmission (DMT) in combination with intensity modulation and direct-detection are two promising approaches for a low-power and low-cost solution for the next generation of data center interconnect applications. We...... experimentally investigate and compare both modulation formats at a data rate of 56 Gb/s and a transmission wavelength of 1544 nm using the same experimental setup. We show that PAM-4 outperforms double sideband DMT and also vestigial sideband DMT for the optical back-to-back (b2b) case and also...... for a transmission distance of 80 km SSMF in terms of required OSNR at a FEC-threshold of 3.8e-3. However, it is also pointed out that both versions of DMT do not require any optical dispersion compensation to transmit over 80 km SSMF while this is essential for PAM-4. Thus, implementation effort and cost may...

Breast imaging using the Twente photoacoustic mammoscope (PAM): new clinical measurements

Science.gov (United States)

Heijblom, Michelle; Piras, Daniele; Ten Tije, Ellen; Xia, Wenfeng; van Hespen, Johan; Klaase, Joost; van den Engh, Frank; van Leeuwen, Ton; Steenbergen, Wiendelt; Manohar, Srirang

2011-07-01

Worldwide, yearly about 450,000 women die from the consequences of breast cancer. Current imaging modalities are not optimal in discriminating benign from malignant tissue. Visualizing the malignancy-associated increased hemoglobin concentration might significantly improve early diagnosis of breast cancer. Since photoacoustic imaging can visualize hemoglobin in tissue with optical contrast and ultrasound-like resolution, it is potentially an ideal method for early breast cancer imaging. The Twente Photoacoustic Mammoscope (PAM) has been developed specifically for breast imaging. Recently, a large clinical study has been started in the Medisch Spectrum Twente in Oldenzaal using PAM. In PAM, the breast is slightly compressed between a window for laser light illumination and a flat array ultrasound detector. The measurements are performed using a Q-switched Nd:YAG laser, pulsed at 1064 nm and a 1 MHz unfocused ultrasound detector array. Three-dimensional data are reconstructed using a delay and sum reconstruction algorithm. Those reconstructed images are compared with conventional imaging and histopathology. In the first phase of the study 12 patients with a malignant lesion and 2 patients with a benign cyst have been measured. The results are used to guide developments in photoacoustic mammography in order to pave the way towards an optimal technique for early diagnosis of breast cancer.

Characterization of the proton binding sites of extracellular polymeric substances in an anaerobic membrane bioreactor.

Science.gov (United States)

Liu, Yi; Chang, Sheng; Defersha, Fantahun M

2015-07-01

This paper focuses on the characterization of the chemical compositions and acidic constants of the extracellular polymeric substances (EPSs) in an anaerobic membrane bioreactor treating synthetic brewery wastewater by using chemical analysis, linear programming analysis (LPA) of titration data, and FT-IR analysis. The linear programming analysis of titration data revealed that the EPSs have proton binding sites with pKa values from pKa ≤ 6, between 6 and 7, and approximately 9.8. The strong acidic sites (pKa ≤ 6) and some weak acidic sites (7.5 membrane filtration. In addition, the FT-IR analysis confirmed the presence of proteins, carbohydrates, nucleic acids, and lipids in the EPS samples. Based on the FT-IR analysis and the main chemical functional groups at the bacterial cell surfaces, the identified proton binding sites were related to carboxyl, phosphate, and hydroxyl/amine groups with pKa values of 4.6 ± 0.7, 6.6 ± 0.01, and 9.7 ± 0.1, respectively, with the corresponding respective intensities of 0.31 ± 0.05, 0.96 ± 0.3, and 1.53 ± 0.3 mmole/g-EPS. The pKa values and intensities of the proton binding sites are the fundamental molecular properties of EPSs that affect the EPS charge, molecular interactions, and metal complexation characteristics. Determination of such properties can advance Derjaguin-Landau-Verwey-Overbeek (DLVO)-based concentration polarization modeling, facilitate the estimation of the osmotic pressure of the EPS concentration polarization layers, and lead to a deeper understanding of the role of metal complexation in membrane fouling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Application of Polyacrylamide (PAM) through Lay-Flat Polyethylene Tubing: Effects on Infiltration, Erosion, N and P Transport, and Corn Yield.

Science.gov (United States)

McNeal, J P; Krutz, L J; Locke, M A; Kenty, M M; Atwill, R L; Pickelmann, D M; Bryant, C J; Wood, C W; Golden, B R; Cox, M S

2017-07-01

Polyacrylamides (PAMs), when applied as a soil amendment, purportedly improve soil infiltration, decrease erosion, and reduce offsite agrochemical transport. The effect of PAM on infiltration, erosion, agrochemical transport, and crop yield when applied in furrow to mid-southern US production systems has not been evaluated. The objective of this study was to assess PAM effects on infiltration, erosion, corn ( L.) grain yield, and nitrogen (N) and phosphorus (P) transport when applied at 10 mg L through lay-flat polyethylene tubing. A 2-yr field study was conducted at the Mississippi State Delta Research and Extension Center in Stoneville, MS, on a Dundee silt loam and a Forestdale silty clay loam. The experimental design was a randomized complete block with four replications of each treatment: irrigated plus no PAM (control) and irrigated plus PAM at 10 mg L. Each irrigation event delivered 102 mm of water at 18.9 L m per furrow, and runoff was captured in a holding tank on the lower end of each plot. Pooled over year and soil texture, PAM increased infiltration and corn grain yield by 6% ( ≤ 0.0398). Polyacrylamide effects on the offsite transport of sediment and N and P were inconsistent, varying across year and soil texture. Results indicate that PAM improves infiltration and corn grain yield on silt loam and silty clay loam textured soils; however, further research is required before PAM can be recommended as a best management practice for mitigating erosion and offsite agrochemical transport in mid-southern production systems. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

PAM, OLA, and LNA are Differentially Taken Up and Trafficked Via Different Metabolic Pathways in Porcine Adipocytes.

Science.gov (United States)

Yu, Caihua; Xi, Lingling; Chen, Jin; Jiang, Qin; Yi, Hongbo; Wang, Yizhen; Wang, Xinxia

2017-11-01

Dietary fatty acids have different effects on fat deposition in pigs. To clarify the underlying mechanisms of this difference, we compared the metabolism of palmitic (PAM, saturated), oleic (OLA, monounsaturated) and linoleic acid (LNA, polyunsaturated) in porcine adipocytes treated with 100 μM PAM, OLA or LNA. We observed that the adipocytes incubated with LNA accumulated more lipids compared with those treated with PAM and OLA. We then probed the metabolism of these fatty acids in porcine adipocytes by using isotope-labelled fatty acids. The results showed that 42% of the [1- 14 C] LNA, 34% of the [1- 14 C] PAM and 28% of the [1- 14 C] OLA were recovered in the cellular lipids. The gene expression analyses showed that LNA significantly increased the expression of adipogenesis- and oxidation-related genes including PPARγ, C/EBPα, ap2 and NRF1. In addition, the cells incubated with LNA showed a decreased Ser 112 phosphorylation in PPARγ compared to those incubated with PAM and OLA. Furthermore, when PPARγ Ser 112 phosphorylation was inhibited, no significant difference in the triacylglycerol contents in the adipocytes was observed. These results showed the dietary fatty acids had different metabolism pathways in porcine adipocytes, and LNA significantly promoted lipid accumulation, probably by regulating PPARγ phosphorylation in adipocytes.

Concurrent validity of the PAM accelerometer relative to the MTI Actigraph using oxygen consumption as a reference

NARCIS (Netherlands)

Slootmaker, S.M.; Chin A Paw, M.J.M.; Schuit, A.J.; Mechelen, W. van; Koppes, L.L.J.

2009-01-01

The purpose of this study was to examine the concurrent validity of the Personal Activity Monitor (PAM) accelerometer relative to the Actigraph accelerometer using oxygen consumption as a reference, and to assess the test-retest reliability of the PAM. Thirty-two fit, normal weight adults (aged

The oxime pro-2-PAM provides minimal protection against the CNS effects of the nerve agents sarin, cyclosarin, and VX in guinea pigs.

Science.gov (United States)

Shih, Tsung-Ming; Guarisco, John A; Myers, Todd M; Kan, Robert K; McDonough, John H

2011-01-01

This study examined whether pro-2-PAM, a pro-drug dihydropyridine derivative of the oxime 2-pralidoxime (2-PAM) that can penetrate the brain, could prevent or reverse the central toxic effects of three nerve agents; sarin, cyclosarin, and VX. The first experiment tested whether pro-2-PAM could reactivate guinea pig cholinesterase (ChE) in vivo in central and peripheral tissues inhibited by these nerve agents. Pro-2-PAM produced a dose-dependent reactivation of sarin- or VX-inhibited ChE in both peripheral and brain tissues, but with substantially greater reactivation in peripheral tissues compared to brain. Pro-2-PAM produced 9-25% reactivation of cyclosarin-inhibited ChE in blood, heart, and spinal cord, but no reactivation in brain or muscle tissues. In a second experiment, the ability of pro-2-PAM to block or terminate nerve agent-induced electroencephalographic seizure activity was evaluated. Pro-2-PAM was able to block sarin- or VX-induced seizures (16-33%) over a dose range of 24-32 mg/kg, but was ineffective against cyclosarin-induced seizures. Animals that were protected from seizures showed significantly less weight loss and greater behavioral function 24 h after exposure than those animals that were not protected. Additionally, brains were free from neuropathology when pro-2-PAM prevented seizures. In summary, pro-2-PAM provided modest reactivation of sarin- and VX-inhibited ChE in the brain and periphery, which was reflected by a limited ability to block or terminate seizures elicited by these agents. Pro-2-PAM was able to reactivate blood, heart, and spinal cord ChE inhibited by cyclosarin, but was not effective against cyclosarin-induced seizures.

Homology Modelling of the GABA Transporter and Analysis of Tiagabine Binding

DEFF Research Database (Denmark)

Skovstrup, S.; Taboureau, Olivier; Bräuner-Osborne, H.

2010-01-01

by Phe 294) to the extracellular vestibule, where the side chain is stabilised by aliphatic residues. The tiagabine binding mode, reaching from the substrate binding site to the extracellular vestibule, forces the side chain of Phe 294 to adopt a distinct conformation from that found in the occluded...

High-Throughput Characterization of Cascade type I-E CRISPR Guide Efficacy Reveals Unexpected PAM Diversity and Target Sequence Preferences.

Science.gov (United States)

Fu, Becky Xu Hua; Wainberg, Michael; Kundaje, Anshul; Fire, Andrew Z

2017-08-01

Interactions between Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNAs and CRISPR-associated (Cas) proteins form an RNA-guided adaptive immune system in prokaryotes. The adaptive immune system utilizes segments of the genetic material of invasive foreign elements in the CRISPR locus. The loci are transcribed and processed to produce small CRISPR RNAs (crRNAs), with degradation of invading genetic material directed by a combination of complementarity between RNA and DNA and in some cases recognition of adjacent motifs called PAMs (Protospacer Adjacent Motifs). Here we describe a general, high-throughput procedure to test the efficacy of thousands of targets, applying this to the Escherichia coli type I-E Cascade (CRISPR-associated complex for antiviral defense) system. These studies were followed with reciprocal experiments in which the consequence of CRISPR activity was survival in the presence of a lytic phage. From the combined analysis of the Cascade system, we found that (i) type I-E Cascade PAM recognition is more expansive than previously reported, with at least 22 distinct PAMs, with many of the noncanonical PAMs having CRISPR-interference abilities similar to the canonical PAMs; (ii) PAM positioning appears precise, with no evidence for tolerance to PAM slippage in interference; and (iii) while increased guanine-cytosine (GC) content in the spacer is associated with higher CRISPR-interference efficiency, high GC content (>62.5%) decreases CRISPR-interference efficiency. Our findings provide a comprehensive functional profile of Cascade type I-E interference requirements and a method to assay spacer efficacy that can be applied to other CRISPR-Cas systems. Copyright © 2017 by the Genetics Society of America.

4-PAM Dispersion-Uncompensated Transmission with Micro-Ring Resonator Enhanced 1.55-µm DML

DEFF Research Database (Denmark)

Da Ros, Francesco; Cristofori, Valentina; Ozolins, Oskars

2017-01-01

Real-time transmission of 14-GBd 4-PAM signal is demonstrated by combining a commercial 1.55-µm DML with a silicon MRR. BER below the HD-FEC threshold is measured after 26-km SSMF transmission without offline digital signal processing.......Real-time transmission of 14-GBd 4-PAM signal is demonstrated by combining a commercial 1.55-µm DML with a silicon MRR. BER below the HD-FEC threshold is measured after 26-km SSMF transmission without offline digital signal processing....

PAM in the Netherlands: activation level of diabetic patients and the relation with quality of health care.

NARCIS (Netherlands)

Hendriks, M.; Rademakers, J.

2011-01-01

Background: The PAM has been proven to be a reliable and valid instrument to measure patient activation in the US and other countries and is used as a first step towards fully informed and involved patients. Given these promising results, the PAM has recently been introduced in the Netherlands. In

Chirality of TLR-2 ligand Pam3CysSK4 in fully synthetic peptide conjugates critically influences the induction of specific CD8+ T-cells.

Science.gov (United States)

Khan, Selina; Weterings, Jimmy J; Britten, Cedrik M; de Jong, Ana R; Graafland, Dirk; Melief, Cornelis J M; van der Burg, Sjoerd H; van der Marel, Gijs; Overkleeft, Hermen S; Filippov, Dmitri V; Ossendorp, Ferry

2009-03-01

Covalent conjugation of synthetic Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides provides well-defined constructs that have significantly improved capacity to induce efficient priming of CD8(+) T lymphocytes in vivo. We have recently explored the cellular mechanisms underlying the efficient induction of a CD8(+) cytotoxic T lymphocyte response by such synthetic model vaccines [Khan, S., Bijker, M.S., Weterings, J.J., Tanke, H.J., Adema, G.J., van, H.T., Drijfhout, J.W., Melief, C.J., Overkleeft, H.S., van der Marel, G.A., Filippov, D.V., van der Burg, S.H., Ossendorp, F., 2007. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells. J. Biol. Chem. 282, 21145-21159.]. In the current study we have investigated the behaviour of two diastereomers of the TLR-2 ligand Pam(3)CSK(4) (Pam) derivatives, namely the R- and S-epimers at C-2 of the glycerol moiety. Other studies have shown that the Pam(3)Cys based lipopeptides of R-configuration (Pam(R)) in the glycerol moiety enhanced macrophage and B-cell activation compared to those with S-configuration (Pam(S)). Here we report that Pam(R)-conjugates lead to better activation of dendritic cells than the Pam(S)-conjugates as judged by higher IL-12 secretion, upregulation of relevant markers for dendritic cell maturation. In contrast both epimers were internalized equally efficient in a clathrin-dependent manner indicating no qualitative difference in the uptake of the two stereoisomeric Pam-conjugates. We conclude that the enhanced DC activation is due to enhanced TLR-2 triggering by the Pam(R)-conjugate in contrast to the Pam(S)-conjugate. Importantly, induction of specific CD8(+) T-cells was significantly higher in mice injected with the Pam(R)-conjugates compared to mice injected with the Pam(S)-conjugate. In summary we show that the favourable effects of the Pam(R)-configuration of TLR-2 ligand can be attributed to

Ocular Tumours in Childhood | Pam | Nigerian Journal of Surgical ...

African Journals Online (AJOL)

Nigerian Journal of Surgical Research. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 3, No 1 (2001) >. Log in or Register to get access to full text downloads. Username, Password, Remember me, or Register. Ocular Tumours in Childhood. V. Pam. Abstract.

Functional Elements on SIRPα IgV domain Mediate Cell Surface Binding to CD47

Science.gov (United States)

Liu, Yuan; Tong, Qiao; Zhou, Yubin; Lee, Hsiau-Wei; Yang, Jenny J.; Bühring, Hans-Jörg; Chen, Yi-Tien; Ha, Binh; Chen, Celia X-J.; Zen, Ke

2007-01-01

Summary SIRPα and SIRPβ1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPα with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPβ1 shares highly homologous extracellular IgV structure with SIRPα, it does not bind to CD47. In this study, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPα, but not SIRPβ1, which determine the extracellular binding interaction of SIRPα to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPα directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPα extracellular binding mediated cell interactions and cell migration. Another SIRPα-specific residue, Met102, appears to assist SIRPα IgV binding through Gln67 and Ala/Val57. An essential role of these amino acids in SIRPα binding to CD47 was further confirmed by introducing these residues into the SIRPβ1 IgV domain, which dramatically converts SIRPβ1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPα selectively binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses. PMID:17070842

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

Science.gov (United States)

Liu, Yuan; Tong, Qiao; Zhou, Yubin; Lee, Hsiau-Wei; Yang, Jenny J; Bühring, Hans-Jörg; Chen, Yi-Tien; Ha, Binh; Chen, Celia X-J; Yang, Yang; Zen, Ke

2007-01-19

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

Re-exploration of the mGlu₁ PAM Ro 07-11401 scaffold: Discovery of analogs with improved CNS penetration despite steep SAR.

Science.gov (United States)

Garcia-Barrantes, Pedro M; Cho, Hyekyung P; Starr, Tahj M; Blobaum, Anna L; Niswender, Colleen M; Conn, P Jeffrey; Lindsley, Craig W

2016-05-01

This letter describes the re-exploration of the mGlu1 PAM Ro 07-11401 scaffold through a multi-dimensional, iterative parallel synthesis approach. Unlike recent series of mGlu1 PAMs with robust SAR, the SAR around the Ro 07-11401 structure was incredibly steep (only ∼6 of 200 analogs displayed mGlu1 PAM activity), and reminiscent of the CPPHA mGlu5 PAM scaffold. Despite the steep SAR, two new thiazole derivatives were discovered with improved in vitro DMPK profiles and ∼3- to 4-fold improvement in CNS exposure (Kps 1.01-1.19); albeit, with a ∼3-fold diminution in mGlu1 PAM potency, yet comparable efficacy (∼5-fold leftward shift of the glutamate concentration-response curve at 10μM). Thus, this effort has provided additional CNS penetrant mGlu1 PAM tools in a different chemotype than the VU0486321 scaffold. These compounds will permit a better understanding of the pharmacology and therapeutic potential of selective mGlu1 activation, while highlighting the steep SAR challenges that can often be encountered in GPCR allosteric modulator discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

Science.gov (United States)

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

Single-lane 180  Gbit/s PAM-4 signal transmission over 2  km SSMF for short-reach applications.

Science.gov (United States)

Zhang, Qiang; Stojanovic, Nebojsa; Prodaniuc, Cristian; Xie, Changsong; Koenigsmann, Michael; Laskowski, Piotr

2016-10-01

We experimentally demonstrate the generation and transmission of a single-lane 180  Gbit/s (90 GBaud) four-level pulse-amplitude modulation (PAM-4) signal in an intensity-modulation direct-detection system with a 7.5 GHz 3 dB bandwidth. The generated signal is transmitted over a 2 km standard single-mode fiber with, to the best of our knowledge, the highest reported net data rate in the C-band: 150  Gbit/s. A net data rate of 168  Gbit/s is also reachable with 1 km reach. The PAM-4 and duobinary (DB) PAM-4 modulation schemes are compared; the obtained results show that DB-PAM-4 significantly outperforms PAM-4 in the considered strong bandwidth-constrained system. Both a feed-forward equalizer and a maximum-likelihood sequence estimator are investigated for data recovery.

Measuring patient activation in Italy: Translation, adaptation and validation of the Italian version of the patient activation measure 13 (PAM13-I).

Science.gov (United States)

Graffigna, Guendalina; Barello, Serena; Bonanomi, Andrea; Lozza, Edoardo; Hibbard, Judith

2015-12-23

The Patient Activation Measure (PAM13) is an instrument that assesses patient knowledge, skills, and confidence for disease self-management. This cross-sectional study was aimed to validate a culturally-adapted Italian Patient Activation Measure (PAM13-I) for patients with chronic conditions. 519 chronic patients were involved in the Italian validation study and responded to PAM13-I. The PAM 13 was translated into Italian by a standardized forward-backward translation. Data quality was assessed by mean, median, item response, missing values, floor and ceiling effects, internal consistency (Cronbach's alpha and average inter-item correlation), item-rest correlations. Rasch Model and differential item functioning assessed scale properties. Mean PAM13-I score was 66.2. Rasch analysis showed that the PAM13-I is a good measure of patient activation. The level of internal consistency was good (α = 0.88). For all items, the distribution of answers was left-skewed, with a small floor effect (range 1.7-4.5 %) and a moderate ceiling effect (range 27.6-55.0 %). The Italian version formed a unidimensional, probabilistic Guttman-like scale explaining 41 % of the variance. The PAM13-I has been demonstrated to be a valid and reliable measure of patient activation and the present study suggests its applicability to the Italian-speaking chronic patient population. The measure has good psychometric properties and appears to be consistent with the developmental nature of the patient activation phenomenon, although it presents a different ranking order of the items comparing to the American version. PAM13-I can be a useful assessment tool to evaluate interventions aimed at improving patient engagement in healthcare and to train doctors in attuning their communication to the level of patients' activation. Future research could be conducted to further confirm the validity of the PAM13-I.

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

DEFF Research Database (Denmark)

Sasaki, T; Brakebusch, C; Engel, J

1998-01-01

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

40 CFR 52.430 - Photochemical Assessment Monitoring Stations (PAMS) Program.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 3 2010-07-01 2010-07-01 false Photochemical Assessment Monitoring Stations (PAMS) Program. 52.430 Section 52.430 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Natural Resources & Environmental Control submitted a plan for the establishment and implementation of a...

40 CFR 52.480 - Photochemical Assessment Monitoring Stations (PAMS) Program.

Science.gov (United States)

2010-07-01

... of Columbia's Department of Consumer and Regulatory Affairs submitted a plan for the establishment... 40 Protection of Environment 3 2010-07-01 2010-07-01 false Photochemical Assessment Monitoring Stations (PAMS) Program. 52.480 Section 52.480 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function

International Nuclear Information System (INIS)

Nagayama, Yuji; Wadsworth, H.L.; Chazenbalk, G.D.; Russo, D.; Seto, Pui; Rapoport, B.

1991-01-01

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction the authors constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites

Measuring patient activation in The Netherlands: translation and validation of the American short form Patient Activation Measure (PAM13).

Science.gov (United States)

Rademakers, Jany; Nijman, Jessica; van der Hoek, Lucas; Heijmans, Monique; Rijken, Mieke

2012-07-31

The American short form Patient Activation Measure (PAM) is a 13-item instrument which assesses patient (or consumer) self-reported knowledge, skills and confidence for self-management of one's health or chronic condition. In this study the PAM was translated into a Dutch version; psychometric properties of the Dutch version were established and the instrument was validated in a panel of chronically ill patients. The translation was done according to WHO guidelines. The PAM 13-Dutch was sent to 4178 members of the Dutch National Panel of people with Chronic illness or Disability (NPCD) in April 2010 (study A) and again to a sub sample of this group (N = 973) in June 2010 (study B). Internal consistency, test-retest reliability and cross-validation with the SBSQ-D (a measure for Health literacy) were computed. The Dutch results were compared to similar Danish and American data. The psychometric properties of the PAM 13-Dutch were generally good. The level of internal consistency is good (α = 0.88) and item-rest correlations are moderate to strong. The Dutch mean PAM score (61.3) is comparable to the American (61.9) and lower than the Danish (64.2). The test-retest reliability was moderate. The association with Health literacy was weak to moderate. The PAM-13 Dutch is a reliable instrument to measure patient activation. More research is needed into the validity of the Patient Activation Measure, especially with respect to a more comprehensive measure of Health literacy.

Comparison of PAM Systems for Acoustic Monitoring and Further Risk Mitigation Application.

Science.gov (United States)

Ludwig, Stefan; Kreimeyer, Roman; Knoll, Michaela

2016-01-01

We present results of the SIRENA 2011 research cruises conducted by the NATO Undersea Research Centre (NURC) and joined by the Research Department for Underwater Acoustics and Geophysics (FWG), Bundeswehr Technical Centre (WTD 71) and the Universities of Kiel and Pavia. The cruises were carried out in the Ligurian Sea. The main aim of the FWG was to test and evaluate the newly developed towed hydrophone array as a passive acoustic monitoring (PAM) tool for risk mitigation applications. The system was compared with the PAM equipment used by the other participating institutions. Recorded sounds were used to improve an automatic acoustic classifier for marine mammals, and validated acoustic detections by observers were compared with the results of the classifier.

Mutational analysis of the extracellular disulphide bridges of the atypical chemokine receptor ACKR3/CXCR7 uncovers multiple binding and activation modes for its chemokine and endogenous non-chemokine agonists.

Science.gov (United States)

Szpakowska, Martyna; Meyrath, Max; Reynders, Nathan; Counson, Manuel; Hanson, Julien; Steyaert, Jan; Chevigné, Andy

2018-07-01

The atypical chemokine receptor ACKR3/CXCR7 plays crucial roles in numerous physiological processes but also in viral infection and cancer. ACKR3 shows strong propensity for activation and, unlike classical chemokine receptors, can respond to chemokines from both the CXC and CC families as well as to the endogenous peptides BAM22 and adrenomedullin. Moreover, despite belonging to the G protein coupled receptor family, its function appears to be mainly dependent on β-arrestin. ACKR3 has also been shown to continuously cycle between the plasma membrane and the endosomal compartments, suggesting a possible role as a scavenging receptor. So far, the molecular basis accounting for these atypical binding and signalling properties remains elusive. Noteworthy, ACKR3 extracellular domains bear three disulphide bridges. Two of them lie on top of the two main binding subpockets and are conserved among chemokine receptors, and one, specific to ACKR3, forms an intra-N terminus four-residue-loop of so far unknown function. Here, by mutational and functional studies, we examined the impact of the different disulphide bridges for ACKR3 folding, ligand binding and activation. We showed that, in contrast to most classical chemokine receptors, none of the extracellular disulphide bridges was essential for ACKR3 function. However, the disruption of the unique ACKR3 N-terminal loop drastically reduced the binding of CC chemokines whereas it only had a mild impact on CXC chemokine binding. Mutagenesis also uncovered that chemokine and endogenous non-chemokine ligands interact and activate ACKR3 according to distinct binding modes characterized by different transmembrane domain subpocket occupancy and N-terminal loop contribution, with BAM22 mimicking the binding mode of CC chemokine N terminus. Copyright © 2018 Elsevier Inc. All rights reserved.

LAYER STRUCTURES FORMED BY SILICA NANOPARTICLES AND CELLULOSE NANOFIBRILS WITH CATIONIC POLYACRYLAMIDE (C-PAM ON CELLULOSE SURFACE AND THEIR INFLUENCE ON INTERACTIONS

Directory of Open Access Journals (Sweden)

Jani Salmi

2009-05-01

Full Text Available A quartz crystal microbalance with dissipation monitoring (QCM-D was used to study the adsorption of the layer formed by silica nanoparticles (SNP and cellulose nanofibrils (NFC together with cationic polyacrylamide (C-PAM on cellulose surface, accompanied by use of atomic force microscope (AFM to study the interactions between cellulose surfaces. The purpose was to understand the multilayer build-up compared to complex structure adsorption. The layer thickness and consequently also the repulsion between surfaces increased with each addition step during layer formation in the SNP-C-PAM systems, whereas the second addition of C-PAM decreased the repulsion in the case of NFC-C-PAM multilayer formation. An exceptionally high repulsion between surfaces was observed when nanofibrillar cellulose was added. This together with the extremely high dissipation values recorded with QCM-D indicated that nanofibrillar cellulose formed a loose and thick layer containing a lot of water. The multilayer systems formed fully and uniformly covered the surfaces. Silica nanoparticles were able to penetrate inside the loose C-PAM structure due to their small size. In contrast, NFC formed individual layers between C-PAM layers. The complex of C-PAM and SNP formed only a partly covered surface, leading to long-ranged pull-off force. This might explain the good flocculation properties reported for polyelectrolyte-nanoparticle systems.

Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors.

Science.gov (United States)

Wang, Jingyi; Lindstrom, Jon

2018-06-01

Heteromeric nicotinic ACh receptors (nAChRs) were thought to have two orthodox agonist-binding sites at two α/β subunit interfaces. Highly selective ligands are hard to develop by targeting orthodox agonist sites because of high sequence similarity of this binding pocket among different subunits. Recently, unorthodox ACh-binding sites have been discovered at some α/α and β/α subunit interfaces, such as α4/α4, α5/α4 and β3/α4. Targeting unorthodox sites may yield subtype-selective ligands, such as those for (α4β2) 2 α5, (α4β2) 2 β3 and (α6β2) 2 β3 nAChRs. The unorthodox sites have unique pharmacology. Agonist binding at one unorthodox site is not sufficient to activate nAChRs, but it increases activation from the orthodox sites. NS9283, a selective agonist for the unorthodox α4/α4 site, was initially thought to be a positive allosteric modulator (PAM). NS9283 activates nAChRs with three engineered α4/α4 sites. PAMs, on the other hand, act at allosteric sites where ACh cannot bind. Known PAM sites include the ACh-homologous non-canonical site (e.g. morantel at β/α), the C-terminus (e.g. Br-PBTC and 17β-estradiol), a transmembrane domain (e.g. LY2087101) or extracellular and transmembrane domain interfaces (e.g. NS206). Some of these PAMs, such as Br-PBTC and 17β-estradiol, require only one subunit to potentiate activation of nAChRs. In this review, we will discuss differences between activation from orthosteric and allosteric sites, their selective ligands and clinical implications. These studies have advanced understanding of the structure, assembly and pharmacology of heteromeric neuronal nAChRs. This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc. © 2017 The British Pharmacological Society.

Characterization of plasticized PEO-PAM blend polymer electrolyte system

Science.gov (United States)

Dave, Gargi; Kanchan, Dinesh

2017-05-01

Present study reports characterization studies of NaCF3SO3 based PEO-PAM Blend Polymer Electrolyte (BPE) system with varying amount of EC+PC as plasticizer prepared by solution cast technique. Structural analysis and surface topography have been performed using FTIR and SEM studies. To understand, thermal properties, DSC studies have been undertaken in the present paper

Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor*

Science.gov (United States)

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722

Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

Science.gov (United States)

Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

2011-01-01

G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process. Copyright © 2010 Elsevier Ltd. All rights reserved.

Translation, adaptation and validation of the American short form Patient Activation Measure (PAM13) in a Danish version.

Science.gov (United States)

Maindal, Helle Terkildsen; Sokolowski, Ineta; Vedsted, Peter

2009-06-29

The Patient Activation Measure (PAM) is a measure that assesses patient knowledge, skill, and confidence for self-management. This study validates the Danish translation of the 13-item Patient Activation Measure (PAM13) in a Danish population with dysglycaemia. 358 people with screen-detected dysglycaemia participating in a primary care health education study responded to PAM13. The PAM13 was translated into Danish by a standardised forward-backward translation. Data quality was assessed by mean, median, item response, missing values, floor and ceiling effects, internal consistency (Cronbach's alpha and average inter-item correlation) and item-rest correlations. Scale properties were assessed by Rasch Rating Scale models. The item response was high with a small number of missing values (0.8-4.2%). Floor effect was small (range 0.6-3.6%), but the ceiling effect was above 15% for all items (range 18.6-62.7%). The alpha-coefficient was 0.89 and the average inter-item correlation 0.38. The Danish version formed a unidimensional, probabilistic Guttman-like scale explaining 43.2% of the variance. We did however, find a different item sequence compared to the original scale. A Danish version of PAM13 with acceptable validity and reliability is now available. Further development should focus on single items, response categories in relation to ceiling effects and further validation of reproducibility and responsiveness.

Inter-data center 28 Gbaud 4-PAM transmission over 240 km standard single mode fiber

DEFF Research Database (Denmark)

Madsen, Peter; Suhr, Lau Frejstrup; Tafur Monroy, Idelfonso

2018-01-01

We report on achieving 28 Gbaud 4-PAM transmission with post-equalization over a 240 km SSMF link without re-engineering the transmission link design. The results demonstrate the prospect of re-using conventional links for inter data center connections.......We report on achieving 28 Gbaud 4-PAM transmission with post-equalization over a 240 km SSMF link without re-engineering the transmission link design. The results demonstrate the prospect of re-using conventional links for inter data center connections....

Measuring patient activation in the Netherlands: translation and validation of the American short form Patient Activation Measure (PAM13

Directory of Open Access Journals (Sweden)

Rademakers Jany

2012-07-01

Full Text Available Abstract Background The American short form Patient Activation Measure (PAM is a 13-item instrument which assesses patient (or consumer self-reported knowledge, skills and confidence for self-management of one’s health or chronic condition. In this study the PAM was translated into a Dutch version; psychometric properties of the Dutch version were established and the instrument was validated in a panel of chronically ill patients. Methods The translation was done according to WHO guidelines. The PAM 13-Dutch was sent to 4178 members of the Dutch National Panel of people with Chronic illness or Disability (NPCD in April 2010 (study A and again to a sub sample of this group (N = 973 in June 2010 (study B. Internal consistency, test-retest reliability and cross-validation with the SBSQ-D (a measure for Health literacy were computed. The Dutch results were compared to similar Danish and American data. Results The psychometric properties of the PAM 13-Dutch were generally good. The level of internal consistency is good (α = 0.88 and item-rest correlations are moderate to strong. The Dutch mean PAM score (61.3 is comparable to the American (61.9 and lower than the Danish (64.2. The test-retest reliability was moderate. The association with Health literacy was weak to moderate. Conclusions The PAM-13 Dutch is a reliable instrument to measure patient activation. More research is needed into the validity of the Patient Activation Measure, especially with respect to a more comprehensive measure of Health literacy.

PAM, MJ and Conventional Grills: Operative Experience on FTU

International Nuclear Information System (INIS)

Mirizzi, F.; Calabro, G.; Ridolfini, V.P.; Tuccillo, A.A.

2006-01-01

The Lower Hybrid Current Drive (LHCD) system for FTU (f = 8 GHz, P = 6 MW/1 s) has a modular configuration that gives it a high degree of flexibility in operations. The RF power generated by six gyrotrons is coupled to the plasma through six independent launchers, each one made by 4 superimposed arrays of 12 waveguides. In the last few years this system has had the unique opportunity to work with conventional grills, classical Multijunction (MJ) and Passive Active Multijunction (PAM) launchers on the same plasma's scenarios. The conventional grill gives the possibility to vary the spectrum of the parallel index of refraction within quite wide margins (N parallel = 1.3 - 3.5) and to launch waves with high directivity at low N parallel . In FTU the coupled power can exceed 100 MW/m 2 at the grill mouth for proper launcher-plasma matching (average power reflection coefficient 10 - 15 %). The MJ reduces the coupling problems arising from impedance mismatching at the plasma-launcher interface. In FTU it has shown good performances also with plasma densities much lower than those normally acceptable for a conventional grill; in this condition an average reflection coefficient around 4% has been measured. Conversely the MJ has a lower flexibility in N|| spectrum and a lower power directivity. In FTU a power density close to 60 MW/m 2 in steady conditions and has been obtained. Limitation came from the upstream distribution lines that were not sufficiently conditioned. The PAM is the LH launcher proposed for ITER due to the possibility of installing an active cooling circuit between the active waveguides to extract the high thermal loads arising both from the plasma and from the RF dissipation into the waveguides' walls. In FTU the power density steadily coupled has been 80 MW/m 2 , 1.4 times the target value for ITER. Very low power reflection (around 3%) also with the PAM mouth flush to the walls, thus in presence of an almost vanishing plasma, has been obtained. This

Measuring patient activation in the Netherlands: translation and validation of the American short form Patient Activation Measure (PAM13).

NARCIS (Netherlands)

Rademakers, J.; Nijman, J.; Hoek, L. van der; Heijmans, M.; Rijken, M.

2012-01-01

Background: The American short form Patient Activation Measure (PAM) is a 13-item instrument which assesses patient (or consumer) self-reported knowledge, skills and confidence for self-management of one's health or chronic condition. In this study the PAM was translated into a Dutch version;

Enhancement of the optical, thermal and electrical properties of PEO/PAM:Li polymer electrolyte films doped with Ag nanoparticles

Science.gov (United States)

Morsi, M. A.; El-Khodary, Sherif A.; Rajeh, A.

2018-06-01

Both lithium bromide (LiBr) and biosynthesized silver nanoparticles (Ag NPs) with average size 2-30 nm have been incorporated into the polymeric matrix of polyethylene oxide and polyacrylamide (PEO/PAM) blend by the casting method. FT-IR analysis indicates the formation of hydrogen bond between the blend components. Also, LiBr and Ag NPs interact with the functional groups of PEO/PAM matrix. The results of XRD analysis depict the semi-crystalline nature of these polymer samples and the degree of crystallinity is decreased due to the addition process. The values of optical energy gap from UV-Vis. data are decreased from 3.55 eV for blend to 3.26 for the nanocomposite sample in the indirect transition. LiBr/Ag NPs assist the improvement of the thermal stability of the PEO/PAM blend, as evidenced by TGA and DTA techniques. Upon the addition of LiBr and Ag NPs, an improvement for the conductivity, dielectric permittivity (έ) and dielectric loss (ἕ) of PEO/PAM solid polymer electrolytes are observed. It's clear that the improvement of the electrical conductivity and dielectric parameters for PEO/PAM: Li+/Ag NPs polymer electrolyte system makes it as a promising candidate for solid-state Li battery applications.

Effect of conditioning by PAM polymers with different charges on the structural and characteristic evolutions of water treatment residuals.

Science.gov (United States)

Yan, W L; Wang, Y L; Chen, Y J

2013-11-01

Three types of polyacrylamide (PAM) flocculants with different charges (cationic PAM WD4960, nonionic PAM M351, and anionic PAM WDA110) were used for water treatment residuals (WTRs) conditioning, and the physicochemical, morphological and structural characteristics of raw and conditioned WTRs were investigated. Rheological methods were employed to analyze the internal structural transition between the raw and conditioned WTRs under a typical dosage of WD4960. Results showed that when the raw WTRs were conditioned with the polymers, the optimum dosage of WD4960 was 4.82 g/kg total suspended solid (TSS) while that of both M351 and WDA110 was 7.24 g/kg TSS. The residual PAM content in the supernatant of the WTR matrix conditioned at the optimum WD4960 dosage was 5.59 mg/L, which is the least among the supernatants obtained with the three types of PAM. Furthermore, the visible fulvic acid (FA) in the supernatant disappeared and the intensity of the ultraviolet FA decreased. The average diameter of irregularly shaped aggregates in the WTR suspensions increased from 35.73 μm to several hundred micrometers with increasing PAM dosage. The size of WTR aggregates conditioned at the optimum WD4960 dosage was much larger than that of aggregates obtained at the optimum M351 or WDA110 dosages. Two-dimensional fractal dimension (D2) values presented an increasing trend with increasing PAM dosage. D2 values of the conditioned WTR aggregates were 1.87, 1.76, and 1.83 at optimum WD4960, M351, and WDA110 dosages, respectively. The cationic PAM (CPAM) WD4960 thus appears to be a more ideal conditioner for WTRs. Consistent relationships were observed among the capillary suction time (CST), average particle size, and D2 values of the conditioned WTR aggregates at the optimum WD4960 dosage. Mass fractal dimensions (D(f)) indicated that both the raw and WD4960-conditioned WTRs behave like weak-link flocs/aggregates. D(f) values (log G'-log TSS) of the WTR aggregates before and after

Extracellular distribution of radiolabel obscures specific binding of diethylstilbestrol in mouse skeletal muscle

International Nuclear Information System (INIS)

Gruber, B.; Blix, P.M.; Cohen, L.

1985-01-01

The extracellular distribution of 3 H-diethylstilbestrol ( 3 H-DES) in mouse skeletal muscle was assessed following intraperitoneal injection. Total muscle extracellular space was measured with 14 C-inulin, and the vascular space with 125 I-albumin. A significant difference in the distribution of native 3 H-DES and its metabolites in muscle and blood was found. This could only be explained if these compounds distributed with the albumin space and not the inulin space

Estimation of human absorbed dose for (166)Ho-PAM: comparison with (166)Ho-DOTMP and (166)Ho-TTHMP.

Science.gov (United States)

Vaez-Tehrani, Mahdokht; Zolghadri, Samaneh; Yousefnia, Hassan; Afarideh, Hossein

2016-10-01

In this study, the human absorbed dose of holmium-166 ((166)Ho)-pamidronate (PAM) as a potential agent for the management of multiple myeloma was estimated. (166)Ho-PAM complex was prepared at optimized conditions and injected into the rats. The equivalent and effective absorbed doses to human organs after injection of the complex were estimated by radiation-absorbed dose assessment resource and methods proposed by Sparks et al based on rat data. The red marrow to other organ absorbed dose ratios were compared with these data for (166)Ho-DOTMP, as the only clinically used (166)Ho bone marrow ablative agent, and (166)Ho-TTHMP. The highest absorbed dose amounts are observed in the bone surface and bone marrow with 1.11 and 0.903 mGy MBq(-1), respectively. Most other organs would receive approximately insignificant absorbed dose. While (166)Ho-PAM demonstrated a higher red marrow to total body absorbed dose ratio than (166)Ho-1,4,7,10-tetraazacyclo dodecane-1,4,7,10 tetra ethylene phosphonic acid (DOTMP) and (166)Ho-triethylene tetramine hexa (methylene phosphonic acid) (TTHMP), the red marrow to most organ absorbed dose ratios for (166)Ho-TTHMP and (166)Ho-PAM are much higher than the ratios for (166)Ho-DOTMP. The result showed that (166)Ho-PAM has significant characteristics than (166)Ho-DOTMP and therefore, this complex can be considered as a good agent for bone marrow ablative therapy. In this work, two separate points have been investigated: (1) human absorbed dose of (166)Ho-PAM, as a potential bone marrow ablative agent, has been estimated; and (2) the complex has been compared with (166)Ho-DOTMP, as the only clinically used bone marrow ablative radiopharmaceutical, showing significant characteristics.

Design of test kits for the RF characterization of the PAM antenna of LHCD system for Aditya-upgrade Tokamak

International Nuclear Information System (INIS)

Jain, Yogesh M.; Sharma, P.K.; Parmar, P.R.; Ambulkar, K.K.

2017-01-01

The Lower Hybrid Current Drive (LHCD) system of the ADITYA-Upgrade tokamak will employ a Passive Active Multijunction (PAM) antenna to launch 250 kW of RF power at 3.7 GHz to drive plasma current non inductively in the tokamak. To evaluate the RF performance of the designed PAM antenna, it is characterized with the help of VNA measurements. The performance of the PAM antenna is mainly decided by the integrated performance of the entire antenna (with a differential phase shift of 270° and equal power distribution between each of the output waveguides) and the performance of mode converter, which transforms input TE 10 mode to TE 30 mode (with a mode purity of 98.5% at the output). This poster thus reports the design and analysis of these testing kits. Also, the test results of PAM antenna obtained by using these test kits would also be presented and discussed in this poster

Disease activity in and quality of life of patients with psoriatic arthritis mutilans: the Nordic PAM Study.

Science.gov (United States)

Lindqvist, U; Gudbjornsson, B; Iversen, L; Laasonen, L; Ejstrup, L; Ternowitz, T; Ståhle, M

2017-11-01

To describe the social status and health-related quality of life of patients with psoriatic arthritis mutilans (PAM) in the Nordic countries. Patients with at least one mutilated joint confirmed by radiology were studied. Disease activity involving joints and skin, physician-assessed disease activity, and patient's education and work status were recorded. Data from the 36-item Short Form Health Survey, Health Assessment Questionnaire and Dermatology Life Quality Index questionnaire were gathered and correlated with disease duration, pain, and general well-being (visual analogue scale). The controls were 58 Swedish patients with long-standing psoriatic arthritis sine PAM. Sixty-seven patients were included. Patients with PAM had a protracted disease history (33 ± 14 years) and disease onset at a relatively early age (30 ± 12 years). Overall inflammatory activity at inclusion was mild to moderate. The mean number of mutilated joints was 8.2 and gross deformity was found in 16% of patients. Forty per cent were treated with biological and 32% with conventional synthetic disease-modifying anti-rheumatic drugs. Forty-two per cent had retired early or were on sick leave. Impaired functional capacity with little or no ability to perform self-care or everyday tasks was reported by 21% of the patients. Patients between 45 and 60 years of age reported the most impaired quality of life in comparison to the control group. PAM seriously affects social functioning. Whether early recognition of PAM and new forms of therapy can improve disease outcome and quality of life remains to be studied.

40 Gb/s Lane Rate NG-PON using Electrical/Optical Duobinary, PAM-4 and Low Complex Equalizations

DEFF Research Database (Denmark)

Wei, J. L.; Grobe, Klaus; Wagner, Christoph

2016-01-01

We present the first numerical investigation and comparison of 40-Gb/s lane rate electrical Duobinary, optical Duobinary and PAM-4 for NG-PONs incorporating low complex linear and nonlinear post-equalizations.......We present the first numerical investigation and comparison of 40-Gb/s lane rate electrical Duobinary, optical Duobinary and PAM-4 for NG-PONs incorporating low complex linear and nonlinear post-equalizations....

Human pentraxin 3 binds to the complement regulator c4b-binding protein.

Directory of Open Access Journals (Sweden)

Anne Braunschweig

Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

Translation, adaptation and validation of the American short form Patient Activation Measure (PAM13 in a Danish version

Directory of Open Access Journals (Sweden)

Sokolowski Ineta

2009-06-01

Full Text Available Abstract Background The Patient Activation Measure (PAM is a measure that assesses patient knowledge, skill, and confidence for self-management. This study validates the Danish translation of the 13-item Patient Activation Measure (PAM13 in a Danish population with dysglycaemia. Methods 358 people with screen-detected dysglycaemia participating in a primary care health education study responded to PAM13. The PAM13 was translated into Danish by a standardised forward-backward translation. Data quality was assessed by mean, median, item response, missing values, floor and ceiling effects, internal consistency (Cronbach's alpha and average inter-item correlation and item-rest correlations. Scale properties were assessed by Rasch Rating Scale models. Results The item response was high with a small number of missing values (0.8–4.2%. Floor effect was small (range 0.6–3.6%, but the ceiling effect was above 15% for all items (range 18.6–62.7%. The α-coefficient was 0.89 and the average inter-item correlation 0.38. The Danish version formed a unidimensional, probabilistic Guttman-like scale explaining 43.2% of the variance. We did however, find a different item sequence compared to the original scale. Conclusion A Danish version of PAM13 with acceptable validity and reliability is now available. Further development should focus on single items, response categories in relation to ceiling effects and further validation of reproducibility and responsiveness.

Further optimization of the M1 PAM VU0453595: Discovery of novel heterobicyclic core motifs with improved CNS penetration.

Science.gov (United States)

Panarese, Joseph D; Cho, Hykeyung P; Adams, Jeffrey J; Nance, Kellie D; Garcia-Barrantes, Pedro M; Chang, Sichen; Morrison, Ryan D; Blobaum, Anna L; Niswender, Colleen M; Stauffer, Shaun R; Conn, P Jeffrey; Lindsley, Craig W

2016-08-01

This Letter describes the continued chemical optimization of the VU0453595 series of M1 positive allosteric modulators (PAMs). By surveying alternative 5,6- and 6,6-heterobicylic cores for the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-5-one core of VU453595, we found new cores that engendered not only comparable or improved M1 PAM potency, but significantly improved CNS distribution (Kps 0.3-3.1). Moreover, this campaign provided fundamentally distinct M1 PAM chemotypes, greatly expanding the available structural diversity for this valuable CNS target, devoid of hydrogen-bond donors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Discovery of a novel 2,4-dimethylquinoline-6-carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

Science.gov (United States)

Long, Madeline F; Engers, Julie L; Chang, Sichen; Zhan, Xiaoyan; Weiner, Rebecca L; Luscombe, Vincent B; Rodriguez, Alice L; Cho, Hyekyung P; Niswender, Colleen M; Bridges, Thomas M; Conn, P Jeffrey; Engers, Darren W; Lindsley, Craig W

2017-11-15

This Letter details our efforts to replace the 3-amino moiety, an essential pharmacophore for M 4 PAM activity in most M 4 PAMs to date, within the thieno[2,3-b]pyridine core, as the β-amino carboxamide motif has been shown to engender poor solubility, varying degrees of P-gp efflux and represents a structural alert. A scaffold hopping exercise identified a novel 2,4-dimethylquinoline carboxamide core that provided M 4 PAM activity and good CNS penetration without an amino moiety. In addition, MacMillan photoredox catalysis chemistry was essential for construction of the 2,4-dimethylquinoline core. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

International Nuclear Information System (INIS)

Pollack, A.E.; Wooten, G.F.

1987-01-01

The binding of the opiate antagonist 3 H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3 H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

Biological applications of an LCoS-BASED PROGRAMMABLE ARRAY MICROSCOPE (PAM)

NARCIS (Netherlands)

Hagen, G.M.; Caarls, W.; Thomas, M.; Hill, A.; Lidke, K.A.; Rieger, B.; Fritsch, C.; Van Geest, B.; Jovin, T.M.; Arndt-Jovin, D.J.

2007-01-01

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a

Microfibril-associated Protein 4 Binds to Surfactant Protein A (SP-A) and Colocalizes with SP-A in the Extracellular Matrix of the Lung

DEFF Research Database (Denmark)

Schlosser, Anders; Thomsen, Theresa H.; Shipley, J. Michael

2006-01-01

for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4......-A composed of the neck region and carbohydrate recognition domain of SP-A indicating that the interaction between MFAP4 and SP-A is mediated via the collagen domain of SP-A. Monoclonal antibodies directed against MFAP4 and SP-A were used for immunohistochemical analysis, which demonstrates that the two...... molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP-A via the collagen region in vitro, and that MFAP4 and SP-A colocates in different lung compartments...

Experimental Demonstration of 32 Gbaud 4-PAM for Data Center Interconnections of up to 320 km

DEFF Research Database (Denmark)

Madsen, Peter; Suhr, Lau Frejstrup; Clausen, Anders

2017-01-01

This paper presents experimental results demonstrating a 64 Gbps 4-PAM transmission over a 320 km SSMF span employing standard 80 km fiber spans for metro links. The receiver consists of a LPF and a DFE utilizing the DD-LMS algorithm.......This paper presents experimental results demonstrating a 64 Gbps 4-PAM transmission over a 320 km SSMF span employing standard 80 km fiber spans for metro links. The receiver consists of a LPF and a DFE utilizing the DD-LMS algorithm....

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

Science.gov (United States)

Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-17

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Comparing 52 Gbps Duobinary and 4-PAM Transmission Over 100m OM-3 Fiber With 25GHz Class VCSELs

DEFF Research Database (Denmark)

Suhr, Lau Frejstrup; Lyubomirsky, Ilya; Daghighian, Henry M.

This paper compares VCSEL based transmission of 52 Gbps duobinary-NRZ and 4-PAM over 100m OM-3 fiber employing a linear equalizer in the receiver.......This paper compares VCSEL based transmission of 52 Gbps duobinary-NRZ and 4-PAM over 100m OM-3 fiber employing a linear equalizer in the receiver....

Regeneration of Red Cell Cholinesterase Activity Following Pralidoxime (2-PAM) Infusion in First 24 h in Organophosphate Poisoned Patients.

Science.gov (United States)

Goel, Parul; Gupta, Nidhi; Singh, Surjit; Bhalla, Ashish; Sharma, Navneet; Gill, K D

2012-01-01

Oximes such as pralidoxime chloride reactivate acetylcholinesterase. However their role in management of organophosphate poisoning is controversial. The study was carried out to find effectiveness of pralidoxime chloride (2-PAM) in regenerating red cell acetyl cholinesterase in first 24 h following administration of it in dose recommended by WHO. Eight patients with OPP [chlorpyriphos (3), phorate (3), dichlorvos (1) and monocrotophos (1) who fulfilled the criteria for inclusion were investigated. In addition to decontamination and atropine, all these patients were administered 30 mg/kg body wt of 2-PAM as bolus dose followed by 7.5 mg/kg body wt/h with maximum dose being 500 mg/h as continuous infusion till first 24 h. Red cell AChE activity was estimated every 15 min for first 4 h, one hourly for next 4 h and then 2 hourly till 24 h and subsequently without 2-PAM every 12 h till 7 days or discharge or death which ever earlier. In all the patients maximum increase in activity was observed in first 4 h following which rise was very slow despite continued 2-PAM infusion and reaching a steady state in 20 h in all the cases. The increase in red cell AChE activity observed in diethyl group at 24 h of 2-PAM infusion was 154% vs. 81% in dimethyl group. At 7 days the increase in activity was 215% vs. 118% respectively. However on multiple repeated ANOVA, no statistically significant difference was observed between diethyl and dimethyl groups at admission and discharge (P > 0.05). Similarly no significant difference was observed in three groups when patients were categorized according to WHO classification of organophosphates (P > 0.05). The maximum increase in red cell AChE activity occurs in first 4 h of 2-PAM administration followed by a slow increase despite 2-PAM infusion till 24 h.

Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

Science.gov (United States)

Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

2014-10-01

To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

The peptidylglycine-α-amidating monooxygenase (PAM) gene rs13175330 A>G polymorphism is associated with hypertension in a Korean population.

Science.gov (United States)

Yoo, Hye Jin; Kim, Minjoo; Kim, Minkyung; Chae, Jey Sook; Lee, Sang-Hyun; Lee, Jong Ho

2017-11-21

Peptidylglycine-α-amidating monooxygenase (PAM) may play a role in the secretion of atrial natriuretic peptide (ANP), which is a hormone involved in the maintenance of blood pressure (BP). The objective of the present study was to determine whether PAM is a novel candidate gene for hypertension (HTN). A total of 2153 Korean participants with normotension and HTN were included. Genotype data were obtained using the Korean Chip. The rs13175330 polymorphism of the PAM gene was selected from the ten single nucleotide polymorphisms (SNPs) most strongly associated with BP. The presence of the G allele of the PAM rs13175330 A>G SNP was associated with a higher risk of HTN after adjustments for age, sex, BMI, smoking, and drinking [OR 1.607 (95% CI 1.220-2.116), p = 0.001]. The rs13175330 G allele carriers in the HTN group treated without antihypertensive therapy (HTN w/o therapy) had significantly higher systolic and diastolic BP than the AA carriers, whereas the G allele carriers in the HTN group treated with antihypertensive therapy (HTN w/ therapy) showed significantly higher diastolic BP. Furthermore, rs13175330 G allele carriers in the HTN w/o therapy group had significantly increased levels of insulin, insulin resistance, and oxidized low-density lipoprotein (LDL) and significantly decreased LDL-cholesterol levels and LDL particle sizes compared to the AA carriers. These results suggest that the PAM rs13175330 A>G SNP is a novel candidate gene for HTN in the Korean population. Additionally, the PAM rs13175330 G allele might be associated with insulin resistance and LDL atherogenicity in patients with HTN.

Application of polyacrylamide (PAM) through lay-flat polyethylene 1 tubing: effects on infiltration, erosion, N and P transport, and corn yield

Science.gov (United States)

Polyacrylamides (PAMs), when applied as a soil amendment, purportedly improve soil infiltration, decrease erosion, and reduce off-site agrochemical transport. The effect of PAM on infiltration, erosion, agrochemical transport, and crop yield when applied in-furrow to Mid-South production systems has...

112-Gbit/s × 4-lane duobinary-4-PAM for 400GBase

DEFF Research Database (Denmark)

Suhr, Lau Frejstrup; Vegas Olmos, Juan José; Mao, B.

2014-01-01

Novel duobinary-4-PAM signaling is experimentally demonstrated to support a 4-lane low-latency 400GbE client side solution. Direct detection of 112 Gbps transmission over a 5 km single wavelength and polarization fiber link is achieved....

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

Science.gov (United States)

Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-01

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

Directory of Open Access Journals (Sweden)

Robert Root-Bernstein

2018-01-01

Full Text Available Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Fabrication and atomic force microscopy/friction force microscopy (AFM/FFM) studies of polyacrylamide-carbon nanotubes (PAM-CNTs) copolymer thin films

International Nuclear Information System (INIS)

Li Xuefeng; Guan Wenchao; Yan Haibiao; Huang Lan

2004-01-01

A novel polyacrylamide-carbon nanotubes (PAM-CNTs) copolymer has been prepared by ultraviolet radiation initiated polymerization. The PAM-CNTs copolymer was characterized by the instruments of Fourier transform infrared spectroscopy, UV-vis absorbance spectra, fluorescence spectra and transmission electron microscope. The morphology and microtribological properties of PAM-CNTs thin films on mica were investigated by atomic force microscopy/friction force microscopy (AFM/FFM). The friction of the films was stable with the change of applied load and the friction coefficient decreased significantly as the CNTs addition. The results show that the rigid rod-like CNTs in polymer would enhance load-bearing and anti-wear properties of the thin films

Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

Directory of Open Access Journals (Sweden)

Deanna M Navaroli

Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

PAM50: Unbiased multimodal template of the brainstem and spinal cord aligned with the ICBM152 space.

Science.gov (United States)

De Leener, Benjamin; Fonov, Vladimir S; Collins, D Louis; Callot, Virginie; Stikov, Nikola; Cohen-Adad, Julien

2018-01-15

Template-based analysis of multi-parametric MRI data of the spinal cord sets the foundation for standardization and reproducibility, thereby helping the discovery of new biomarkers of spinal-related diseases. While MRI templates of the spinal cord have been recently introduced, none of them cover the entire spinal cord. In this study, we introduced an unbiased multimodal MRI template of the spinal cord and the brainstem, called PAM50, which is anatomically compatible with the ICBM152 brain template and uses the same coordinate system. The PAM50 template is based on 50 healthy subjects, covers the full spinal cord (C1 to L2 vertebral levels) and the brainstem, is available for T1-, T2-and T2*-weighted MRI contrasts and includes a probabilistic atlas of the gray matter and white matter tracts. Template creation accuracy was assessed by computing the mean and maximum distance error between each individual spinal cord centerline and the PAM50 centerline, after registration to the template. Results showed high accuracy for both T1- (mean = 0.37 ± 0.06 mm; max = 1.39 ± 0.58 mm) and T2-weighted (mean = 0.11 ± 0.03 mm; max = 0.71 ± 0.27 mm) contrasts. Additionally, the preservation of the spinal cord topology during the template creation process was verified by comparing the cross-sectional area (CSA) profile, averaged over all subjects, and the CSA profile of the PAM50 template. The fusion of the PAM50 and ICBM152 templates will facilitate group and multi-center studies of combined brain and spinal cord MRI, and enable the use of existing atlases of the brainstem compatible with the ICBM space. Copyright © 2017 Elsevier Inc. All rights reserved.

Patient activation in Europe: an international comparison of psychometric properties and patients' scores on the short form Patient Activation Measure (PAM-13).

Science.gov (United States)

Rademakers, Jany; Maindal, Helle Terkildsen; Steinsbekk, Aslak; Gensichen, Jochen; Brenk-Franz, Katja; Hendriks, Michelle

2016-10-12

To allow better assessment of patients' individual competencies for self-management, the Patient Activation Measure (PAM) has been developed in the USA. Because the American studies have shown the PAM to be a valuable tool, several European countries have translated the instrument into their native languages (Danish, Dutch, German, Norwegian). The aim was to compare the psychometric properties in studies from the different countries and establish whether the scores on the PAM vary between the studies. Data from the four separate studies were subjected to the same data cleaning procedures and statistical analyses. The psychometric properties of the instruments were established with measures of data quality and scale structure. The mean patient activation score and distribution across four predefined activation levels were described and the differences between the four studies were tested with ANOVA (unadjusted and adjusted) followed by a post-hoc Tukey HSD test and the Pearson chi-squared test respectively. The total N of the four studies was 5184. The percentage of missing values was low in all datasets, confirming the good quality of the datasets. Factor analyses revealed moderate to strong factor loadings on the first factor in all datasets. Cronbach's α was high for all version, ranging from .80 (German) to .88 (Dutch). Item-rest correlations varied between .32 and .66, indicating a moderate to strong correlation of the individual items to the sum scale. Both the mean PAM score and the distribution across activation levels differed between the four datasets. After adjustment of the PAM score, patients in Norway in particular had a higher patient activation level. The European translations of PAM-13 (into Danish, Dutch, German and Norwegian) resulted in four instruments with good psychometric capabilities for measuring patient activation. The mean PAM score and the distribution across activation levels differed between the four datasets.

Experimental study of 112 Gb/s short reach transmission employing PAM formats and SiP intensity modulator at 1.3 μm.

Science.gov (United States)

Chagnon, Mathieu; Osman, Mohamed; Poulin, Michel; Latrasse, Christine; Gagné, Jean-Frédéric; Painchaud, Yves; Paquet, Carl; Lessard, Stéphane; Plant, David

2014-08-25

We present a Silicon Photonic (SiP) intensity modulator operating at 1.3 μm with pulse amplitude modulation formats for short reach transmission employing a digital to analog converter for the RF signal generator, enabling pulse shaping and precompensation of the transmitter's frequency response. Details of the SiP Mach-Zehnder interfometer are presented. We study the system performance at various bit rates, PAM orders and propagation distances. To the best of our knowledge, we report the first demonstration of a 112 Gb/s transmission over 10 km of SMF fiber operating below pre-FEC BER threshold of 3.8 × 10(-3) employing PAM-8 at 37.4 Gbaud using a fully packaged SiP modulator. An analytical model for the Q-factor metric applicable for multilevel PAM-N signaling is derived and accurately experimentally verified in the case of Gaussian noise limited detection. System performance is experimentally investigated and it is demonstrated that PAM order selection can be optimally chosen as a function of the desired throughput. We demonstrate the ability of the proposed transmitter to exhibit software-defined transmission for short reach applications by selecting PAM order, symbol rate and pulse shape.

Von Willebrand protein binds to extracellular matrices independently of collagen.

OpenAIRE

Wagner, D D; Urban-Pickering, M; Marder, V J

1984-01-01

Von Willebrand protein is present in the extracellular matrix of endothelial cells where it codistributes with fibronectin and types IV and V collagen. Bacterial collagenase digestion of endothelial cells removed fibrillar collagen, but the pattern of fibronectin and of von Willebrand protein remained undisturbed. Exogenous von Willebrand protein bound to matrices of different cells, whether rich or poor in collagen. von Willebrand protein also decorated the matrix of cells grown in the prese...

The toll-like receptor 2 agonist Pam3CSK4 is neuroprotective after spinal cord injury.

Science.gov (United States)

Stivers, Nicole S; Pelisch, Nicolas; Orem, Ben C; Williams, Joshua; Nally, Jacqueline M; Stirling, David P

2017-08-01

Microglia/macrophage activation and recruitment following spinal cord injury (SCI) is associated with both detrimental and reparative functions. Stimulation of the innate immune receptor Toll-like receptor-2 (TLR2) has shown to be beneficial following SCI, and it increases axonal regeneration following optic nerve crush. However, the mechanism(s) remain unclear. As microglia express high levels of TLR2, we hypothesized that modulating the microglial response to injury using a specific TLR2 agonist, Pam3CSK4, would prevent secondary-mediated white matter degeneration following SCI. To test this hypothesis, we documented acute changes in microglia, axons, and oligodendroglia over time using two-photon excitation and an ex vivo laser-induced SCI (LiSCI) model. We utilized double transgenic mice that express GFP in either microglia or oligodendroglia, and YFP in axons, and we applied the lipophilic fluorescent dye (Nile Red) to visualize myelin. We found that treatment with Pam3CSK4 initiated one hour after injury induced a significant increase in the extent and timing of the microglial response to injury compared to vehicle controls. This enhanced response was observed 2 to 4h following SCI and was most prominent in areas closer to the ablation site. In addition, Pam3CSK4 treatment significantly reduced axonal dieback rostral and caudal to the ablation at 6h post-SCI. This protective effect of Pam3CSK4 was also mirrored when assessing secondary bystander axonal damage (i.e., axons spared by the primary injury that then succumb to secondary degeneration), and when assessing the survival of oligodendroglia. Following these imaging experiments, custom microarray analysis of the ex vivo spinal cord preparations revealed that Pam3CSK4-treatment induced an alternative (mixed M1:M2) microglial activation profile. In summary, our data suggest that by providing a second "sterile" activation signal to microglia through TLR2/TLR1 signaling, the microglial response to injury can

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry

International Nuclear Information System (INIS)

Herlory, Olivier; Bonzom, Jean-Marc; Gilbin, Rodolphe

2013-01-01

Highlights: •Our study addressed the toxicity thresholds of uranium on microalgae using PAM fluorometry. •The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium. •Uranium impaired the electron flux between the photosystems until almost complete inhibition. •Non-photochemical quenching was identified as the most sensitive fluorescence parameter. •PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response. -- Abstract: Although ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5 h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F 0 /F v . Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency (F ′ q /F ′ m , EC 50 = 303 ± 64 μg U L −1 after 5 h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC 50 = 142 ± 98 μg U L −1 after 5 h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from

Valid measures of periodic leg movements (PLMs) during a suggested immobilization test using the PAM-RL leg activity monitors require adjusting detection parameters for noise and signal in each recording.

Science.gov (United States)

Yang, Myung Sung; Montplaisir, Jacques; Desautels, Alex; Winkelman, John W; Cramer Bornemann, Michel A; Earley, Christopher J; Allen, Richard P

2014-01-01

Individuals with restless legs syndrome (RLS) (Willis-Ekbom disease [WED]) usually have periodic leg movements (PLMs). The suggested immobilization test (SIT) measures sensory and motor features of WED during wakefulness. Surface electromyogram (EMG) recordings of the anterior tibialis (AT) are used as the standard for counting PLMs. However, due to several limitations, leg activity meters such as the PAM-RL were advanced as a potential substitute. In our study, we assessed the validity of the measurements of PLM during wakefulness (PLMW) in the SIT for PAM-RL using both default and custom detection threshold parameters compared to AT EMG. Data were obtained from 39 participants who were diagnosed with primary WED and who were on stable medication as part of another study using the SIT to repeatedly evaluate WED symptoms over 6-12 months. EMG recordings and PAM-RL, when available, were used to detect PLMW for each SIT. Complete PAM-RL and polysomnography (PSG) EMG data were available for 253 SITs from that study. The default PAM-RL (dPAM-RL) detected leg movements based on manufacturer's noise (resting) and signal (movement) amplitude criteria developed to accurately detect PLM during sleep (PLMS). The custom PAM-RL (cPAM-RL) similarly detected leg movements except the noise and movement detection parameters were adjusted to match the PAM-RL data for each SIT. The distributions of the differences between either dPAM-RL or cPAM-RL and EMG PLMW were strongly leptokurtic (Kurtosis >2) with many small differences and a few unusually large differences. These distributions are better described by median and quartile ranges than mean and standard deviation. Despite an adequate correlation (r=0.66) between the dPAM-RL and EMG recordings, the dPAM-RL on average significantly underscored the number of PLMW (median: quartiles=-13: -51.2, 0.0) and on Bland-Altman plots had a significant magnitude bias with greater underscoring for larger average PLMW/h. There also was an

A designated centre for people with disabilities operated by St John of God Community Services Ltd, Louth

LENUS (Irish Health Repository)

Jones, Robert T

2010-05-12

Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of

Experimental Study of 1.55-μm EML-Based Optical IM/DD PAM-4/8 Short Reach Systems

DEFF Research Database (Denmark)

Pang, Xiaodan; Ozolins, Oskars; Gaiarin, Simone

2017-01-01

We experimentally evaluate high-speed intensity modulation/direct detection (IM/DD) transmissions with a 1.55-μm broadband electro-absorption modulated laser and pulse amplitude modulations (PAM). We demonstrate 80 Gb/s/λ PAM-4 and 96 Gb/s/λ PAM-8 transmissions with low-complexity digital...... equalizers at the receiver. Performance comparison with different types of equalizers are performed, including linear symbol-spaced feed-forward equalizer (FFE), fractional (half-symbol) spaced FFE and decision feedback equalizer (DFE), with different tap number. It is found that for both cases, a 6-tap...... symbol-spaced FFE is sufficient to achieve a stable performance with bit-error-rate below the 7% overhead hard decision forward error correction (7%-OH HD-FEC) threshold over a 4 km standard single mode fiber link. Practical considerations including comparison between adaptive and static equalizer...

Solar Radiation Stress in Natural Acidophilic Biofilms of Euglena mutabilis Revealed by Metatranscriptomics and PAM Fluorometry.

Science.gov (United States)

Puente-Sánchez, Fernando; Olsson, Sanna; Gómez-Rodriguez, Manuel; Souza-Egipsy, Virginia; Altamirano-Jeschke, Maria; Amils, Ricardo; Parro, Victor; Aguilera, Angeles

2016-02-01

The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Río Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday. Copyright © 2016 Elsevier GmbH. All rights reserved.

Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Wenfang; Li, Huan; Hallstrøm, Søren

2013-01-01

-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3......" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show...

Performance assessment methodology (PAM) for low level radioactive waste (LLRW) disposal facilities

International Nuclear Information System (INIS)

Selander, W.N.

1992-01-01

An overview is given for Performance Assessment Methodology (PAM) for Low Level Radioactive Waste (LLRW) disposal technologies, as required for licensing and safety studies. This is a multi-disciplinary activity, emphasizing applied mathematics, mass transfer, geohydrology and radiotoxicity effects on humans. (author). 2 refs

Asynchronous monitoring of the quality of multilevel optical PAM signals

Science.gov (United States)

Siuzdak, J.

2017-08-01

In the paper, there is analyzed the signal quality assessment method based on delay tap asynchronous sampling, both for binary and multilevel PAM signals. The obtained multilevel phase diagrams are far more complicated than binary ones. The phase diagrams are affected by the signal distortions but it is difficult to relate reliably the phase diagram form to the distortion type and its influence on the signal quality.

Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

Science.gov (United States)

Kim, Jung-Hwan; Jeun, Eun-Ji; Hong, Chun-Pyo; Kim, Seong-Hoon; Jang, Min Seong; Lee, Eun-Jung; Moon, Sook Jin; Yun, Chang Ho; Im, Sin-Hyeog; Jeong, Seok-Geun; Park, Beom-Young; Kim, Kyong-Tai; Seoh, Ju-Young; Kim, Yoon-Keun; Oh, Sung-Jong; Ham, Jun-Sang; Yang, Bo-Gie; Jang, Myoung Ho

2016-02-01

The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Optimum quantum receiver for detecting weak signals in PAM communication systems

Science.gov (United States)

Sharma, Navneet; Rawat, Tarun Kumar; Parthasarathy, Harish; Gautam, Kumar

2017-09-01

This paper deals with the modeling of an optimum quantum receiver for pulse amplitude modulator (PAM) communication systems. The information bearing sequence {I_k}_{k=0}^{N-1} is estimated using the maximum likelihood (ML) method. The ML method is based on quantum mechanical measurements of an observable X in the Hilbert space of the quantum system at discrete times, when the Hamiltonian of the system is perturbed by an operator obtained by modulating a potential V with a PAM signal derived from the information bearing sequence {I_k}_{k=0}^{N-1}. The measurement process at each time instant causes collapse of the system state to an observable eigenstate. All probabilities of getting different outcomes from an observable are calculated using the perturbed evolution operator combined with the collapse postulate. For given probability densities, calculation of the mean square error evaluates the performance of the receiver. Finally, we present an example involving estimating an information bearing sequence that modulates a quantum electromagnetic field incident on a quantum harmonic oscillator.

Red Wine administration to Apolipoprotein E-deficient Mice reduces their Macrophage-derived Extracellular Matrix Atherogenic Properties

Directory of Open Access Journals (Sweden)

MARIELLE KAPLAN

2004-01-01

Full Text Available Proteoglycans (PGs from the arterial extracellular matrix (ECM contribute to the trapping of LDL and oxidized LDL (Ox-LDL in the arterial wall, a phenomenon called "lipoprotein retention". Moreover, we have shown that subsequent to their binding to the matrix, LDL and Ox-LDL are taken up by macrophages. Oxidative stress significantly increases macrophage secretion of ECM-PGs, lipoprotein binding to the ECM and the uptake of ECM-retained lipoproteins by macrophages. The aim of the present study was to determine whether red wine administration to atherosclerotic mice would affect their peritoneal macrophage-derived extracellular matrix properties, such as the glycosaminoglycan content and the ability to bind LDL. In addition, we questioned the ability of LDL bound to the mice peritoneal macrophages-derived ECM to be taken up by macrophages. Red wine administration to atherosclerotic mice did not affect the mice peritoneal macrophages-derived ECM glycosaminoglycan content but it significantly reduced the mice peritoneal macrophages-derived ECM ability to bind LDL and the subsequent uptake of ECM-retained LDL by the macrophages. The present study thus clearly demonstrated the inhibitory effect of red wine consumption by E0 mice on their peritoneal macrophage-derived extracellular matrix atherogenic properties.

Clinicians' beliefs and attitudes toward patient self-management in the Netherlands; translation and testing of the American Clinician Support for Patient Activation Measure (CS-PAM).

Science.gov (United States)

Rademakers, Jany; Jansen, Daphne; van der Hoek, Lucas; Heijmans, Monique

2015-04-03

The aim of this study was to test the Dutch version of the Clinician Support for Patient Activation Measure (CS-PAM), to explore the beliefs of Dutch clinicians about patients' self-management, and to establish whether there are differences in this respect between general practitioners and other primary care providers. The CS-PAM was translated in Dutch and data were collected in a sample of 489 general practitioners and other primary care providers. Statistical analyses (RASCH, Cronbach's α) were performed to establish the psychometric properties of the instrument. The psychometric scores of the Dutch CS-PAM were acceptable to good, and the difficulty level and structure was comparable to that of the original instrument. The average score of Dutch clinicians on the CS-PAM was 65.1 (SD 10.7), somewhat lower compared to their colleagues in the US (69; SD 12.1) and the UK (69, SD 12.8). Dutch general practitioners scored significantly lower on the CS-PAM compared to other primary care providers. The Dutch CS-PAM is a reliable instrument to measure beliefs of clinicians regarding patient self-management. Further validation studies are necessary to establish the distribution of scores in specific provider populations and to assess the clinical relevance of the instrument for different outcomes.

Discovery of VU6005649, a CNS Penetrant mGlu7/8 Receptor PAM Derived from a Series of Pyrazolo[1,5-a]pyrimidines.

Science.gov (United States)

Abe, Masahito; Seto, Mabel; Gogliotti, Rocco G; Loch, Matthew T; Bollinger, Katrina A; Chang, Sichen; Engelberg, Eileen M; Luscombe, Vincent B; Harp, Joel M; Bubser, Michael; Engers, Darren W; Jones, Carrie K; Rodriguez, Alice L; Blobaum, Anna L; Conn, P Jeffrey; Niswender, Colleen M; Lindsley, Craig W

2017-10-12

Herein, we report the structure-activity relationships within a series of mGlu 7 PAMs based on a pyrazolo[1,5- a ]pyrimidine core with excellent CNS penetration ( K p s > 1 and K p,uu s > 1). Analogues in this series proved to display a range of Group III mGlu receptor selectivity, but VU6005649 emerged as the first dual mGlu 7/8 PAM, filling a void in the Group III mGlu receptor PAM toolbox and demonstrating in vivo efficacy in a mouse contextual fear conditioning model.

PAMs ameliorates the imiquimod-induced psoriasis-like skin disease in mice by inhibition of translocation of NF-κB and production of inflammatory cytokines.

Directory of Open Access Journals (Sweden)

Rongkun Dou

Full Text Available Psoriasis is a chronic and persistent inflammatory skin disease seriously affecting the quality of human life. In this study, we reported an ancient formula of Chinese folk medicine, the natural plant antimicrobial solution (PAMs for its anti-inflammatory effects and proposed the primary mechanisms on inhibiting the inflammatory response in TNF-α/IFN-γ-induced HaCaT cells and imiquimod-induced psoriasis-like skin disease mouse model. Two main functional components of hydroxysafflor Yellow A and allantoin in PAMs were quantified by HPLC to be 94.2±2.2 and 262.9±12.5 μg/mL respectively. PAMs could significantly reduce the gene expression and inflammatory cytokines production of Macrophage-Derived Chemokine (MDC, IL-8 and IL-6 in TNF-α/IFN-γ-induced HaCaT cells. PAMs also significantly ameliorates the psoriatic-like symptoms in a mouse model with the evaluation scores for both the single (scales, thickness, erythema and cumulative features were in the order of blank control < Dexamethasone < PAMs < 50% ethanol < model groups. The results were further confirmed by hematoxylin-eosin staining, RT-qPCR and immunohistochemistry. The down-regulated gene expression of IL-8, TNF-α, ICAM-1 and IL-23 in mouse tissues was consistent with the results from those of the HaCaT cells. The inhibition of psoriasis-like skin inflammation by PAMs was correlated with the inactivation of the translocation of P65 protein into cellular nucleus, indicating the inhibition of the inflammatory NF-κB signaling pathway. Taken together, these findings suggest that PAMs may be a promising drug candidate for the treatment of inflammatory skin disorders, such as psoriasis.

A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

Directory of Open Access Journals (Sweden)

Yu Zhu

2015-11-01

Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

Directory of Open Access Journals (Sweden)

Adam Bajinting

2017-06-01

Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

KUALITAS FISIK DAN KIMIA AIR PAM DI JAKARTA, BOGOR, TANGERANG, BEKASI TAHUN 1999 - 2001

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Mariana Raini

2012-09-01

Full Text Available Drinking water quality of PAM in Jakarta, Bogor, Tangerang and Bekasi between the year of 1999 - 2001 has been studied. Water quality was determined by a series of physical and chemical test at Chemical Laboratory Pusat Penelitian dan Pengembangan Farmasi dan Obat Tradisional as stated in Permenkes no.416/Menkes/Per/IX/1990 on condition and water quality control including smell, color, turbidity, contents of manganese, iron, nitrite, sulphate, organic subtance, turbidity, hardness and pH. Total sampling was done on all drinking water samples from DKI Jakarta, Bogor, Tangerang and Bekasi delivered to be tested at Pusat Penelitian dan Pengembangan Farmasi dan Obat Tradisional between 1999-2001 and destilled water as control. Result of the study from 431 drinking water samples shows that samples that met the requirements as drinking water is 91,65% and 8,35% sub standard, most of the the sub standard drinking water is cause by the contents of manganese (4,41%, iron (2,09%, organic subtance (1,62%, chloride (0,93% and high water hardness (0,23% as well as pH (3,15%, turbidity (1,86% and colors (1,62% Statistically, the water quality of PAM in Jakarta, Bogor, Tangerang and Bekasi at 1999, 2000 and 2001 is not different.  Key word : water quality of PAM, drinking water, physical test, chemical test.

Improvement of an automated protein crystal exchange system PAM for high-throughput data collection

International Nuclear Information System (INIS)

Hiraki, Masahiko; Yamada, Yusuke; Chavas, Leonard M. G.; Wakatsuki, Soichi; Matsugaki, Naohiro

2013-01-01

A special liquid-nitrogen Dewar with double capacity for the sample-exchange robot has been created at AR-NE3A at the Photon Factory, allowing continuous fully automated data collection. In this work, this new system is described and the stability of its calibration is discussed. Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable

Improvement of an automated protein crystal exchange system PAM for high-throughput data collection

Energy Technology Data Exchange (ETDEWEB)

Hiraki, Masahiko, E-mail: masahiko.hiraki@kek.jp; Yamada, Yusuke; Chavas, Leonard M. G. [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); Wakatsuki, Soichi [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, CA 94025-7015 (United States); Stanford University, Beckman Center B105, Stanford, CA 94305-5126 (United States); Matsugaki, Naohiro [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan)

2013-11-01

A special liquid-nitrogen Dewar with double capacity for the sample-exchange robot has been created at AR-NE3A at the Photon Factory, allowing continuous fully automated data collection. In this work, this new system is described and the stability of its calibration is discussed. Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

From the Field: Speech Therapy Outcome Measures--Interview with Dr. Pam Enderby

Science.gov (United States)

Montgomery, Judy K.

2015-01-01

This article is an interview with Dr. Pam Enderby--a speech language therapist and professor at the Institute of General Practice and Primary Care at the University of Sheffield, Community Sciences Centre, Northern General Hospital, in the United Kingdom--conducted by Judy Montgomery, Editor in Chief, of "Communication Disorders…

Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain.

Science.gov (United States)

Kim, Il-Doo; Lim, Chae-Moon; Kim, Jung-Bin; Nam, Hye Yeong; Nam, Kihoon; Kim, Seung-Woo; Park, Jong-Sang; Lee, Ja-Kyeong

2010-03-19

Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of

Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs Formation and Arterial Thrombosis.

Directory of Open Access Journals (Sweden)

Daniella M Mizurini

Full Text Available The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs, a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability.This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method.Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host's hemostatic responses and innate immune system.

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry

Energy Technology Data Exchange (ETDEWEB)

Herlory, Olivier, E-mail: olivier.herlory@gmail.com [IRSN-Laboratoire d’Ecotoxicologie des Radionucléides, Centre de Cadarache, BP3, 13115 Saint Paul lez Durance (France); Bonzom, Jean-Marc, E-mail: jean-marc.bonzom@irsn.fr [IRSN-Laboratoire d’Ecotoxicologie des Radionucléides, Centre de Cadarache, BP3, 13115 Saint Paul lez Durance (France); Gilbin, Rodolphe, E-mail: rodolphe.gilbin@irsn.fr [IRSN-Laboratoire de Biogéochimie, Biodisponibilité et Transferts des Radionucléides, Centre de Cadarache, BP3, 13115 Saint Paul lez Durance (France)

2013-09-15

Highlights: •Our study addressed the toxicity thresholds of uranium on microalgae using PAM fluorometry. •The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium. •Uranium impaired the electron flux between the photosystems until almost complete inhibition. •Non-photochemical quenching was identified as the most sensitive fluorescence parameter. •PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response. -- Abstract: Although ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5 h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F{sub 0}/F{sub v}. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency (F{sup ′}{sub q}/F{sup ′}{sub m}, EC{sub 50} = 303 ± 64 μg U L{sup −1} after 5 h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC{sub 50} = 142 ± 98 μg U L{sup −1} after 5 h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown

ANTS/PAM: Future Exploration of the Asteroid Belt

Science.gov (United States)

Clark, P. E.; Curtis, S. A.; Rilee, M. L.; Cheung, C. Y.

2004-05-01

The Autonomous Nano-Technology Swarm (ANTS) is applied to the Prospecting Asteroid Mission (PAM) concept, as part of a NASA RASC study. The ANTS architecture is inspired by success of social insect colonies, based on the division of labor within the colonies: 1) within their specialties, individual specialists generally outperform general-ists, and 2) with sufficiently efficient social interaction and coordination, the group of specialists generally outper-forms the group of generalists. ANTS as applied to PAM involves a thousand individual specialist `sciencecraft', one subswarm per target, in an environment where detection and tracking of irregular, infrequent targets is a major chal-lenge. Workers, carry and operate eight to nine different scientific instruments, including spectrometers, ranging and radio science devices, imagers. The remaining specialists, Messenger/Rulers, provide communication and coordina-tion. The non-expendable propulsion system is based on autonomously deployable and configurable solar sails, a system suitable to a low gravity environment. The design of the neural basis function requires a minimum of 4 or 5 specialists for collective decision making. Allowing for ten instrument specialist teams and compensating for antici-pated high attrition, we calculate an initial minimum of 100 per subswarm should allow characterization of hundreds of asteroids. The difficulty in observing irregular, rapidly moving, poorly illuminated objects is largely overcome by the ANT sciencecraft capability to optimize conditions for each instrument. Components are composed of carbon nanotubules reversibly deployable from NEMS nodes, allowing 100 times decrease in packaging volume. 1000 smart 10 centimeter, 1 kg cubic boxes create a 1000 kg 1 meter cube.

Comparing Longitudinal Profile Patterns of Mathematics and Reading in Early Child Longitudinal Study, Kindergarten: The Profile Analysis via Multidimensional Scaling (PAMS) Approach

Science.gov (United States)

Kim, Se-Kang

2010-01-01

The aim of the study is to compare longitudinal patterns from Mathematics and Reading data from the direct child assessment of Early Child Longitudinal Study, Kindergarten (ECLS-K, US Department of Education, National Center for Education Statistics 2006), utilizing Profile Analysis via Multidimensional Scaling (PAMS). PAMS has been used initially…

Time skewing and amplitude nonlinearity mitigation by feedback equalization for 56 Gbps VCSEL-based PAM-4 links

Science.gov (United States)

You, Yue; Zhang, Wenjia; Sun, Lin; Du, Jiangbing; Liang, Chenyu; Yang, Fan; He, Zuyuan

2018-03-01

The vertical cavity surface emitting laser (VCSEL)-based multimode optical transceivers enabled by pulse amplitude modulation (PAM)-4 will be commercialized in near future to meet the 400-Gbps standard short reach optical interconnects. It is still challenging to achieve over 56/112-Gbps with the multilevel signaling as the multimode property of the device and link would introduce the nonlinear temporal response for the different levels. In this work, we scrutinize the distortions that relates to the multilevel feature of PAM-4 modulation, and propose an effective feedback equalization scheme for 56-Gbps VCSEL-based PAM-4 optical interconnects system to mitigate the distortions caused by eye timing-skew and nonlinear power-dependent noise. Level redistribution at Tx side is theoretically modeled and constructed to achieve equivalent symbol error ratios (SERs) of four levels and improved BER performance. The cause of the eye skewing and the mitigation approach are also simulated at 100-Gbps and experimentally investigated at 56-Gbps. The results indicate more than 2-dB power penalty improvement has been achieved by using such a distortion aware equalizer.

Upper Limb Rehabilitation Robot Powered by PAMs Cooperates with FES Arrays to Realize Reach-to-Grasp Trainings

Science.gov (United States)

Su, Chen; Jiang, Xiaobo

2017-01-01

The reach-to-grasp activities play an important role in our daily lives. The developed RUPERT for stroke patients with high stiffness in arm flexor muscles is a low-cost lightweight portable exoskeleton rehabilitation robot whose joints are unidirectionally actuated by pneumatic artificial muscles (PAMs). In order to expand the useful range of RUPERT especially for patients with flaccid paralysis, functional electrical stimulation (FES) is taken to activate paralyzed arm muscles. As both the exoskeleton robot driven by PAMs and the neuromuscular skeletal system under FES possess the highly nonlinear and time-varying characteristics, iterative learning control (ILC) is studied and is taken to control this newly designed hybrid rehabilitation system for reaching trainings. Hand function rehabilitation refers to grasping. Because of tiny finger muscles, grasping and releasing are realized by FES array electrodes and matrix scan method. By using the surface electromyography (EMG) technique, the subject's active intent is identified. The upper limb rehabilitation robot powered by PAMs cooperates with FES arrays to realize active reach-to-grasp trainings, which was verified through experiments. PMID:29065566

Upper Limb Rehabilitation Robot Powered by PAMs Cooperates with FES Arrays to Realize Reach-to-Grasp Trainings

Directory of Open Access Journals (Sweden)

Xikai Tu

2017-01-01

Full Text Available The reach-to-grasp activities play an important role in our daily lives. The developed RUPERT for stroke patients with high stiffness in arm flexor muscles is a low-cost lightweight portable exoskeleton rehabilitation robot whose joints are unidirectionally actuated by pneumatic artificial muscles (PAMs. In order to expand the useful range of RUPERT especially for patients with flaccid paralysis, functional electrical stimulation (FES is taken to activate paralyzed arm muscles. As both the exoskeleton robot driven by PAMs and the neuromuscular skeletal system under FES possess the highly nonlinear and time-varying characteristics, iterative learning control (ILC is studied and is taken to control this newly designed hybrid rehabilitation system for reaching trainings. Hand function rehabilitation refers to grasping. Because of tiny finger muscles, grasping and releasing are realized by FES array electrodes and matrix scan method. By using the surface electromyography (EMG technique, the subject’s active intent is identified. The upper limb rehabilitation robot powered by PAMs cooperates with FES arrays to realize active reach-to-grasp trainings, which was verified through experiments.

Photo-Activated Localization Microscopy of Single Carbohydrate Binding Modules on Cellulose Nanofibers

Science.gov (United States)

Hor, Amy; Dagel, Daryl; Luu, Quocanh; Savaikar, Madhusudan; Ding, Shi-You; Smith, Steve

2015-03-01

Photo Activated Localization Microscopy (PALM) is used to conduct an in vivo study of the binding affinity of polysaccharide-specific Carbohydrate Binding Modules (CBMs) to insoluble cellulose substrates. Two families of CBMs, namely TrCBM1 and CtCBM3, were modified to incorporate photo-activatable mCherry fluorescent protein (PAmCherry), and exposed to highly crystalline Valonia cellulose nano-fibrils. The resulting PALM images show CBMs binding along the nano-fibril long axis in a punctuated linear array, localized with, on average, 10 nm precision. Statistical analysis of the binding events results in nearest neighbor distributions between CBMs. A comparison between TrCBM1 and CtCBM3 reveals a similarity in the nearest neighbor distribution peaks but differences in the overall binding density. The former is attributed to steric hindrance among the CBMs on the nano-fibril whereas the latter is attributed to differences in the CBMs' binding strength. These results are compared to similar distributions derived from TEM measurements of dried samples of CtCBM3-CdSs quantum dot bioconjugates and AFM images of CtCBM3-GFP bound to similar Valonia nano-fibrils. Funding provided by NSF MPS/DMR/BMAT Award # 1206908.

Experimental Demonstration of 84 Gb/s PAM-4 Over up to 1.6 km SSMF Using a 20-GHz VCSEL at 1525 nm

DEFF Research Database (Denmark)

Eiselt, Nicklas; Griesser, Helmut; Wei, Jinlong

2017-01-01

We demonstrate 84-Gb/s four-level pulse amplitude modulation (PAM-4) over up to 1.6-km standard single mode fiber using a 20-GHz single mode short cavity vertical cavity surface emitting laser diode at a transmission wavelength of 1525 nm. Different equalizer approaches including a common...... feedforward equalizer, a nonlinear Volterra equalizer (NLVE), a maximum likelihood sequence estimator (MLSE) and their combinations are evaluated working either as an equalizer for a standard PAM-4 or a partial response PAM-4 signal with seven levels. It is demonstrated that a standard FFE is not enough...... for a transmission distance of >0.6 km, while the use of an NLVE or FFE + MLSE is able to improve the transmission distance towards 1 km. The use of partial-response PAM-4 FFE in combination with a short memory MLSE is able to efficiently equalize the bandwidth limitations, showing more than 10-times BER improvement...

The Role of Extracellular Histones in Influenza Virus Pathogenesis.

Science.gov (United States)

Ashar, Harshini K; Mueller, Nathan C; Rudd, Jennifer M; Snider, Timothy A; Achanta, Mallika; Prasanthi, Maram; Pulavendran, Sivasami; Thomas, Paul G; Ramachandran, Akhilesh; Malayer, Jerry R; Ritchey, Jerry W; Rajasekhar, Rachakatla; Chow, Vincent T K; Esmon, Charles T; Teluguakula, Narasaraju

2018-01-01

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Extracellular enzyme kinetics scale with resource availability

Science.gov (United States)

Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

2014-01-01

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

Functional Elements on SIRPα IgV domain Mediate Cell Surface Binding to CD47

OpenAIRE

Liu, Yuan; Tong, Qiao; Zhou, Yubin; Lee, Hsiau-Wei; Yang, Jenny J.; Bühring, Hans-Jörg; Chen, Yi-Tien; Ha, Binh; Chen, Celia X-J.; Zen, Ke

2006-01-01

SIRPα and SIRPβ1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPα with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPβ1 ...

Dynamic bending of bionic flexible body driven by pneumatic artificial muscles(PAMs) for spinning gait of quadruped robot

Science.gov (United States)

Lei, Jingtao; Yu, Huangying; Wang, Tianmiao

2016-01-01

The body of quadruped robot is generally developed with the rigid structure. The mobility of quadruped robot depends on the mechanical properties of the body mechanism. It is difficult for quadruped robot with rigid structure to achieve better mobility walking or running in the unstructured environment. A kind of bionic flexible body mechanism for quadruped robot is proposed, which is composed of one bionic spine and four pneumatic artificial muscles(PAMs). This kind of body imitates the four-legged creatures' kinematical structure and physical properties, which has the characteristic of changeable stiffness, lightweight, flexible and better bionics. The kinematics of body bending is derived, and the coordinated movement between the flexible body and legs is analyzed. The relationship between the body bending angle and the PAM length is obtained. The dynamics of the body bending is derived by the floating coordinate method and Lagrangian method, and the driving force of PAM is determined. The experiment of body bending is conducted, and the dynamic bending characteristic of bionic flexible body is evaluated. Experimental results show that the bending angle of the bionic flexible body can reach 18°. An innovation body mechanism for quadruped robot is proposed, which has the characteristic of flexibility and achieve bending by changing gas pressure of PAMs. The coordinated movement of the body and legs can achieve spinning gait in order to improve the mobility of quadruped robot.

Laboratory algal bioassays using PAM fluorometry: effects of test conditions on the determination of herbicide and field sample toxicity.

Science.gov (United States)

Sjollema, Sascha B; van Beusekom, Sebastiaan A M; van der Geest, Harm G; Booij, Petra; de Zwart, Dick; Vethaak, A Dick; Admiraal, Wim

2014-05-01

Pulse Amplitude Modulation (PAM) fluorometry, based on chlorophyll a fluorescence, is a frequently used technique in algal bioassays to assess toxicity of single compounds or complex field samples. Several test conditions can influence the test results, and because a standardized test protocol is currently lacking, linking the results of different studies is difficult. Therefore, the aim of the present study was to gain insight into the effects of test conditions of laboratory algal bioassays using PAM fluorometry on the outcome of toxicity tests. To this purpose, we described the results from several pilot studies on test development in which information is provided on the effects of the main test factors during the pretest phase, the test preparation, the exposure period, and the actual measurement. The experiments were focused on individual herbicides and complex field samples and included the effects of culturing conditions, cell density, solvent concentration, exposure time, and the presence of actinic light. Several of these test conditions were found to influence the outcome of the toxicity test, and the presented information provides important background information for the interpretation of toxicity results and describes which test conditions should be taken into account when using an algal bioassay with PAM fluorometry. Finally, the application of PAM fluorometry in algal toxicity testing is discussed. © 2014 SETAC.

Real-Time Evaluation of 26-GBaud PAM-4 Intensity Modulation and Direct Detection Systems for Data-Center Interconnects

DEFF Research Database (Denmark)

Eiselt, Nicklas; Griesser, Helmut; Wei, Jinlong

2016-01-01

Real-time transmission with 26-GBaud PAM-4 as a promising modulation format for data-center interconnects with operation in C-band is evaluated. For an OSNR penalty below 2 dB a dispersion tolerance of up to 10 km of SSMF is achieved......Real-time transmission with 26-GBaud PAM-4 as a promising modulation format for data-center interconnects with operation in C-band is evaluated. For an OSNR penalty below 2 dB a dispersion tolerance of up to 10 km of SSMF is achieved...

Astrocytic autoantibody of neuromyelitis optica (NMO-IgG) binds to aquaporin-4 extracellular loops, monomers, tetramers and high order arrays

Science.gov (United States)

Iorio, Raffaele; Fryer, James P.; Hinson, Shannon R.; Fallier-Becker, Petra; Wolburg, Hartwig; Pittock, Sean J.; Lennon, Vanda A.

2012-01-01

The principal central nervous system (CNS) water channel, aquaporin-4 (AQP4), is confined to astrocytic and ependymal membranes and is the target of a pathogenic autoantibody, neuromyelitis optica (NMO)-IgG. This disease-specific autoantibody unifies a spectrum of relapsing CNS autoimmune inflammatory disorders of which NMO exemplifies the classic phenotype. Multiple sclerosis and other immune-mediated demyelinating disorders of the CNS lack a distinctive biomarker. Two AQP4 isoforms, M1 and M23, exist as homotetrameric and heterotetrameric intramembranous particles (IMPs). Orthogonal arrays of predominantly M23 particles (OAPs) are an ultrastructural characteristic of astrocytic membranes. We used high-titered serum from 32 AQP4-IgG-seropositive patients and 85 controls to investigate the nature and molecular location of AQP4 epitopes that bind NMO-IgG, and the influence of supramolecular structure. NMO-IgG bound to denatured AQP4 monomers (68% of cases), to native tetramers and high order arrays (90% of cases), and to AQP4 in live cell membranes (100% of cases). Disease-specific epitopes reside in extracellular loop C more than in loops A or E. IgG binding to intracellular epitopes lacks disease specificity. These observations predict greater disease specificity and sensitivity for tissue-based and cell-based serological assays employing “native” AQP4 than assays employing denatured AQP4 and fragments. NMO-IgG binds most avidly to plasma membrane surface AQP4 epitopes formed by loop interactions within tetramers and by intermolecular interactions within high order structures. The relative abundance and localization of AQP4 high order arrays in distinct CNS regions may explain the variability in clinical phenotype of NMO spectrum disorders. PMID:22906356

The soluble extracellular domain of E-cadherin interferes with EPEC adherence via interaction with the Tir:intimin complex.

Science.gov (United States)

Login, Frédéric H; Jensen, Helene H; Pedersen, Gitte A; Amieva, Manuel R; Nejsum, Lene N

2018-06-19

Enteropathogenic Escherichia coli (EPEC) causes watery diarrhea when colonizing the surface of enterocytes. The translocated intimin receptor (Tir):intimin receptor complex facilitates tight adherence to epithelial cells and formation of actin pedestals beneath EPEC. We found that the host cell adherens junction protein E-cadherin (Ecad) was recruited to EPEC microcolonies. Live-cell and confocal imaging revealed that Ecad recruitment depends on, and occurs after, formation of the Tir:intimin complex. Combinatorial binding experiments using wild-type EPEC, isogenic mutants lacking Tir or intimin, and E. coli expressing intimin showed that the extracellular domain of Ecad binds the bacterial surface in a Tir:intimin-dependent manner. Finally, addition of the soluble extracellular domain of Ecad to the infection medium or depletion of Ecad extracellular domain from the cell surface reduced EPEC adhesion to host cells. Thus, the soluble extracellular domain of Ecad may be used in the design of intervention strategies targeting EPEC adherence to host cells.-Login, F. H., Jensen, H. H., Pedersen, G. A., Amieva, M. R., Nejsum, L. N. The soluble extracellular domain of E-cadherin interferes with EPEC adherence via interaction with the Tir:intimin complex.

Multiband LTE-A, WiFi ac, and 4-PAM baseband simultaneous transmission over 50 m thick-core POF for in-home network

NARCIS (Netherlands)

Forni, F.; Shi, Y.; Van Den Boom, H.P.A.; Tangdiongga, E.; Koonen, A.M.J.

2017-01-01

We demonstrated the simultaneous transmission of multiband LTE-A signals, a WiFi IEEE802.11ac and a gigabit/s baseband 4-PAM signal over 1mm core diameter PMMA GI-POF. The optical link used a red light 650 nm laser diode and a p-i-n photodiode with a transimpedance amplifier. The 4-PAM transmission

Discovery of a novel, CNS penetrant M4 PAM chemotype based on a 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core.

Science.gov (United States)

Bewley, Blake R; Spearing, Paul K; Weiner, Rebecca L; Luscombe, Vincent B; Zhan, Xiaoyan; Chang, Sichen; Cho, Hyekyung P; Rodriguez, Alice L; Niswender, Colleen M; Conn, P Jeffrey; Bridges, Thomas M; Engers, Darren W; Lindsley, Craig W

2017-09-15

This Letter details the discovery and subsequent optimization of a novel M 4 PAM scaffold based on an 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core, which represents a distinct departure from the classical M 4 PAM chemotypes. Optimized compounds in this series demonstrated improved M 4 PAM potency on both human and rat M 4 (4 to 5-fold relative to HTS hit), and displayed attractive physicochemical and DMPK profiles, including good CNS penetration (rat brain:plasma K p =5.3, K p,uu =2.4; MDCK-MDR1 (P-gp) ER=1.1). Copyright © 2017 Elsevier Ltd. All rights reserved.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

Science.gov (United States)

Xie, Haihua; Tang, Lianchao; He, Xiubin; Liu, Xiexie; Zhou, Chenchen; Liu, Junjie; Ge, Xianglian; Li, Jin; Liu, Changbao; Zhao, Junzhao; Qu, Jia; Song, Zongming; Gu, Feng

2018-04-01

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of VU0467485/AZ13713945: An M4 PAM Evaluated as a Preclinical Candidate for the Treatment of Schizophrenia.

Science.gov (United States)

Wood, Michael R; Noetzel, Meredith J; Melancon, Bruce J; Poslusney, Michael S; Nance, Kellie D; Hurtado, Miguel A; Luscombe, Vincent B; Weiner, Rebecca L; Rodriguez, Alice L; Lamsal, Atin; Chang, Sichen; Bubser, Michael; Blobaum, Anna L; Engers, Darren W; Niswender, Colleen M; Jones, Carrie K; Brandon, Nicholas J; Wood, Michael W; Duggan, Mark E; Conn, P Jeffrey; Bridges, Thomas M; Lindsley, Craig W

2017-02-09

Herein, we report the structure-activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M 4 ) positive allosteric modulators (PAMs). Compound 6c (VU0467485) possesses robust in vitro M 4 PAM potency across species and in vivo efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and suitable predicted human PK, 6c (VU0467485) was evaluated as a preclinical development candidate.

Traffic disruption in PAM DIRAC road (Prévessin Site)

CERN Document Server

2003-01-01

From 8th September to 19th September, ST Division will be doing some road works to install HDPE ducts for optical fibre cables under the PAM DIRAC road. For this reason, the road will be closed during 2 days and alternative arrangements will be put in place to reroute the traffic. We kindly ask all users to respect these temporary arrangements. Thank you for your understanding in this matter. ST-EL Group Tel. 77779 - 160484 / 75498 - 163198

Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture.

Science.gov (United States)

Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-Ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

2010-01-15

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture*

Science.gov (United States)

Kii, Isao; Nishiyama, Takashi; Li, Minqi; Matsumoto, Ken-ichi; Saito, Mitsuru; Amizuka, Norio; Kudo, Akira

2010-01-01

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment. PMID:19887451

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

Science.gov (United States)

Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

2007-01-01

Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

Of Wondrous Places and "Benevolent Neglect": An Interview with Pam Munoz Ryan

Science.gov (United States)

Fabbi, Jennifer; Johnson, Amy

2007-01-01

With her recent book, "Paint the Wind" (2007), hitting the shelves this fall, author Pam Munoz Ryan delivers a welcome addition to the 25 plus books she has written for young people, including her award-winning novels "Esperanza Rising" (2000) and "Riding Freedom" (1998) and picture books "Amelia and Eleanor Go for a Ride" (1999) and "When Marian…

A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

Science.gov (United States)

Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

PAM50 Breast Cancer Subtyping by RT-qPCR and Concordance with Standard Clinical Molecular Markers

Directory of Open Access Journals (Sweden)

Bastien Roy RL

2012-10-01

Full Text Available Abstract Background Many methodologies have been used in research to identify the “intrinsic” subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions. Methods We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER, progesterone receptor (PR, and Her2/neu (HER2 protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH. Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and “intrinsic” subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments. Results ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC ≥ 0.9. Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B subtypes (92%, but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74% and Luminal A tumors were less likely to have high proliferation (11% vs 77%. Seventy-seven percent (30/39 of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57% and HER2-E (30% subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as

IMPLEMENTASI PENGELOLAAN AIR MINUM RUMAH TANGGA (PAM RT DI JAWA BARAT DAN (NUSA TENGGARA TIMUR

Directory of Open Access Journals (Sweden)

Athena Anwar

2016-09-01

Full Text Available Abstract In order to reduce waterborne diseases, the current government and its partners are developing ahousehold water treatment and safety storage (HWTS. This article is part of a study on Development ofAn Evidence-Based Guidelines for Promotion of HWTS which is conducted in three pilot sites: Bandungcity, Bandung district (West Java and Sikka district (East Nusa Tenggara in 2008. The aim of this studywas to find out how the program was implemented and how is the public opinion about the management(processing and storage of drinking water. Data collection was done by interview through a questionnaire.Data source were health officers and partners (qualitative data and the community (quantitative data.Qualitative data processing was performed by the content and the domain method, while quantitative datawas processed through SPSS software. The results show that the government develop the HWTS alongwith its partner: Aman Tirta (Bandung City, Pelita Indonesia (Bandung District, and Dian Desa (SikkaDistrict. Activity of HWTS was done through several stages, such as preparation/dissemination,implementation, monitoring and evaluation. Every partner will have its own way in carrying out the stagesof activity. Not all health officers of district/city states involved in the implementation of the HWTSprogram. It was only Sikka District Health officer that claimed to have fully engaged in the implementationof HWTS in the region. The results of processing and data analysis showed that more than 80% ofrespondents said the HWTS methods were suitable with drinking water treatment for their region. In termsof increasing the water quality, price of materials/tools, and easyness in the water treatment; respondents'opinions very widely.Keywords: HWTS, drinking water, water treatment Abstrak Dalam upaya menurunkan penyakit yang ditularkan melalui air, saat ini pemerintah dan mitrasedang mengembangkan pengelolaan air minum rumah tangga (PAM RT atau household water

Maximizing the effect of an α7 nicotinic receptor PAM in a mouse model of schizophrenia-like sensory inhibition deficits.

Science.gov (United States)

Stevens, Karen E; Zheng, Lijun; Floyd, Kirsten L; Stitzel, Jerry A

2015-06-22

Positive allosteric modulators (PAMs) for the α7 nicotinic receptor hold promise for the treatment of sensory inhibition deficits observed in schizophrenia patients. Studies of these compounds in the DBA/2 mouse, which models the schizophrenia-related deficit in sensory inhibition, have shown PAMs to be effective in improving the deficit. However, the first published clinical trial of a PAM for both sensory inhibition deficits and related cognitive difficulties failed, casting a shadow on this therapeutic approach. The present study used both DBA/2 mice, and C3H Chrna7 heterozygote mice to assess the ability of the α7 PAM, PNU-120596, to improve sensory inhibition. Both of these strains of mice have reduced hippocampal α7 nicotinic receptor numbers and deficient sensory inhibition similar to schizophrenia patients. Low doses of PNU-120596 (1 or 3.33mg/kg) were effective in the DBA/2 mouse but not the C3H Chrna7 heterozygote mouse. Moderate doses of the selective α7 nicotinic receptor agonist, choline chloride (10 or 33mg/kg), were also ineffective in improving sensory inhibition in the C3H Chrna7 heterozygote mouse. However, combining the lowest doses of both PNU-120596 and choline chloride in this mouse model did improve sensory inhibition. We propose here that the difference in efficacy of PNU-120596 between the 2 mouse strains is driven by differences in hippocampal α7 nicotinic receptor numbers, such that C3H Chrna7 heterozygote mice require additional direct stimulation of the α7 receptors. These data may have implications for further clinical testing of putative α7 nicotinic receptor PAMs. Copyright © 2015 Elsevier B.V. All rights reserved.

Probability approaching method (PAM) and its application on fuel management optimization

International Nuclear Information System (INIS)

Liu, Z.; Hu, Y.; Shi, G.

2004-01-01

For multi-cycle reloading optimization problem, a new solving scheme is presented. The multi-cycle problem is de-coupled into a number of relatively independent mono-cycle issues, then this non-linear programming problem with complex constraints is solved by an advanced new algorithm -probability approaching method (PAM), which is based on probability theory. The result on simplified core model shows well effect of this new multi-cycle optimization scheme. (authors)

Utility of 2-Pyridine Aldoxime Methyl Chloride (2-PAM) for Acute Organophosphate Poisoning: A Systematic Review and Meta-Analysis.

Science.gov (United States)

Blumenberg, Adam; Benabbas, Roshanak; deSouza, Ian S; Conigliaro, Alyssa; Paladino, Lorenzo; Warman, Elliot; Sinert, Richard; Wiener, Sage W

2018-03-01

Organophosphates (OP) account for the majority of pesticide-related unintentional or intentional poisonings in lower- and middle-income countries. The therapeutic role of atropine is well-established for patients with acute OP poisoning. The benefit of adding 2-pyridine aldoxime methyl chloride (2-PAM), however, is controversial. We performed a systematic review and meta-analysis of available randomized controlled trials (RCT) to compare 2-PAM plus atropine in comparison to atropine alone for acute OP poisoning. We searched PubMed, EMBASE, and SCOPUS up to March 2017. The Cochrane review handbook was used to assess the risk of bias. Data were abstracted and risk ratios (RR) were calculated for mortality, rate of intubation, duration of intubation, intermediate syndrome, and complications such as hospital-acquired infections, dysrhythmias, and pulmonary edema. We found five studies comprising 586 patients with varying risks of bias. The risk of death (RR = 1.5, 95% CI 0.9-2.5); intubation (RR = 1.3, 95% CI 1.0-1.6); intermediate syndrome (RR = 1.6, 95% CI 1.0-2.6); complications (RR = 1.2, 95% CI 0.8-1.8); and the duration of intubation (mean difference 0.0, 95% CI - 1.6-1.6) were not significantly different between the atropine plus 2-PAM and atropine alone. Based on our meta-analysis of the available RCTs, 2-PAM was not shown to improve outcomes in patients with acute OP poisoning.

Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms

DEFF Research Database (Denmark)

Chiang, Wen-Chi; Nilsson, Martin; Jensen, Peter Østrup

2013-01-01

Within recent years, it has been established that extracellular DNA is a key constituent of the matrix of microbial biofilms. In addition, it has recently been demonstrated that DNA binds positively charged antimicrobials such as aminoglycosides and antimicrobial peptides. In the present study, we...... provide evidence that extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. We show that exogenously supplemented DNA integrates into P. aeruginosa biofilms and increases their tolerance toward aminoglycosides. We provide evidence that biofilms formed by a DNA release......-deficient P. aeruginosa quorum-sensing mutant are more susceptible to aminoglycoside treatment than wild-type biofilms but become rescued from the detrimental action of aminoglycosides upon supplementation with exogenous DNA. Furthermore, we demonstrate that exposure to lysed polymorphonuclear leukocytes...

LHCD and coupling experiments with an ITER-like PAM launcher on the FTU tokamak

International Nuclear Information System (INIS)

Pericoli Ridolfini, V.; Apicella, M.L.; Barbato, E.; Buratti, P.; Calabro, G.; Cardinali, A.; Mirizzi, F.; Panaccione, L.; Podda, S.; Tuccillo, A.A.; Bibet, Ph.; Granucci, G.; Sozzi, C.

2005-01-01

Successful experimental tests on a PAM (passive active multijunction) prototype antenna for the Lower Hybrid (LH) waves similar to that foreseen for ITER have been carried out on FTU. The power level routinely achieved without any fault in the transmission lines for the maximum time allowed by the LH power plant, i.e. 0.9 s, is 250 kW versus a design value of 270. It corresponds to 50 MW/m 2 through the ITER antenna active area if it is scaled for the different LH frequencies (5 GHz in ITER, 8 GHz in FTU) and it is more than 1.4 times the goal of the ITER design (33 MW/m 2 ). The test results validate the main features indicated by the simulation codes, concerning the power handling, the coupling and the launched N parallel spectrum. The power reflection coefficient R c is always ≤ 2.5%, once the PAM launcher has been properly conditioned, even with the grill mouth retracted 2 mm inside the port shadow, with density in front of the launcher very close or even lower than the cut-off value. The current drive efficiency is comparable to a conventional grill in similar conditions, once the lower directivity is taken into account. The flexibility in the N parallel spectrum is confirmed by the HXR and ECE spectra. Conditioning the PAM to operate at the ITER equivalent power level has required only one day of RF operation, without a previous baking of the waveguides. (author)

IM/DD vs. 4-PAM Using a 1550-nm VCSEL over Short-Range SMF/MMF Links for Optical Interconnects

DEFF Research Database (Denmark)

Karinou, Fotini; Rodes Lopez, Roberto; Prince, Kamau

2013-01-01

We experimentally compare the performance of 10.9-Gb/s IM/DD and 5-GBd 4-PAM modulation formats over 5-km SMF and 1-km MMF links, employing a commercially-available 1550-nm VCSEL as an enabling technology for use in optical interconnects.......We experimentally compare the performance of 10.9-Gb/s IM/DD and 5-GBd 4-PAM modulation formats over 5-km SMF and 1-km MMF links, employing a commercially-available 1550-nm VCSEL as an enabling technology for use in optical interconnects....

Binding of recombinant apolipoprotein(a) to extracellular matrix proteins

NARCIS (Netherlands)

van der Hoek, Y. Y.; Sangrar, W.; Côté, G. P.; Kastelein, J. J.; Koschinsky, M. L.

1994-01-01

Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and

Targeting Extracellular Histones with Novel RNA Bio drugs for the Treatment of Acute Lung Injury

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0179 TITLE: Targeting Extracellular Histones with Novel RNA Bio -drugs for the Treatment of Acute Lung Injury...4. TITLE AND SUBTITLE Targeting Extracellular Histones with Novel RNA Bio -drugs for the Treatment of Acute Lung Injury 5a. CONTRACT NUMBER 5b...and field situations. To accomplish this goal, we developed novel bio -reagents (RNA aptamers) that bind to those histones known to cause MODS/ARDS and

Differential effects of the Toll-like receptor 2 agonists, PGN and Pam3CSK4 on anti-IgE induced human mast cell activation.

Directory of Open Access Journals (Sweden)

Yangyang Yu

Full Text Available Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI, mast cells are also activated by Toll-like receptors (TLRs which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN and tripalmitoyl-S-glycero-Cys-(Lys4 (Pam3CSK4. Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Science.gov (United States)

Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

2018-01-01

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

NARCIS (Netherlands)

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

Binding Mode of Insulin Receptor and Agonist Peptide

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

Insulin is a protein hormone secreted by pancreatic β cells. One of its main functions is to keep the balance of glucose inside the body by regulating the absorption and metabolism of glucose in the periphery tissue, as well as the production and storage of hepatic glycogen. The insulin receptor is a transmembrane glycoprotein in which two α subunits with a molecular weight of 135 kD and twoβ subunits with a molecular weight of 95 kD are joined by a disulfide bond to form a β-α-α-β structure. The extracellular α subunit, especially, its three domains near the N-terminal are partially responsible for signal transduction or ligand-binding, as indicated by the experiments. The extracellular α subunits are involved in binding the ligands. The experimental results indicate that the three domains of the N-terminal of the α subunits are the main determinative parts of the insulin receptor to bind the insulin or mimetic peptide.We employed the extracellular domain (PDBID: 1IGR) of the insulin-like growth factor-1 receptor (IGF-1 R ) as the template to simulate and optimize the spatial structures of the three domains in the extracellular domain of the insulin receptor, which includes 468 residues. The work was accomplished by making use of the homology program in the Insight Ⅱ package on an Origin3800 server. The docking calculations of the insulin receptor obtained by homology with hexapeptides were carried out by means of the program Affinity. The analysis indicated that there were hydrogen bonding, and electrostatic and hydrophobic effects in the docking complex of the insulin receptor with hexapeptides.Moreover, we described the spatial orientation of a mimetic peptide with agonist activity in the docking complex. We obtained a rough model of binding of DLAPSQ or STIVYS with the insulin receptor, which provides the powerful theoretical support for designing the minimal insulin mimetic peptide with agonist activity, making it possible to develop oral small

Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis

DEFF Research Database (Denmark)

Tawakoli, Pune Nina; Neu, Thomas R; Busck, Mette Marie

2017-01-01

lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence......The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled......-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates...

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

Science.gov (United States)

Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

2005-10-28

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

Demonstration of the First Real-Time End-to-End 40-Gb/s PAM-4 for Next-Generation Access Applications using 10-Gb/s Transmitter

DEFF Research Database (Denmark)

Wei, J. L.; Eiselt, Nicklas; Griesser, Helmut

2016-01-01

We demonstrate the first known experiment of a real-time end-to-end 40-Gb/s PAM-4 system for next-generation access applications using 10-Gb/s class transmitters only. Based on the measurement of a real-time 40-Gb/s PAM system, low-cost upstream and downstream link power budgets are estimated. Up...

Autocrine signal transmission with extracellular ligand degradation

International Nuclear Information System (INIS)

Muratov, C B; Posta, F; Shvartsman, S Y

2009-01-01

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand–receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers

Autocrine signal transmission with extracellular ligand degradation

Science.gov (United States)

Muratov, C B; Posta, F; Shvartsman, S Y

2009-03-01

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand-receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers.

AL-PAM assisted filtration of mature fine tailings produced from oil sands development

Energy Technology Data Exchange (ETDEWEB)

Alamgir, A.; Masliyah, J.; Xu, Z. [Alberta Univ., Edmonton, AB (Canada). Dept. of Chemical and Materials Engineering

2010-07-01

The inventory of mature fine tailings (MFT) produced by the oil sands extraction process continues to grow, and is considered as a long-term environmental liability. Large amounts of water are trapped in the MFT, and the tailings ponds present a threat to local wildlife and ecosystems. This PowerPoint presentation discussed a research project that is being conducted to identify conditions for producing stackable MFT deposits. The study is investigating various types and dosages of polymers and evaluating the degrees of MFT dilution and process configurations needed to maximize re-usable water recovery. Polymeric flocculants include AL-PAM and Magnafloc 1011. Settling tests have demonstrated that 50 ppm is the optimum dosage for both polymers when MFT is diluted to 5 wt percent solids, while 75 ppm of AL-PAM and 100 ppm of MF1011 are optimum dosages for MFT diluted to 10 wt percent solids. A novel supernatant filtration method was then used to produce filtration cakes and water. The study showed that the supernatant can be used to further dilute the MFTs. tabs., figs.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Science.gov (United States)

Grill, Brock; Chen, Lizhen; Tulgren, Erik D; Baker, Scott T; Bienvenut, Willy; Anderson, Matthew; Quadroni, Manfredo; Jin, Yishi; Garner, Craig C

2012-02-22

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic

Directory of Open Access Journals (Sweden)

An S

2013-02-01

Full Text Available Songhie An,* Kihoon Nam,* Sunghyun Choi, Cheng Z Bai, Yan Lee, Jong-Sang ParkDepartment of Chemistry, Seoul National University, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model.Methods and results: The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4. Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of >40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay, 4´,6-Diamidino-2-phenylindole (DAPI TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining.Conclusion: The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier's high transfection efficiency (35%–40% in glioma cells and the selective apoptosis-inducing activity of

Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus.

Science.gov (United States)

Ul-Hasan, Sabah; Burgess, Daniel M; Gajewiak, Joanna; Li, Qing; Hu, Hao; Yandell, Mark; Olivera, Baldomero M; Bandyopadhyay, Pradip K

2013-11-01

Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

How adults with cardiac conditions in Singapore understand the Patient Activation Measure (PAM-13) items: a cognitive interviewing study.

Science.gov (United States)

Ngooi, Bi Xia; Packer, Tanya L; Warner, Grace; Kephart, George; Koh, Karen Wei Ling; Wong, Raymond Ching Chiew; Lim, Serene Peiying

2018-03-01

Validation studies of the PAM-13 have found differences in scale performance, suggesting that health beliefs embedded in different cultures and/or self-management needs of different client groups influence how people respond to the items. The purpose of this study was to examine how adults with cardiac conditions in Singapore interpreted and responded to the PAM-13, to investigate possible reasons for differences in responses and to propose solutions to overcome them. We conducted retrospective cognitive interviews with 13 participants in an out-patient heart center. Interviews were transcribed and analyzed based on the framework approach to qualitative analysis. The four stages from Tourangeau's cognitive model were used as a framework to index the data from each item. There was variation in comprehension of questions leading to variation in responses. Comprehension issues were due to terms perceived by participants to be vague and the use of English terms uncommon in Singapore. Cultural influences impacted decision processes and problems with response processes of the self-rating Likert scale surfaced. This study reinforces the need to culturally adapt the tool, even when language translation is not necessary. Providing Likert scales with a larger number of may widen the relevance of PAM-13 in Singapore. Implications for rehabilitation Need to culturally adapt assessment tool, even when language translation is not necessary. Consider using Likert scales with a larger number of categories when using in Asian countries such as Singapore. Caution must be taken when using PAM-13 levels to decide interventions for each individual.

Positive and negative metacognitions about alcohol use among university students: Psychometric properties of the PAMS and NAMS French versions.

Science.gov (United States)

Gierski, Fabien; Spada, Marcantonio M; Fois, Eveline; Picard, Aurélie; Naassila, Mickaël; Van der Linden, Martial

2015-08-01

Metacognitions about the positive and negative effects of alcohol use have been associated with various patterns of drinking. The aim of the present study was to validate French versions of the Positive Alcohol Metacognitions Scale (PAMS) and the Negative Alcohol Metacognitions Scale (NAMS) developed by Spada and Wells (2008, Addict. Behav. 33, 515) and to investigate the relationship between metacognitions and patterns of alcohol use among university students. Responses of 1600 university students who participated in an internet survey-based study on alcohol use were submitted to confirmatory (N=800, mean age 20.40 years, 45.50% male) and exploratory (N=800, mean age 20.34 years, 45.38% male) factor analyses in two separate samples. Alcohol use, binge drinking and mood were also assessed. In line with the original versions of the scales, results provided support for a two-factor structure of the French PAMS and NAMS. Both scales revealed adequate internal reliability. Good temporal stability was found for the two factors of the NAMS, whereas one factor of the PAMS showed weakness across time. Predictive validity revealed that negative alcohol metacognitions about the uncontrollability of alcohol use were found to be consistently associated with alcohol use and binge drinking, whereas positive metacognitions about alcohol use were found to be differentially associated with alcohol use and binge drinking. The French versions of the PAMS and NAMS exhibited suitable psychometric properties. This study also emphasized the role of metacognitions about alcohol use in drinking behaviour among university students. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Validation of the German version of the patient activation measure 13 (PAM13-D in an international multicentre study of primary care patients.

Directory of Open Access Journals (Sweden)

Katja Brenk-Franz

Full Text Available The patients' active participation in their medical care is important for patients with chronic diseases. Measurements of patient activation are needed for studies and in clinical practice. This study aims to validate the Patient Activation Measure 13 (PAM13-D in German-speaking primary care patients. This international cross-sectional multicentre study enrolled consecutively patients from primary care practices in three German-speaking countries: Germany, Austria, and Switzerland. Patients completed the PAM13-D questionnaire. General Self-Efficacy scale (GSE was used to assess convergent validity. Furthermore Cronbach's alpha was performed to assess internal consistency. Exploratory factor analysis was used to evaluate the underlying factor structure of the items. We included 508 patients from 16 primary care practices in the final analysis. Results were internally consistent, with a Cronbach's alpha of 0.84. Factor analysis revealed one major underlying factor. The mean values of the PAM13-D correlated significantly (r = 0.43 with those of the GSE. The German PAM13 is a reliable and valid measure of patient activation. Thus, it may be useful in primary care clinical practice and research.

Nonimmune immunoglobulin binding and multiple adhesion characterize Plasmodium falciparum-infected erythrocytes of placental origin

DEFF Research Database (Denmark)

Rasti, Niloofar; Namusoke, Fatuma; Chêne, Arnaud

2006-01-01

The harmful effects of pregnancy-associated malaria (PAM) are engendered by the heavy sequestration of Plasmodium falciparum-parasitized RBCs in the placenta. It is well documented that this process is mediated by interactions of parasite-encoded variant surface antigens and placental receptors...... and adhesion to multiple receptors (IgG/IgM/HA/CSA) rather than the exclusive binding to CSA is a characteristic of fresh Ugandan placental isolates. These findings are of importance for the understanding of the pathogenesis of placental malaria and have implications for the ongoing efforts to develop a global...

Cation binding at the node of Ranvier: I. Localization of binding sites during development.

Science.gov (United States)

Zagoren, J C; Raine, C S; Suzuki, K

1982-06-17

Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation.

Science.gov (United States)

Mueller, Sandra; Kleinau, Gunnar; Jaeschke, Holger; Paschke, Ralf; Krause, Gerd

2008-06-27

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

Energy Technology Data Exchange (ETDEWEB)

Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

2010-01-14

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

The extracellular loop 2 (ECL2 of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.

Directory of Open Access Journals (Sweden)

David Wifling

Full Text Available In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R shows extraordinarily high constitutive activity. In the extracellular loop (ECL, replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.

Synthesis and binding studies of Alzheimer ligands on solid support.

Science.gov (United States)

Rzepecki, Petra; Geib, Nina; Peifer, Manuel; Biesemeier, Frank; Schrader, Thomas

2007-05-11

Aminopyrazole derivatives constitute the first class of nonpeptidic rationally designed beta-sheet ligands. Here we describe a double solid-phase protocol for both synthesis and affinity testing. The presented solid-phase synthesis of four types of hybrid compounds relies on the Fmoc strategy and circumvents subsequent HPLC purification by precipitating the final product from organic solution in pure form. Hexa- and octapeptide pendants with internal di- and tetrapeptide bridges are now amenable in high yields to combinatorial synthesis of compound libraries for high-throughput screening purposes. Solid-phase peptide synthesis (SPPS) on an acid-resistant PAM allows us, after PMB deprotection, to subject the free aminopyrazole binding sites in an immobilized state to on-bead assays with fluorescence-labeled peptides. From the fluorescence emission intensity decrease, individual binding constants can be calculated via reference curves by simple application of the law of mass action. Gratifyingly, host/guest complexation can be monitored quantitatively even for those ligands, which are almost insoluble in water.

User Instructions for the Policy Analysis Modeling System (PAMS)

Energy Technology Data Exchange (ETDEWEB)

McNeil, Michael A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Letschert, Virginie E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Van Buskirk, Robert D. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

2018-03-13

PAMS uses country-specific and product-specific data to calculate estimates of impacts of a Minimum Efficiency Performance Standard (MEPS) program. The analysis tool is self-contained in a Microsoft Excel spreadsheet, and requires no links to external data, or special code additions to run. The analysis can be customized to a particular program without additional user input, through the use of the pull-down menus located on the Summary page. In addition, the spreadsheet contains many areas into which user-generated input data can be entered for increased accuracy of projection. The following is a step-by-step guide for using and customizing the tool.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

Science.gov (United States)

Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

2012-04-01

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Current status and future prospects of an automated sample exchange system PAM for protein crystallography

Science.gov (United States)

Hiraki, M.; Yamada, Y.; Chavas, L. M. G.; Matsugaki, N.; Igarashi, N.; Wakatsuki, S.

2013-03-01

To achieve fully-automated and/or remote data collection in high-throughput X-ray experiments, the Structural Biology Research Centre at the Photon Factory (PF) has installed PF automated mounting system (PAM) for sample exchange robots at PF macromolecular crystallography beamlines BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. We are upgrading the experimental systems, including the PAM for stable and efficient operation. To prevent human error in automated data collection, we installed a two-dimensional barcode reader for identification of the cassettes and sample pins. Because no liquid nitrogen pipeline in the PF experimental hutch is installed, the users commonly add liquid nitrogen using a small Dewar. To address this issue, an automated liquid nitrogen filling system that links a 100-liter tank to the robot Dewar has been installed on the PF macromolecular beamline. Here we describe this new implementation, as well as future prospects.

Detection of weed algae in open pond cultures of Cyanobacterium aponinum using PAM

Directory of Open Access Journals (Sweden)

Dominik Winckelmann

2016-02-01

Full Text Available Abstract The potential use of non-arable land in the al-Wusta region of the Sultanate of Oman for the production of algae biomass was examined. Brackish cleaned production water from oil production supplemented with commercial fertilizer was used as growth medium. The indigenous isolate Cyanobacterium aponinum WP7(1 was grown in open ponds using batch or semi-continuous cultivation. Biomass production rates of 15–24 g/m2/day were achieved. The change of salinity due to evaporation, which was thought to be a major challenge, did not exceed 35 ppt. All cultures showed contaminations with weed algae. Contaminations with green algae or diatoms were detectable using fluorescence pattern excited by four different wavelengths using a pulse-amplitude-modulation chlorophyll fluorometer (PAM. It is possible to estimate the health level and the mayor groups of which a culture is composed using the PAM method. Therefore, the fluorescence of the photosynthetically inactive sample is compared with the fluorescence after all copies of photosystem II were closed by exposing the sample to a high-intensity light beam. A detection limit of one weed algae cell in a hundred cells was achieved.

The Toll-like receptor 1/2 agonists Pam(3) CSK(4) and human β-defensin-3 differentially induce interleukin-10 and nuclear factor-κB signalling patterns in human monocytes.

Science.gov (United States)

Funderburg, Nicholas T; Jadlowsky, Julie K; Lederman, Michael M; Feng, Zhimin; Weinberg, Aaron; Sieg, Scott F

2011-10-01

Human β-defensin 3 (hBD-3) activates antigen-presenting cells through Toll-like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD-3 with those of another TLR1/2 agonist, Pam(3) CSK(4) , in human monocytes. Monocytes incubated with hBD-3 or Pam(3) CSK(4) produced interleukin-6 (IL-6), IL-8 and IL-1β, but only Pam(3) CSK(4) induced IL-10. The IL-10 induction by Pam(3) CSK(4) caused down-modulation of the co-stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD-3. Assessment of signalling pathways linked to IL-10 induction indicated that mitogen-activated protein kinases were activated similarly by hBD-3 or Pam(3) CSK(4) , whereas the non-canonical nuclear factor-κB pathway was only induced by Pam(3) CSK(4) . Our data suggest that the lack of non-canonical nuclear factor-κB signalling by hBD-3 could contribute to the failure of this TLR agonist to induce production of the anti-inflammatory cytokine, IL-10, in human monocytes. © 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

Isolation and Characterization of Polyacrylamide-Degrading Bacteria from Dewatered Sludge

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Feng Yu

2015-04-01

Full Text Available Polyacrylamide (PAM is a water-soluble polymer that is widely used as a flocculant in sewage treatment. The accumulation of PAM affects the formation of dewatered sludge and potentially produces hazardous monomers. In the present study, the bacterial strain HI47 was isolated from dewatered sludge. This strain could metabolize PAM as its sole nutrient source and was subsequently identified as Pseudomonas putida. The efficiency of PAM degradation was 31.1% in 7 days and exceeded 45% under optimum culture condition (pH 7.2, 39 °C and 100 rpm. The addition of yeast extract and glucose improved the bacterial growth and PAM degradation. The degraded PAM samples were analyzed by gel-filtration chromatography, Fourier transform infrared and high-performance liquid chromatography. The results showed that high-molecular-weight PAM was partly cleaved to small molecular oligomer derivatives and part of the amide groups of PAM had been converted to carboxyl groups. The biodegradation did not accumulate acrylamide monomers. Based on the SDS-PAGE and N-terminal sequencing results, the PAM amide groups were converted into carboxyl groups by a PAM-induced extracellular enzyme from the aliphatic amidase family.

The concentration of extracellular superoxide dismutase in plasma is maintained by LRP-mediated endocytosis

DEFF Research Database (Denmark)

Petersen, Steen V; Thøgersen, Ida B; Valnickova, Zuzana

2010-01-01

In this study, we show that human extracellular superoxide dismutase (EC-SOD) binds to low-density lipoprotein receptor-related protein (LRP). This interaction is most likely responsible for the removal of EC-SOD from the blood circulation via LRP expressed in liver tissue. The receptor recognition...

Transmission of 2 × 56 Gb/s PAM-4 signal over 100 km SSMF using 18 GHz DMLs.

Science.gov (United States)

Zhou, Shiwei; Li, Xiang; Yi, Lilin; Yang, Qi; Fu, Songnian

2016-04-15

We experimentally demonstrate C-band 2 × 56 Gb/s pulse-amplitude modulation (PAM)-4 signal transmission over 100 km standard single-mode fiber (SSMF) using 18 GHz direct-modulated lasers (DMLs) and direct detection, without inline optical amplifier. A delay interferometer (DI) at the transmitter side is used to extend the transmission reach from 40 to 100 km. A digital Volterra filter at the receiver side is used to mitigate the nonlinear distortions. We obtain an average bit error ratio (BER) of 1.5 × 10(-3) for 2 × 56 Gb/s PAM-4 signal after 100 km SSMF transmission at the optimal input power, which is below the 7% forward error correction (FEC) threshold (3.8 × 10(-3)).

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

Directory of Open Access Journals (Sweden)

Hyong Woo Choi

2016-03-01

Full Text Available Damage-associated molecular pattern molecules (DAMPs signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3 is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i MAPK activation, ii defense-related gene expression, iii callose deposition, and iv enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast. Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA, which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

Expression of PAM50 Genes in Lung Cancer: Evidence that Interactions between Hormone Receptors and HER2/HER3 Contribute to Poor Outcome

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Jill M. Siegfried

2015-11-01

Full Text Available Non–small cell lung cancers (NSCLCs frequently express estrogen receptor (ER β, and estrogen signaling is active in many lung tumors. We investigated the ability of genes contained in the prediction analysis of microarray 50 (PAM50 breast cancer risk predictor gene signature to provide prognostic information in NSCLC. Supervised principal component analysis of mRNA expression data was used to evaluate the ability of the PAM50 panel to provide prognostic information in a stage I NSCLC cohort, in an all-stage NSCLC cohort, and in The Cancer Genome Atlas data. Immunohistochemistry was used to determine status of ERβ and other proteins in lung tumor tissue. Associations with prognosis were observed in the stage I cohort. Cross-validation identified seven genes that, when analyzed together, consistently showed survival associations. In pathway analysis, the seven-gene panel described one network containing the ER and progesterone receptor, as well as human epidermal growth factor receptor (HER2/HER3 and neuregulin-1. NSCLC cases also showed a significant association between ERβ and HER2 protein expression. Cases positive for HER2 expression were more likely to express HER3, and ERβ-positive cases were less likely to be both HER2 and HER3 negative. Prognostic ability of genes in the PAM50 panel was verified in an ERβ-positive cohort representing all NSCLC stages. In The Cancer Genome Atlas data sets, the PAM50 gene set was prognostic in both adenocarcinoma and squamous cell carcinoma, whereas the seven-gene panel was prognostic only in squamous cell carcinoma. Genes in the PAM50 panel, including those linking ER and HER2, identify lung cancer patients at risk for poor outcome, especially among ERβ-positive cases and squamous cell carcinoma.

Extracellular histones induce erythrocyte fragility and anemia.

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Kordbacheh, Farzaneh; O'Meara, Connor H; Coupland, Lucy A; Lelliott, Patrick M; Parish, Christopher R

2017-12-28

Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients. © 2017 by The American Society of Hematology.

PAM-OBG: A monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma

Science.gov (United States)

Sharpe, Martyn A.; Raghavan, Sudhir; Baskin, David S.

2018-01-01

Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O6-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome. We have designed and tested both in vitro and in vivo, a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O6-benzylguanine (O6BG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to O6BG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER). In mouse intracranial models of primary human glioma, we show that PAM-OBG increases survival of mice treated with either BCNU or CCNU by a factor of six and that in a chemoradiation model utilizing six rounds of TMZ/2Gy radiation, pre-treatment with PAM-OBG more than doubled survival time. PMID:29844863

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

Science.gov (United States)

Bozzi, Manuela; Bianchi, Marzia; Sciandra, Francesca; Paci, Maurizio; Giardina, Bruno; Brancaccio, Andrea; Cicero, Daniel O

2003-11-25

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

Evaluation of High-Speed EML-based IM/DD links with PAM Modulations and Low-Complexity Equalization

DEFF Research Database (Denmark)

Pang, Xiaodan; Ozolins, Oskars; Gaiarin, Simone

2017-01-01

We experimentally evaluated up to 96 Gb/s/lambda PAM IM/DD transmissions with an EML and digital equalizations. Symbol-spaced FFE/DFEs with fewer than 10 taps are shown being sufficient for a high and stable performance over a 4 km SMF link....

Dual-wavelength OR-PAM with compressed sensing for cell tracking in a 3D cell culture system

Science.gov (United States)

Huang, Rou-Xuan; Fu, Ying; Liu, Wang; Ma, Yu-Ting; Hsieh, Bao-Yu; Chen, Shu-Ching; Sun, Mingjian; Li, Pai-Chi

2018-02-01

Monitoring dynamic interactions of T cells migrating toward tumor is beneficial to understand how cancer immunotherapy works. Optical-resolution photoacoustic microscope (OR-PAM) can provide not only high spatial resolution but also deeper penetration than conventional optical microscopy. With the aid of exogenous contrast agents, the dual-wavelength OR-PAM can be applied to map the distribution of CD8+ cytotoxic T lymphocytes (CTLs) with gold nanospheres (AuNS) under 523nm laser irradiation and Hepta1-6 tumor spheres with indocyanine green (ICG) under 800nm irradiation. However, at 1K laser PRF, it takes approximately 20 minutes to obtain a full sample volume of 160 × 160 × 150 μm3 . To increase the imaging rate, we propose a random non-uniform sparse sampling mechanism to achieve fast sparse photoacoustic data acquisition. The image recovery process is formulated as a low-rank matrix recovery (LRMR) based on compressed sensing (CS) theory. We show that it could be stably recovered via nuclear-norm minimization optimization problem to maintain image quality from a significantly fewer measurement. In this study, we use the dual-wavelength OR-PAM with CS to visualize T cell trafficking in a 3D culture system with higher temporal resolution. Data acquisition time is reduced by 40% in such sample volume where sampling density is 0.5. The imaging system reveals the potential to understand the dynamic cellular process for preclinical screening of anti-cancer drugs.

Extracellular matrix structure.

Science.gov (United States)

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

Science.gov (United States)

Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

2008-04-01

Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

Extracellular matrix organization in various regions of rat brain grey matter.

Science.gov (United States)

Brückner, G; Härtig, W; Kacza, J; Seeger, J; Welt, K; Brauer, K

1996-05-01

Previous studies revealed the concentration of extracellular matrix proteoglycans in the so-called perineuronal nets on the one hand and in certain zones of the neuropil on the other. This nonhomogeneous distribution suggested a non-random chemical and spatial heterogeneity of the extracellular space. In the present investigation, regions dominated by one of both distribution patterns, i.e. piriform and parietal cortex, reticular thalamic nucleus, medial septum/diagonal band complex and cerebellar nuclei, were selected for correlative light and electron microscopic analysis. The labelling was performed by the use of the N-acetylgalactosamine-binding plant lectin Wisteria floribunda agglutinin visualized by peroxidase staining and additionally by photoconversion of red carbocyanine fluorescence labelling for electron microscopy. The intense labelling of the neuropil of a superficial piriform region, presumably identical with sublayer Ia, was confined to a fine meshwork spreading over the extracellular space between non-myelinated axons, dendrites and glial profiles. In the reticular thalamic nucleus the neuronal cell bodies were embedded in zones of labelled neuropil. In contrast to these patterns, the labelled extracellular matrix in different cortical layers and in the other subcortical regions was concentrated in perineuronal nets as large accumulations at surface areas of the neuronal perikarya and dendrites and the attached presynaptic boutons. Astrocytic processes usually were separated from the neuronal surface by the interposed extracellular material. Despite a great variability, the width of the extracellular space containing the labelled matrix components in all perineuronal nets appeared to be considerably larger than that in the labelled zones of neuropil and the non-labelled microenvironment of other neurons. Our results support the view that differences expressed in topographical and spatial peculiarities of the extracellular matrix constituents are

Binding of sFRP-3 to EGF in the extra-cellular space affects proliferation, differentiation and morphogenetic events regulated by the two molecules.

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Raffaella Scardigli

Full Text Available BACKGROUND: sFRP-3 is a soluble antagonist of Wnts, widely expressed in developing embryos. The Wnt gene family comprises cysteine-rich secreted ligands that regulate cell proliferation, differentiation, organogenesis and oncogenesis of different organisms ranging from worms to mammals. In the canonical signal transduction pathway Wnt proteins bind to the extracellular domain of Frizzled receptors and consequently recruit Dishevelled (Dsh to the cell membrane. In addition to Wnt membrane receptors belonging to the Frizzled family, several other molecules have been described which share homology in the CRD domain and lack the putative trans-membrane domain, such as sFRP molecules (soluble Frizzled Related Protein. Among them, sFRP-3 was originally isolated from bovine articular cartilage and also as a component of the Spemann organizer. sFRP-3 blocks Wnt-8 induced axis duplication in Xenopus embryos and binds to the surface of cells expressing a membrane-anchored form of Wnt-1. Injection of sFRP-3 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that sFRP-3 specifically blocks EGF-induced fibroblast proliferation and foci formation. Over-expression of sFRP-3 reverts EGF-mediated inhibition of hair follicle development in the mouse ectoderm while its ablation in Xenopus maintains EGF-mediated inhibition of ectoderm differentiation. Conversely, over-expression of EGF reverts the inhibition of somitic myogenesis and axis truncation in Xenopus and mouse embryos caused by sFRP-3. In vitro experiments demonstrated a direct binding of EGF to sFRP-3 both on heparin and on the surface of CHO cells where the molecule had been membrane anchored. CONCLUSIONS/SIGNIFICANCE: sFRP-3 and EGF reciprocally inhibit their effects on cell proliferation, differentiation and morphogenesis and indeed are expressed in contiguous domains of the embryo, suggesting that in

Combined Atropine and 2-PAM Cl Effects on Tracking Performance and Visual, Physiological, and Psychological Functions

Science.gov (United States)

1988-12-01

tracking task reveals the magnitude Akitrihm. Spare. and Environmental Medicine • December. I$ II I ANTIDOTE EFFECTS--PEN ETAR ET AL. and duration of the... marihuana on dynamic visual acu- blood pressure following the combination of 2-PAM Cl ity: I. Threshold measurements. Perception Psychophys. 1975

Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

Energy Technology Data Exchange (ETDEWEB)

Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

2006-05-25

Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

Science.gov (United States)

Wise, John G

2012-06-26

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

Capacity upgrade in short-reach optical fibre networks: simultaneous 4-PAM 20 Gbps data and polarization-modulated PPS clock signal using a single VCSEL carrier

Science.gov (United States)

Isoe, G. M.; Wassin, S.; Gamatham, R. R. G.; Leitch, A. W. R.; Gibbon, T. B.

2017-11-01

In this work, a four-level pulse amplitude modulation (4-PAM) format with a polarization-modulated pulse per second (PPS) clock signal using a single vertical cavity surface emitting laser (VCSEL) carrier is for the first time experimentally demonstrated. We propose uncomplex alternative technique for increasing capacity and flexibility in short-reach optical communication links through multi-signal modulation onto a single VCSEL carrier. A 20 Gbps 4-PAM data signal is directly modulated onto a single mode 10 GHz bandwidth VCSEL carrier at 1310 nm, therefore, doubling the network bit rate. Carrier spectral efficiency is further maximized by exploiting the inherent orthogonal polarization switching of the VCSEL carrier with changing bias in transmission of a PPS clock signal. We, therefore, simultaneously transmit a 20 Gbps 4-PAM data signal and a polarization-based PPS clock signal using a single VCSEL carrier. It is the first time a signal VCSEL carrier is reported to simultaneously transmit a directly modulated 20 Gbps 4-PAM data signal and a polarization-based PPS clock signal. We further demonstrate on the design of a software-defined digital signal processing (DSP)-assisted receiver as an alternative to costly receiver hardware. Experimental results show that a 3.21 km fibre transmission with simultaneous 20 Gbps 4-PAM data signal and polarization-based PPS clock signal introduced a penalty of 3.76 dB. The contribution of polarization-based PPS clock signal to this penalty was found out to be 0.41 dB. Simultaneous distribution of data and timing clock signals over shared network infrastructure significantly increases the aggregated data rate at different optical network units (ONUs), without costly investment.

ITER-like PAM launcher for tore supra's LHCD system

International Nuclear Information System (INIS)

Belo, J.H.; Bibet, Ph.; Missirlian, M.; Achard, J.; Beaumont, B.; Bertrand, B.; Chantant, M.; Chappuis, Ph.; Doceul, L.; Durocher, A.; Gargiulo, L.; Saille, A.; Samaille, F.; Villedieu, E.

2004-01-01

A new launcher based upon the PAM concept (Passive-Active Multijunction) already proposed for ITER has been developed and is currently under realisation at Tore Supra. It was designed for an injection power capability of 2.7 MW CW at 3.7 GHz, a power density of 25 MW/m 2 , to radiate a power spectrum peaked at N || = 1.7 with a maximum power directivity near the electron cut-off density and with very good coupling properties on a wide range of electron densities. In this paper an overall description of the antenna as well as the foreseen manufacturing and assembling processes are given, followed by the results from studies and optimizations of its RF components, and by a stress analysis of its thermomechanical behaviour. (authors)

The validity of the PAM-RL device for evaluating periodic limb movements in sleep and an investigation on night-to-night variability of periodic limb movements during sleep in patients with restless legs syndrome or periodic limb movement disorder using this system.

Science.gov (United States)

Kobayashi, Mina; Namba, Kazuyoshi; Ito, Eiki; Nishida, Shingo; Nakamura, Masaki; Ueki, Yoichiro; Furudate, Naomichi; Kagimura, Tatsuo; Usui, Akira; Inoue, Yuichi

2014-01-01

The status of night-to-night variability for periodic limb movements in sleep (PLMS) has not been clarified. With this in mind, we investigated the validity of PLMS measurement by actigraphy with the PAM-RL device in Japanese patients with suspected restless legs syndrome (RLS) or periodic limb movement disorder (PLMD) and the night-to-night variability of PLMS among the subjects. Forty-one subjects (mean age, 52.1±16.1 years) underwent polysomnography (PSG) and PAM-RL measurement simultaneously. Thereafter, subjects used the PAM-RL at home on four more consecutive nights. The correlation between PLMS index on PSG (PLMSI-PSG) and PLM index on PAM-RL (PLMI-PAM) was 0.781 (PPAM-RL. PAM-RL is thought to be valuable for assessing PLMS even in Japanese subjects. Recording of PAM-RL for three or more consecutive nights may be required to ensure the screening reliability of a patient with suspected pathologically frequent PLMS. Copyright © 2013 Elsevier B.V. All rights reserved.

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

Energy Technology Data Exchange (ETDEWEB)

Lu, Jun; Byrne, Noel; Wang, John; Bricogne, Gerard; Brown, Frank K.; Chobanian, Harry R.; Colletti, Steven L.; Di Salvo, Jerry; Thomas-Fowlkes, Brande; Guo, Yan; Hall, Dawn L.; Hadix, Jennifer; Hastings, Nicholas B.; Hermes, Jeffrey D.; Ho, Thu; Howard, Andrew D.; Josien, Hubert; Kornienko, Maria; Lumb, Kevin J.; Miller, Michael W.; Patel, Sangita B.; Pio, Barbara; Plummer, Christopher W.; Sherborne, Bradley S.; Sheth, Payal; Souza, Sarah; Tummala, Srivanya; Vonrhein, Clemens; Webb, Maria; Allen, Samantha J.; Johnston, Jennifer M.; Weinglass, Adam B.; Sharma, Sujata; Soisson, Stephen M. (Merck); (Globel Phasing)

2017-06-05

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Multiband LTE-A and 4-PAM signals over large-core plastic fibers for in-home networks

NARCIS (Netherlands)

Forni, F.; Shi, Y.; van den Boom, H.P.A.; Tangdiongga, E.; Koonen, A.M.J.

2016-01-01

This letter presents the transmission of eight standard compliant 64-QAM long term evolution advanced (LTE-A) bands and 1.4 Gb/s 4-pulse amplitude modulation (PAM) signals over 20 m of 1 mm core diameter graded-index polymethyl methacrylate plastic optical fiber. The optical transceiver consists of

Cognitive enhancement and antipsychotic-like activity following repeated dosing with the selective M4 PAM VU0467154.

Science.gov (United States)

Gould, Robert W; Grannan, Michael D; Gunter, Barak W; Ball, Jacob; Bubser, Michael; Bridges, Thomas M; Wess, Jurgen; Wood, Michael W; Brandon, Nicholas J; Duggan, Mark E; Niswender, Colleen M; Lindsley, Craig W; Conn, P Jeffrey; Jones, Carrie K

2018-01-01

Although selective activation of the M 1 muscarinic acetylcholine receptor (mAChR) subtype has been shown to improve cognitive function in animal models of neuropsychiatric disorders, recent evidence suggests that enhancing M 4 mAChR function can also improve memory performance. Positive allosteric modulators (PAMs) targeting the M 4 mAChR subtype have shown therapeutic potential for the treatment of multiple symptoms observed in schizophrenia, including positive and cognitive symptoms when assessed in acute preclinical dosing paradigms. Since the cholinergic system has been implicated in multiple stages of learning and memory, we evaluated the effects of repeated dosing with the highly selective M 4 PAM VU0467154 on either acquisition and/or consolidation of learning and memory when dosed alone or after pharmacologic challenge with the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR) antagonist MK-801. MK-801 challenge represents a well-documented preclinical model of NMDAR hypofunction that is thought to underlie some of the positive and cognitive symptoms observed in schizophrenia. In wildtype mice, 10-day, once-daily dosing of VU0467154 either prior to, or immediately after daily testing enhanced the rate of learning in a touchscreen visual pairwise discrimination task; these effects were absent in M 4 mAChR knockout mice. Following a similar 10-day, once-daily dosing regimen of VU0467154, we also observed 1) improved acquisition of memory in a cue-mediated conditioned freezing paradigm, 2) attenuation of MK-801-induced disruptions in the acquisition of memory in a context-mediated conditioned freezing paradigm and 3) reversal of MK-801-induced hyperlocomotion. Comparable efficacy and plasma and brain concentrations of VU0467154 were observed after repeated dosing as those previously reported with an acute, single dose administration of this M 4 PAM. Together, these studies are the first to demonstrate that cognitive enhancing and antipsychotic

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry.

Science.gov (United States)

Herlory, Olivier; Bonzom, Jean-Marc; Gilbin, Rodolphe

2013-09-15

Although ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F0/Fv. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency ( [Formula: see text] , EC50=303 ± 64 μg UL(-1) after 5h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC50=142 ± 98 μg UL(-1) after 5h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from fluorescence induction kinetics are valuable indicators for evaluating the impact of uranium on PSII in green algae. PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response to uranium in microalgae. Copyright © 2013 Elsevier B.V. All rights reserved.

Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic.

Science.gov (United States)

An, Songhie; Nam, Kihoon; Choi, Sunghyun; Bai, Cheng Z; Lee, Yan; Park, Jong-Sang

2013-01-01

Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model. The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG) by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4). Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of .40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, 4',6-Diamidino-2-phenylindole (DAPI) TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining. The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier's high transfection efficiency (35%-40%) in glioma cells and the selective apoptosis-inducing activity of apoptin in tumor cells.

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Control of extracellular matrix assembly by syndecan-2 proteoglycan

DEFF Research Database (Denmark)

Klass, C M; Couchman, J R; Woods, A

2000-01-01

Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably...... to rearrange laminin or fibronectin substrates into fibrils and to bind exogenous fibronectin. Transfection of activated alphaIIbalphaLdeltabeta3 integrin into alpha(5)-deficient CHO B2 cells resulted in reestablishment of the previously lost fibronectin matrix. However, cotransfection of this cell line with S...

Analog-based duobinary-4-PAM for electrical bandwidth limited optical fiber links

DEFF Research Database (Denmark)

Suhr, Lau Frejstrup; Tafur Monroy, Idelfonso; Vegas Olmos, Juan José

2016-01-01

This paper presents a demonstration of a seven level duobinary-4-PAM signal operating at 10.16 Gbit/s. The polybinary generation is achieved by a simple electrical Bessel filter of 5th order, with a frequency cut-off of 1.8 GHz. We assess the impact of the filter placement, either at the transmit...... at the transmitter or the receiver, and find the impact is minimal in terms of bit error rate performance in transmissions of up to 40 km of single mode fiber....

First Demonstration of Real-Time End-to-End 40 Gb/s PAM-4 System using 10-G Transmitter for Next Generation Access Applications

DEFF Research Database (Denmark)

Wei, Jinlong; Eiselt, Nicklas; Griesser, Helmut

We demonstrate the first known experiment of a real-time end-to-end 40-Gb/s PAM-4 system for next generation access applications using 10G class transmitters only. Up to 25-dB upstream link budget for 20 km SMF is achieved.......We demonstrate the first known experiment of a real-time end-to-end 40-Gb/s PAM-4 system for next generation access applications using 10G class transmitters only. Up to 25-dB upstream link budget for 20 km SMF is achieved....

Extracellular gadolinium-based contrast media: Differences in diagnostic efficacy

Energy Technology Data Exchange (ETDEWEB)

Molen, Aart J. van der [Department of Radiology C-2S, Leiden University Medical Centre, Albinusdreef 2, NL-2333 ZA Leiden (Netherlands)], E-mail: molen@lumc.nl; Bellin, Marie-France [Universite Paris-Sud XI, AP-HP, Service de Radiologie, Hopital Paul Brousse, 12-14 Avenue Paul Vaillant Couturier, F-94804 Villejuif Cedex (France)

2008-05-15

Since the introduction of the first gadolinium-based contrast agent (Gd-CA) in 1988 it has become clear that these agents significantly improve the diagnostic efficacy of MRI. Studies on single agents have shown that, in comparison to unenhanced sequences, all agents help to improve the detection and delineation of lesions which can alter diagnosis in up to 40% of patients. Doubling or tripling the standard dose of 0.1 mmol/kg body weight may be beneficial for selected indications (e.g. brain perfusion, equivocal single dose study in MRI for brain metastasis, small vessel MR angiography). A more limited number of studies have compared the various agents. These studies do not show clinically significant differences in diagnostic efficacy between the various extracellular Gd-CA. Agents with higher concentration or protein binding may be relatively better suitable for selected applications (e.g. perfusion MRI). The higher relaxivity agents may be used in somewhat lower doses than the extracellular agents.

Extracellular gadolinium-based contrast media: Differences in diagnostic efficacy

International Nuclear Information System (INIS)

Molen, Aart J. van der; Bellin, Marie-France

2008-01-01

Since the introduction of the first gadolinium-based contrast agent (Gd-CA) in 1988 it has become clear that these agents significantly improve the diagnostic efficacy of MRI. Studies on single agents have shown that, in comparison to unenhanced sequences, all agents help to improve the detection and delineation of lesions which can alter diagnosis in up to 40% of patients. Doubling or tripling the standard dose of 0.1 mmol/kg body weight may be beneficial for selected indications (e.g. brain perfusion, equivocal single dose study in MRI for brain metastasis, small vessel MR angiography). A more limited number of studies have compared the various agents. These studies do not show clinically significant differences in diagnostic efficacy between the various extracellular Gd-CA. Agents with higher concentration or protein binding may be relatively better suitable for selected applications (e.g. perfusion MRI). The higher relaxivity agents may be used in somewhat lower doses than the extracellular agents

Extracellular vesicles in Alzheimer's disease: friends or foes? Focus on aβ-vesicle interaction.

Science.gov (United States)

Joshi, Pooja; Benussi, Luisa; Furlan, Roberto; Ghidoni, Roberta; Verderio, Claudia

2015-03-03

The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer's disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific molecules which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathology.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

Science.gov (United States)

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Curriculation and Competence Based Education Training (CBET in Tanzania: A Critical Assessment of Public Administration and Management (PAM Curricula at Tanzania Public Service College (TPSC

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Ramadhani Marijani

2017-08-01

Full Text Available The article directs the discourse in Public Administration and Management (PAM curricula at Tanzania Public Service College. The motive for this attempt is based on the fact that Public Administration (PA is itself recognized as a profession whose teaching aims at producing competent professionals and therefore needs constant review to conform to the current work complexities. Five big questions and content analysis are used as the basis for directing the discourse. The findings indicate that curriculation for PAM is complex and dynamic. Moreover, the findings indicate that the two curricula are more vocational than educational as there is little weight allocated for occupational course modules (36 out of 360 in PA and (126 out of 360 in HRM. This may pose the danger of “pourtpouri” problem in curriculum design. The article proposes rethinking interventions on curriculation for PAM at TPSC to benefit from Public Administration discipline.

Carrier of Wingless (Cow), a Secreted Heparan Sulfate Proteoglycan, Promotes Extracellular Transport of Wingless

Science.gov (United States)

Chang, Yung-Heng; Sun, Yi Henry

2014-01-01

Morphogens are signaling molecules that regulate growth and patterning during development by forming a gradient and activating different target genes at different concentrations. The extracellular distribution of morphogens is tightly regulated, with the Drosophila morphogen Wingless (Wg) relying on Dally-like (Dlp) and transcytosis for its distribution. However, in the absence of Dlp or endocytic activity, Wg can still move across cells along the apical (Ap) surface. We identified a novel secreted heparan sulfate proteoglycan (HSPG) that binds to Wg and promotes its extracellular distribution by increasing Wg mobility, which was thus named Carrier of Wg (Cow). Cow promotes the Ap transport of Wg, independent of Dlp and endocytosis, and this function addresses a previous gap in the understanding of Wg movement. This is the first example of a diffusible HSPG acting as a carrier to promote the extracellular movement of a morphogen. PMID:25360738

Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins.

Science.gov (United States)

Schiera, Gabriella; Di Liegro, Carlo Maria; Puleo, Veronica; Colletta, Oriana; Fricano, Anna; Cancemi, Patrizia; Di Cara, Gianluca; Di Liegro, Italia

2016-11-01

Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2.

Experimental investigation of impulse response shortening for low-complexity MLSE of a 112-Gbit/s PAM-4 transceiver

NARCIS (Netherlands)

van der Heide, S.; Eiselt, N.; Griesser, H.; Olmos, J. J.V.; Tafur Monroy, I.; Okonkwo, C.

2016-01-01

An optimized channel shortening method for reduced complexity MLSE detection enables the compensation of severe inter-symbol interference. Experimental investigations demonstrate the benefit on the performance for a severely bandwidth limited 112-Gbit/s PAM-4 transmission system with residual

Experimental investigation of impulse response shortening for low-complexity MLSE of a 112-Gbit/s PAM-4 transceiver

DEFF Research Database (Denmark)

Van Der Heide, Sjoerd; Eiselt, Nicklas; Griesser, Helmut

2016-01-01

An optimized channel shortening method for reduced complexity MLSE detection enables the compensation of severe inter-symbol interference. Experimental investigations demonstrate the benefit on the performance for a severely bandwidth limited 112-Gbit/s PAM-4 transmission system with residual chr...

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

Science.gov (United States)

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Science.gov (United States)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

PamR, a new MarR-like regulator affecting prophages and metabolic genes expression in Bacillus subtilis.

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Alba De San Eustaquio-Campillo

Full Text Available B. subtilis adapts to changing environments by reprogramming its genetic expression through a variety of transcriptional regulators from the global transition state regulators that allow a complete resetting of the cell genetic expression, to stress specific regulators controlling only a limited number of key genes required for optimal adaptation. Among them, MarR-type transcriptional regulators are known to respond to a variety of stresses including antibiotics or oxidative stress, and to control catabolic or virulence gene expression. Here we report the characterization of the ydcFGH operon of B. subtilis, containing a putative MarR-type transcriptional regulator. Using a combination of molecular genetics and high-throughput approaches, we show that this regulator, renamed PamR, controls directly its own expression and influence the expression of large sets of prophage-related and metabolic genes. The extent of the regulon impacted by PamR suggests that this regulator reprograms the metabolic landscape of B. subtilis in response to a yet unknown signal.

Porphyromonas endodontalis binds, reduces and grows on human hemoglobin.

Science.gov (United States)

Zerr, M; Drake, D; Johnson, W; Cox, C D

2001-08-01

Porphyromonas endodontalis is a black-pigmented, obligate anaerobic rod-shaped bacterium implicated as playing a major role in endodontic infections. We have previously shown that P. endodontalis requires the porphyrin nucleus, preferably supplied as hemoglobin, as a growth supplement. The bacteria also actively transport free iron, although this activity does not support growth in the absence of a porphyrin source. The purpose of this study was to further investigate the binding and subsequent utilization of human hemoglobin by P. endodontalis. P. endodontalis binds hemoglobin and reduces the Fe(III) porphyrin, resulting in a steady accumulation of ferrous hemoglobin. Reduction of methemoglobin was similar to the extracellular reduction of nitrobluetetrazolium in the presence of oxidizable substrate. Turbidimetric and viable cell determinations showed that P. endodontalis grew when supplied only hemoglobin. Therefore, we conclude that hemoglobin appears to serve as a sole carbon and nitrogen source, and that these bacteria reduce extracellular compounds at the expense of oxidized substrates.

3.375-Gb/s RGB-LED based WDM visible light communication system employing PAM-8 modulation with phase shifted Manchester coding.

Science.gov (United States)

Chi, Nan; Zhang, Mengjie; Zhou, Yingjun; Zhao, Jiaqi

2016-09-19

Optical background noise and second-order nonlinear distortions are two main challenges faced by indoor high-speed VLC system. In this paper, a novel phase shifted Manchester (PS-Manchester) coding based on PAM-8 is proposed and experimentally demonstrated to mitigate these noise and distortions. With the aid of PS-Manchester coding and WDM, a total data rate of 3.375-Gb/s can be successfully achieved in the RGB-LED based VLC system. The BER is under 7% HD-FEC limit of 3.8x10-3 after 1-m indoor free space transmission. To the best of our knowledge, this is the highest data rate ever achieved in PAM VLC systems.

Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.

Science.gov (United States)

Delpech, B; Maingonnat, C; Girard, N; Chauzy, C; Maunoury, R; Olivier, A; Tayot, J; Creissard, P

1993-01-01

Hyaluronan (HA) and the hyaluronan-binding glycoprotein hyaluronectin (HN) were measured in 23 gliomas and 8 meningiomas and their location was revisited in 35 tumours. A clear-cut difference was found in the HN/HA ratio values of glioblastomas (below 0.5) and that of astrocytomas (above 0.5 P edification of the extracellular matrix. In meningiomas only the stroma would be responsible for HA and HN production.

Identification and Molecular Typing of Naegleria fowleri from a Patient of Primary Amebic Meningoencephalitis (PAM) in China.

Science.gov (United States)

Zhang, Ling-Ling; Wu, Mao; Hu, Bang-Chuan; Chen, Hua-Liang; Pan, Jin-Ren; Ruan, Wei; Yao, Li-Nong

2018-05-08

Naegleria fowleri (N. fowleri) is the only Naegleria spp. known to cause an acute, fulminant, and rapidly fatal central nervous system infection called primary amebic meningoencephalitis (PAM) in human. In 2016, a suspected PAM patient was found in Zhejiang Province of China. The pathogen was identified by microscopic examination and PCR. The positive PCR products were sequenced and the sequences were aligned using NCBI BLAST programme. The homologous and phylogenetic analysis was conducted using the MEGA 6 programme. Under the microscopy, the motile cells with pseudopodia were observed in the direct smear, the motion characteristics of pseudopodia as well as the cell morphology suggested that the pathogen were amoeba trophozoites. The smears stained with Wright-Giemsa showed amoeba trophozoites with various sharps, which were measured of 10-25μm and characterized by the prominent, centrally placed nucleolus and the vacuolated cytoplasm. The PCR showed negative for E. histolytica and E. dispar, while positive for Naegleria spp.and N. fowleri. The nucleotide sequences acquired from this study were submitted to the Genbank with accession numbers of KX909928 and KX909927, respectively. The Blast analysis revealed that the sequences of KX909928 and KX909927 have 100% similarity with the sequence of N. fowleri gene (KT375442.1). Sequence alignment and phylogenetic tree reavealed that N. fowleri collected from this study was classified as genotype 2 and had a closest relative with N. lovaniensis. This study confirms N. fowleri as the agent responsible for this patient, however, PAM normally progresses fast and universally fatal within a week, so the patient still died at two weeks after the onset of symptoms. Copyright © 2018. Published by Elsevier Ltd.

Analysis of the Performance of a PAM/PPM/OOK System Operating with OCDMA, under Nonlinear Optical Effects in Optical Fiber Propagation

Science.gov (United States)

Correia, D. G.; Sales, J. C.; Pinto, P. V. F.; Moura, L. P.; Ferreira, A. C.; Menezes, J. W. M.; Guimarães, G. F.; Sombra, A. S. B.

2016-06-01

In this article, we present a numerical simulation study of encoding, decoding and propagation performance of short optical pulses and words with modulations OOK, PAM and PPM in OCDMA systems (Optical Code Division Multiple Access). The encoding and decoding of short pulses are obtained through fiber Bragg grating(FBG - FBG optical) devices, where the codes are inserted through discrete jumps in the optical phase (±π) where Gold codes were used. A figure of merit (SNR - Signal to Noise Ratio) was obtained to quantify the interference in propagation of short optical pulses. An increase in the temporal width was observed. For decoded pulses due to the nonlinearity effect, we observed an increase of 1.3 ps considering the propagation with γ=3 W-1 km-1 and γ=24 W-1 km-1. Analysis of coding and decoding words "a" and "w" was done. Considering the propagation (with γ=9 W-1 km-1) of a word "w", an error occurred in all modulations except for simultaneous PPM/PAM modulation, which is associated to the better autocorrelation characteristics obtained with the OOK, PAM and PPM modulations alone, and could double the transmission rate. The nonlinear effects directly affect the process of the autocorrelation codes due to interference from adjacent chip components of the code.

Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

International Nuclear Information System (INIS)

Mecham, R.P.; Hinek, A.; Entwistle, R.; Wrenn, D.S.; Griffin, G.L.; Senior, R.M.

1989-01-01

Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [ 3 H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. The authors propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment

Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin

Science.gov (United States)

Mandelin, Jami; Lin, Emme C. K.; Hu, Dana D.; Knowles, Susan K.; Do, Kim-Anh; Wang, Xuemei; Sage, E. Helene; Smith, Jeffrey W.; Arap, Wadih; Pasqualini, Renata

2009-01-01

BACKGROUND Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSION Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies. PMID:19224553

Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

Directory of Open Access Journals (Sweden)

Chen Zhang

2016-09-01

Full Text Available Ca2+-sensing receptors (CaSRs play a central role in regulating extracellular calcium concentration ([Ca2+]o homeostasis and many (pathophysiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor.

Science.gov (United States)

Ragnarsson, Lotten; Wang, Ching-I Anderson; Andersson, Åsa; Fajarningsih, Dewi; Monks, Thea; Brust, Andreas; Rosengren, K Johan; Lewis, Richard J

2013-01-18

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Extracellular polymeric substances from copper-tolerance Sinorhizobium meliloti immobilize Cu{sup 2+}

Energy Technology Data Exchange (ETDEWEB)

Hou, Wenjie; Ma, Zhanqiang; Sun, Liangliang; Han, Mengsha; Lu, Jianjun; Li, Zhenxiu; Mohamad, Osama Abdalla; Wei, Gehong, E-mail: weigehong@nwsuaf.edu.cn

2013-10-15

Highlights: • EPS produced by Sinorhizobium meliloti CCNWSX0020 restricts uptake of Cu{sup 2+}. • We focused on the EPS, which is divided into three main parts. • LB-EPS played a more important role than S-EPS and TB-EPS in Cu{sup 2+} immobilization. • Proteins and carbohydrates were the main extracellular compounds which had functional groups such as carboxyl (-COOH), hydroxyl (-OH), and amide (N-H), primarily involved in metal ion binding. -- Abstract: The copper tolerance gene of wild-type heavy metal-tolerance Sinorhizobium meliloti CCNWSX0020 was mutated by transposon Tn5-a. The mutant was sensitive up to 1.4 mM Cu{sup 2+}. Production, components, surface morphology, and functional groups of extracellular polymeric substances (EPS) of the wild-type strains were compared with sensitive mutant in immobilization of Cu{sup 2+}. EPS produced by S. meliloti CCNWSX0020 restricts uptake of Cu{sup 2+}. The cell wall EPS were categorized based on the compactness and fastness: soluble EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS). LB-EPS played a more important role than S-EPS and TB-EPS in Cu{sup 2+} immobilization. Scanning electron microscopy (SEM) analysis LB-EPS had rough surface and many honeycomb pores, making them conducive to copper entry; therefore, they may play a role as a microbial protective barrier. Fourier transform-infrared (FT-IR) analysis further confirm that proteins and carbohydrates were the main extracellular compounds which had functional groups such as carboxyl (-COOH), hydroxyl (-OH), and amide (N-H), primarily involved in metal ion binding.

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

Science.gov (United States)

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Stimulation of Suicidal Erythrocyte Death by Increased Extracellular Phosphate Concentrations

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Jakob Voelkl

2014-02-01

Full Text Available Background/Aim: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca2+-activity ([Ca2+]i. Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca2+]i from Fluo3-fluorescence. Results: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM. The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca2+]i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca2+ to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca2+, by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. Conclusion: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca2+ concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO4.

Intrinsic subtypes from PAM50 gene expression assay in a population-based breast cancer cohort: differences by age, race, and tumor characteristics.

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Sweeney, Carol; Bernard, Philip S; Factor, Rachel E; Kwan, Marilyn L; Habel, Laurel A; Quesenberry, Charles P; Shakespear, Kaylynn; Weltzien, Erin K; Stijleman, Inge J; Davis, Carole A; Ebbert, Mark T W; Castillo, Adrienne; Kushi, Lawrence H; Caan, Bette J

2014-05-01

Data are lacking to describe gene expression-based breast cancer intrinsic subtype patterns for population-based patient groups. We studied a diverse cohort of women with breast cancer from the Life After Cancer Epidemiology and Pathways studies. RNA was extracted from 1 mm punches from fixed tumor tissue. Quantitative reverse-transcriptase PCR was conducted for the 50 genes that comprise the PAM50 intrinsic subtype classifier. In a subcohort of 1,319 women, the overall subtype distribution based on PAM50 was 53.1% luminal A, 20.5% luminal B, 13.0% HER2-enriched, 9.8% basal-like, and 3.6% normal-like. Among low-risk endocrine-positive tumors (i.e., estrogen and progesterone receptor positive by immunohistochemistry, HER2 negative, and low histologic grade), only 76.5% were categorized as luminal A by PAM50. Continuous-scale luminal A, luminal B, HER2-enriched, and normal-like scores from PAM50 were mutually positively correlated. Basal-like score was inversely correlated with other subtypes. The proportion with non-luminal A subtype decreased with older age at diagnosis, P Trend < 0.0001. Compared with non-Hispanic Whites, African American women were more likely to have basal-like tumors, age-adjusted OR = 4.4 [95% confidence intervals (CI), 2.3-8.4], whereas Asian and Pacific Islander women had reduced odds of basal-like subtype, OR = 0.5 (95% CI, 0.3-0.9). Our data indicate that over 50% of breast cancers treated in the community have luminal A subtype. Gene expression-based classification shifted some tumors categorized as low risk by surrogate clinicopathologic criteria to higher-risk subtypes. Subtyping in a population-based cohort revealed distinct profiles by age and race. ©2014 AACR.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

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Varrault, Annie; Dantec, Christelle; Le Digarcher, Anne; Chotard, Laëtitia; Bilanges, Benoit; Parrinello, Hugues; Dubois, Emeric; Rialle, Stéphanie; Severac, Dany; Bouschet, Tristan; Journot, Laurent

2017-10-13

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

DEFF Research Database (Denmark)

Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

2010-01-01

extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known...... conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive...... about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic...

Binding of [125I]iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

International Nuclear Information System (INIS)

Jones, J.I.; Fitzpatrick, L.A.

1990-01-01

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor*♦

Science.gov (United States)

Ragnarsson, Lotten; Wang, Ching-I Anderson; Andersson, Åsa; Fajarningsih, Dewi; Monks, Thea; Brust, Andreas; Rosengren, K. Johan; Lewis, Richard J.

2013-01-01

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β1-adrenergic receptor crystal structure, we modeled the α1B-adrenoceptor (α1B-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α1B-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α1-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs. PMID:23184947

Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

Science.gov (United States)

Lam, Charlton; Jamerson, Melissa; Cabral, Guy; Carlesso, Ana Maris; Marciano-Cabral, Francine

2017-10-01

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

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O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Coherency Identification of Generators Using a PAM Algorithm for Dynamic Reduction of Power Systems

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Seung-Il Moon

2012-11-01

Full Text Available This paper presents a new coherency identification method for dynamic reduction of a power system. To achieve dynamic reduction, coherency-based equivalence techniques divide generators into groups according to coherency, and then aggregate them. In order to minimize the changes in the dynamic response of the reduced equivalent system, coherency identification of the generators should be clearly defined. The objective of the proposed coherency identification method is to determine the optimal coherent groups of generators with respect to the dynamic response, using the Partitioning Around Medoids (PAM algorithm. For this purpose, the coherency between generators is first evaluated from the dynamic simulation time response, and in the proposed method this result is then used to define a dissimilarity index. Based on the PAM algorithm, the coherent generator groups are then determined so that the sum of the index in each group is minimized. This approach ensures that the dynamic characteristics of the original system are preserved, by providing the optimized coherency identification. To validate the effectiveness of the technique, simulated cases with an IEEE 39-bus test system are evaluated using PSS/E. The proposed method is compared with an existing coherency identification method, which uses the K-means algorithm, and is found to provide a better estimate of the original system. 

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.

Science.gov (United States)

Courtney, D G; Moore, J E; Atkinson, S D; Maurizi, E; Allen, E H A; Pedrioli, D M L; McLean, W H I; Nesbit, M A; Moore, C B T

2016-01-01

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

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Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna

2012-03-01

Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.

Association of Bordetella dermonecrotic toxin with the extracellular matrix

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Miyake Masami

2010-09-01

Full Text Available Abstract Background Bordetella dermonecrotic toxin (DNT causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. Results Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. Conclusions Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains.

Science.gov (United States)

Han, Sangyeul; Kim, Sun; Bahl, Samira; Li, Lin; Burande, Clara F; Smith, Nicole; James, Marianne; Beauchamp, Roberta L; Bhide, Pradeep; DiAntonio, Aaron; Ramesh, Vijaya

2012-08-31

Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.

Low-cost and miniaturized 100-Gb/s (2 × 50 Gb/s) PAM-4 TO-packaged ROSA for data center networks.

Science.gov (United States)

Kang, Sae-Kyoung; Huh, Joon Young; Lee, Jie Hyun; Lee, Joon Ki

2018-03-05

We design and implement a cost-effective and compact 100-Gb/s (2 × 50 Gb/s) PAM-4 receiver optical sub-assembly (ROSA) by using a TO-can package instead of an expensive box-type package. It consists of an optical demultiplexer, two PIN-PDs and a 2-channel linear transimpedance amplifier. The components are passively aligned and assembled using alignment marks engraved on each part. With a real-time PAM-4 DSP chip, we measured the back-to-back receiver sensitivities of the 100-Gb/s ROSA based on TO-56 to be less than -13.2 dBm for both channels at a bit error rate of 2.4e-4. The crosstalk penalty due to the adjacent channel interference was observed around 0.1 dB.

Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

Directory of Open Access Journals (Sweden)

Jamal Stie

2009-06-01

Full Text Available The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS, resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface.The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen.The results of this study provide evidence for the

Challenges in the development of an M4 PAM in vivo tool compound: The discovery of VU0467154 and unexpected DMPK profiles of close analogs.

Science.gov (United States)

Wood, Michael R; Noetzel, Meredith J; Poslusney, Michael S; Melancon, Bruce J; Tarr, James C; Lamsal, Atin; Chang, Sichen; Luscombe, Vincent B; Weiner, Rebecca L; Cho, Hyekyung P; Bubser, Michael; Jones, Carrie K; Niswender, Colleen M; Wood, Michael W; Engers, Darren W; Brandon, Nicholas J; Duggan, Mark E; Conn, P Jeffrey; Bridges, Thomas M; Lindsley, Craig W

2017-01-15

This letter describes the chemical optimization of a novel series of M 4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration). Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytoplasmic binding and disposition kinetics of diclofenac in the isolated perfused rat liver

Science.gov (United States)

Weiss, Michael; Kuhlmann, Olaf; Hung, Daniel Y; Roberts, Michael S

2000-01-01

The binding kinetics of diclofenac to hepatocellular structures were evaluated in the perfused rat liver using the multiple indicator dilution technique and a stochastic model of organ transit time density.The single-pass, in situ rat liver preparation was perfused with buffer solution (containing 2% albumin) at 30 ml min−1. Diclofenac and [14C]-sucrose (extracellular reference) were injected simultaneously as a bolus dose into the portal vein (six experiments in three rats). An analogous series of experiments was performed with [14C]-diclofenac and [3H]-sucrose.The diclofenac outflow data were analysed using three models of intracellular distribution kinetics, assuming (1) instantaneous distribution and binding (well-mixed model), (2) ‘slow' binding at specific intracellular sites after instantaneous distribution throughout the cytosol (slow binding model), and (3) ‘slowing' of cytoplasmic diffusion due to instantaneous binding (slow diffusion model).The slow binding model provided the best description of the data. The rate constants for cellular influx and sequestration were 0.126±0.026 and 0.013±0.009 s−1, respectively. The estimated ratio of cellular initial distribution volume to extracellular volume of 2.82 indicates an almost instantaneous distribution in the cellular water space, while the corresponding ratio of 5.54 estimated for the apparent tissue distribution volume suggests a relatively high hepatocellular binding. The non-instantaneous intracellular equilibration process was characterized by time constants of the binding and unbinding process of 53.8 and 49.5 s, respectively. The single-pass availability of diclofenac was 86%. The results obtained with [14C]-diclofenac and [3H]-sucrose were not statistically different. PMID:10903973

Imaging c-PAM-induced flocculation of paper fibers.

Science.gov (United States)

Hartley, William H; Banerjee, Sujit

2008-04-01

The flocculation of paper fibers by cationic polyacrylamides (c-PAM) was studied by imaging the fibers that remain free during flocculation. Studies with fibers of different lengths showed that the degree of flocculation increases with fiber length, with the best flocs being formed with mixtures of short and long fibers. Short fibers did not flocculate by themselves but were captured by flocs formed with longer fibers. The short fibers strengthen the floc and give it shear resistance. Shear had the expected effect of promoting flocculation at low Reynolds number but disrupting it at higher values. For a given polymer the maximum floc size for a mixture of fibers is dictated by the length distribution of the fibers. The polymer dose governs the rate of flocculation. The technique is especially useful in following the tail end of the flocculation process. At this stage a floc is almost fully grown and a small increase in its size would be very difficult to measure by conventional techniques. In contrast, the number of free fibers measured by single fiber imaging decreases rapidly at this point.

Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

DEFF Research Database (Denmark)

Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.

2006-01-01

Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage...

A Conserved Salt Bridge between Transmembrane Segment 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

DEFF Research Database (Denmark)

Pedersen, Anders Vingborg; Andreassen, Thorvald Faurschou; Løland, Claus Juul

2014-01-01

into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the "thin gate" formed by an interaction between Arg-30 and Asp-404. In the human dopamine...

Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells : a plug-and-play approach

NARCIS (Netherlands)

Kooijmans, Sander A A; Gitz-Francois, Jerney J J M; Schiffelers, Raymond M; Vader, Pieter

2018-01-01

Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, manipulation of targeting properties of EVs through engineering of the producer cells can be challenging and

The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the natural occurring R213G substitution

DEFF Research Database (Denmark)

Gottfredsen, Randi Heidemann; Goldstrohm, David; Hartney, John

2014-01-01

and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand......-binding capacity, this could not be correlated to proteolytic removal of the ECM-binding region, because the integrity of the material recovered from the medium was comparable to that of the cell surface-associated protein. The naturally occurring R213G amino acid substitution located in the ECM-binding region...

Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

Science.gov (United States)

Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

2007-10-01

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

Large-Scale Investigation of Leishmania Interaction Networks with Host Extracellular Matrix by Surface Plasmon Resonance Imaging

Science.gov (United States)

Fatoux-Ardore, Marie; Peysselon, Franck; Weiss, Anthony; Bastien, Patrick; Pratlong, Francine

2014-01-01

We have set up an assay to study the interactions of live pathogens with their hosts by using protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging. We have used this assay to characterize the interactions of Leishmania promastigotes with ∼70 mammalian host biomolecules (extracellular proteins, glycosaminoglycans, growth factors, cell surface receptors). We have identified, in total, 27 new partners (23 proteins, 4 glycosaminoglycans) of procyclic promastigotes of six Leishmania species and 18 partners (15 proteins, 3 glycosaminoglycans) of three species of stationary-phase promastigotes for all the strains tested. The diversity of the interaction repertoires of Leishmania parasites reflects their dynamic and complex interplay with their mammalian hosts, which depends mostly on the species and strains of Leishmania. Stationary-phase Leishmania parasites target extracellular matrix proteins and glycosaminoglycans, which are highly connected in the extracellular interaction network. Heparin and heparan sulfate bind to most Leishmania strains tested, and 6-O-sulfate groups play a crucial role in these interactions. Numerous Leishmania strains bind to tropoelastin, and some strains are even able to degrade it. Several strains interact with collagen VI, which is expressed by macrophages. Most Leishmania promastigotes interact with several regulators of angiogenesis, including antiangiogenic factors (endostatin, anastellin) and proangiogenic factors (ECM-1, VEGF, and TEM8 [also known as anthrax toxin receptor 1]), which are regulated by hypoxia. Since hypoxia modulates the infection of macrophages by the parasites, these interactions might influence the infection of host cells by Leishmania. PMID:24478075

Comparison of PAM and CAP modulations robustness against mode partition noise in optical links

Science.gov (United States)

Stepniak, Grzegorz

2017-08-01

Mode partition noise (MPN) of the laser employed at the transmitter can significantly degrade the transmission performance. In the paper, we introduce a simulation model of MPN in vertical cavity surface emitting laser (VCSEL) and simulate transmission of pulse amplitude modulation (PAM) and carrierless amplitude phase (CAP) signals in multimode fiber (MMF) link. By turning off other effects, like relative intensity noise (RIN), we focus solely on the influence of MPN on transmission performance degradation. Robustness of modulation and equalization type against MPN is studied.

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

Energy Technology Data Exchange (ETDEWEB)

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

Directory of Open Access Journals (Sweden)

Yumiko Obayashi

2017-10-01

Full Text Available Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO and 2-methoxyethanol (MTXE. The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube, protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In

Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition

Science.gov (United States)

Datsenko, Kirill A.; Jackson, Ryan N.; Wiedenheft, Blake; Severinov, Konstantin; Brouns, Stan J. J.

2013-01-01

Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism. PMID:24039596

A study of photochemical againg of ambient air using Potential Aerosol Mass (PAM) chamber under the different sources and types of emissions

Science.gov (United States)

Lee, T.; Son, J.; Kim, J.; Kim, S.; Sung, K.; Park, G.; Link, M.; Park, T.; Kim, K.; Kang, S.; Ban, J.; Kim, D. S.

2016-12-01

Recent research proposed that Secondary Aerosol (SA) is important class of predicting future climate change scenarios, health effect, and a general air quality. However, there has been lack of studies to investigate SA formation all over the world. This study tried to focus on understanding potential secondary aerosol formation and its local impact by the photochemical aging of inorganic and organic aerosols in the ambient air using the Potential Aerosol Mass (PAM) chamber under the different sources and types of emissions. PAM chamber manufactured by Aerodyne make an oxidizing environment that simulates oxidation processes on timescales of 12-15 hrs in the atmosphere. Chemical compositions of ambient aerosol and aerosol that was aged in the PAM chamber were alternately measured every 2-minutes using the High Resolution-Time of Flight-Aerosol Mass Spectrometer (HR-ToF-AMS). HR-ToF-AMS provides non-refractory aerosol mass concentrations including nitrate, sulfate, hydrocarbon-like and oxygenated organic aerosol in real time. This study includes a residence area of mixture of sources, a forest site of dominant source of biogenic VOCs, an underground parking lot of dominant vehicle emission, and laboratory experiment of vehicle emissions under different fuels and speeds using the chassis dynamometer. As a result, it was revealed that gasoline and LPG vehicle relatively made more potential SA than diesel vehicle.

Extracellular pH regulates zinc signaling via an Asp residue of the zinc-sensing receptor (ZnR/GPR39).

Science.gov (United States)

Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

2012-09-28

Zinc activates a specific Zn(2+)-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca(2+) responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na(+)/H(+) exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn(2+) binding site, His(17) or His(19), or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp(313) with alanine resulted in similar Ca(2+) responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na(+)/H(+) exchange at pH 7.4 and pH 6.5. Substitution of Asp(313) to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp(313), which was shown to modulate Zn(2+) binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.

Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)*

Science.gov (United States)

Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

2012-01-01

Zinc activates a specific Zn2+-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca2+ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na+/H+ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn2+ binding site, His17 or His19, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp313 with alanine resulted in similar Ca2+ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na+/H+ exchange at pH 7.4 and pH 6.5. Substitution of Asp313 to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp313, which was shown to modulate Zn2+ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity. PMID:22879599

Extracellular determinants of anion discrimination of the Cl-/H+ antiporter protein CLC-5.

Science.gov (United States)

De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

2011-12-23

Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.

Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

Directory of Open Access Journals (Sweden)

Cristina Antonella Nadalutti

Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.

Science.gov (United States)

Myers, Stephanie M; Bawn, Ruth H; Bisset, Louise C; Blackburn, Timothy J; Cottyn, Betty; Molyneux, Lauren; Wong, Ai-Ching; Cano, Celine; Clegg, William; Harrington, Ross W; Leung, Hing; Rigoreau, Laurent; Vidot, Sandrine; Golding, Bernard T; Griffin, Roger J; Hammonds, Tim; Newell, David R; Hardcastle, Ian R

2016-08-08

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

Comparison of OOK- and PAM-4 modulation for 10 Gbit/s transmission over up to 300 m polymer optical fiber

NARCIS (Netherlands)

Breyer, F.; Lee, S.C.J.; Randel, S.; Hanik, N.

2008-01-01

10 Gbit/s Transmission over up to 300 m of multimode 62.5 µm core-diameter perfluorinated graded-index polymer optical fiber is compared using on-off-keying (OOK) or 4-level pulse amplitude modulation (PAM-4) and feed-forward or decision-feedback equalization.

Seasonal and diel changes in photosynthetic activity of the snow algae Chlamydomonas nivalis (Chlorophyceae) from Svalbard determined by PAM fluorometry

Czech Academy of Sciences Publication Activity Database

Stibal, Marek; Elster, Josef; Šabacká, Marie; Kaštovská, Klára

2007-01-01

Roč. 59, - (2007), s. 265-273 ISSN 0168-6496 R&D Projects: GA AV ČR KJB6005409 Institutional research plan: CEZ:AV0Z60050516 Keywords : Chlamydomonas nivalis * photosynthetic activity * PAM fluorometry Subject RIV: EF - Botanics Impact factor: 3.039, year: 2007

Experimental demonstration of 112-Gbit/s PAM-4 over up to 80 km SSMF at 1550 nm for Inter-DCI applications

NARCIS (Netherlands)

Eiselt, N.; van der Heide, S.; Griesser, H.; Eiselt, M.; Okonkwo, C.; Olmos, J.J.V.; Tafur Monroy, I.

2016-01-01

We experimentally demonstrate 112-Gbit/s PAM-4 over 80 km SSMF at 1550 nm. It is shown, that a channel shortening filter (CSF) matched to the memory of a subsequent MLSE significantly improves the performance while keeping complexity manageable.

Experimental demonstration of 112-Gbit/s PAM-4 over up to 80 km SSMF at 1550 nm for inter-DCI applications

DEFF Research Database (Denmark)

Eiselt, Nicklas; Van Der Heide, Sjoerd; Griesser, Helmut

2016-01-01

We experimentally demonstrate 112-Gbit/s PAM-4 over 80 km SSMF at 1550 nm. It is shown that a channel shortening filter (CSF) matched to the memory of a subsequent MLSE significantly improves the performance while keeping complexity manageable....

Ligand binding and activation mechanism og the glucagon-like peptide-1 receptor

DEFF Research Database (Denmark)

Underwood, Christina Rye

GLP-1R interacts with receptor agonists. The thesis includes four studies, which investigate different aspects of these interactions. The first study elucidates GLP-1 binding to the extracellular domain of GLP-1R (ECD) (Study I), whereas the second study identifies receptor domains important for small...

Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

Directory of Open Access Journals (Sweden)

Thomas Stockner

Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2017-01-01

Roč. 7, Jan 16 (2017), č. článku 40381. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * N-methylscopolamine * ligand binding * molecular dynamics Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 4.259, year: 2016

Exploring with PAM: Prospecting ANTS Missions for Solar System Surveys

Science.gov (United States)

Clark, P. E.; Rilee, M. L.; Curtis, S. A.

2003-01-01

ANTS (Autonomous Nano-Technology Swarm), a large (1000 member) swarm of nano to picoclass (10 to 1 kg) totally autonomous spacecraft, are being developed as a NASA advanced mission concept. ANTS, based on a hierarchical insect social order, use an evolvable, self-similar, hierarchical neural system in which individual spacecraft represent the highest level nodes. ANTS uses swarm intelligence attained through collective, cooperative interactions of the nodes at all levels of the system. At the highest levels this can take the form of cooperative, collective behavior among the individual spacecraft in a very large constellation. The ANTS neural architecture is designed for totally autonomous operation of complex systems including spacecraft constellations. The ANTS (Autonomous Nano Technology Swarm) concept has a number of possible applications. A version of ANTS designed for surveying and determining the resource potential of the asteroid belt, called PAM (Prospecting ANTS Mission), is examined here.

Diagnose Test-Taker's Profile in Terms of Core Profile Patterns: Principal Component (PC) vs. Profile Analysis via MDS (PAMS) Approaches.

Science.gov (United States)

Kim, Se-Kang; Davison, Mark L.

A study was conducted to examine how principal components analysis (PCA) and Profile Analysis via Multidimensional Scaling (PAMS) can be used to diagnose individuals observed score profiles in terms of core profile patterns identified by each method. The standardization sample from the Wechsler Intelligence Scale for Children, Third Edition…

Focusing on the adult attachment style in schizophrenia in community mental health centres: validation of the Psychosis Attachment Measure (PAM) in a German-speaking sample.

Science.gov (United States)

Kvrgic, Sara; Beck, Eva-Marina; Cavelti, Marialuisa; Kossowsky, Joe; Stieglitz, Rolf-Dieter; Vauth, Roland

2012-07-01

Assessing attachment style in people with schizophrenia may be important to identify a risk factor in building a strong therapeutic relationship and so indirectly to understand the development of mal-compliance as one of the major obstacles in the treatment of schizophrenia. The present study analysed the psychometric properties of the German version of the Psychosis Attachment Measure (PAM), which assesses avoidant and anxious attachment style. A sample of 127 patients suffering from chronic schizophrenia or schizoaffective disorder participated in this study. In testing discriminant validity, we assessed psychopathology, depression, therapeutic relationship and service engagement. Internal consistency, test-retest reliability and factor structure were analysed. The German version of PAM exhibited acceptable to good internal and test-retest reliabilities and the two-factor structure of the English version could be replicated. Avoidant attachment style was related to higher levels of positive symptoms and to a poorer therapeutic relationship. In the context of external validation, a regression analysis revealed that a poor therapeutic relationship correlated with avoidant attachment style, independent of anxious attachment style and depressive symptoms. Anxious attachment was associated with higher treatment adherence. Both insecure attachment styles (avoidant and anxious) were found to be correlated with higher levels of depression, but only attachment anxiety had an independent predictive value for self-reported depression in regression analysis. The German version of PAM displayed satisfactory psychometric properties and seems to be a reliable measure for assessing attachment style in individuals with schizophrenia. Validation of PAM led to the finding that only the avoidant attachment style might be a risk factor when building a strong therapeutic relationship in schizophrenia. In future studies, other factors influencing therapeutic relationship should be

Challenges in the development of an M4 PAM preclinical candidate: The discovery, SAR, and in vivo characterization of a series of 3-aminoazetidine-derived amides.

Science.gov (United States)

Tarr, James C; Wood, Michael R; Noetzel, Meredith J; Bertron, Jeanette L; Weiner, Rebecca L; Rodriguez, Alice L; Lamsal, Atin; Byers, Frank W; Chang, Sichen; Cho, Hyekyung P; Jones, Carrie K; Niswender, Colleen M; Wood, Michael W; Brandon, Nicholas J; Duggan, Mark E; Conn, P Jeffrey; Bridges, Thomas M; Lindsley, Craig W

2017-07-01

This letter details the continued chemical optimization of a novel series of M 4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core by incorporating a 3-amino azetidine amide moiety. The analogs described within this work represent the most potent M 4 PAMs reported for this series to date. The SAR to address potency, clearance, subtype selectivity, CNS exposure, and P-gp efflux are described. This work culminated in the discovery of VU6000918, which demonstrated robust efficacy in a rat amphetamine-induced hyperlocomotion reversal model at a minimum efficacious dose of 0.3mg/kg. Copyright © 2017 Elsevier Ltd. All rights reserved.

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

OpenAIRE

L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field co...

First Real-Time 400G PAM-4 Demonstration for Inter-Data Center Transmission over 100 km of SSMF at 1550 nm

DEFF Research Database (Denmark)

Eiselt, Nicklas; Wei, Jinlong; Griesser, Helmut

2016-01-01

Real-time transmission of 400G (8x50G DWDM) PAM-4 signals for data center interconnects up to 100 km SSMF is successfully demonstrated. All channels stay well below the 802.3bj KR4 FEC limit, thus allowing error free transmission...

Lipopeptide-Induced Suicidal Erythrocyte Death Correlates with the Degree of Acylation

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Abdulla Al Mamun Bhuyan

2017-01-01

Full Text Available Background/Aims: Consequences of bacterial infection include anemia, which could result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Bacterial components known to stimulate eryptosis include lipopeptides. Signaling mediating the triggering of eryptosis include increased cytosolic Ca2+ activity ([Ca2+]i, oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling. Methods: Human erythrocytes were incubated for 48 hours in the absence and presence of 1 or 5 µg/ml of the synthetic lipopeptides Pam1 (lipopeptide with one fatty acid, Pam2 (lipopeptide with two fatty acids, or Pam3 (lipopeptide with three fatty acids. In the following phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies. Results: Pam1 (5 µg/ml, Pam2 (5 µg/ml and Pam3 (1 and 5 µg/ml significantly increased the percentage of annexin-V-binding to erythrocytes in a dose dependent manner, which was largely independent of Ca2+. Pam1-3 increased the percentage of both, swollen and shrunken erythrocytes without significantly modifying the average forward scatter. They also increased reactive oxygen species (ROS and ceramide abundance. In all assays the effect on eryptosis increased with increasing number of fatty acids, with Pam3 showing always the strongest effect. In contrast, a comparison of the effect of Pam1-3 on TLR2 dependent immune stimulation showed that not Pam3 but Pam2 displayed the strongest activity, and that immune stimulation was triggered at much lower concentrations than eryptosis. Conclusions: Lipopeptides are not only important

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

Science.gov (United States)

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.

Science.gov (United States)

Zhang, Yanlin; Guan, Li; Yu, Jie; Zhao, Zanmei; Mao, Lijun; Li, Shuqiang; Zhao, Jinyuan

2016-11-21

During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating

[3H]nitrendipine binding to adrenal capsular membranes

International Nuclear Information System (INIS)

Finkel, M.S.; Aguilera, G.; Catt, K.J.; Keiser, H.R.

1984-01-01

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. The authors therefore studied the [ 3 H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with K/sub D/ = .26 +/- .04 nM and B/sub max/ = 105 +/- 5.7 fmol/mg protein. Specific binding of [ 3 H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine >> verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [ 3 H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production. 17 references, 2 figures, 1 table

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

Science.gov (United States)

Singh, Digvijay; Mallon, John; Poddar, Anustup; Wang, Yanbo; Tippana, Ramreddy; Yang, Olivia; Bailey, Scott; Ha, Taekjip

2018-05-22

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

DEFF Research Database (Denmark)

Rummel, Pia Cwarzko; Thiele, Stefanie; Hansen, Laerke Smidt

2013-01-01

interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other...... chemokine receptors, high affinity CCL3 chemokine binding was maintained in the absence of either bridge. In CCR5, the closest homolog to CCR1, a completely different dependency was observed as neither chemokine activation nor binding was retained in the absence of either bridge. In contrast, both bridges...... where dispensable for small-molecule activation. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1, preserved folding of ECL2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup, does not necessarily...

Extracellular Determinants of Anion Discrimination of the Cl−/H+ Antiporter Protein CLC-5*

Science.gov (United States)

De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

2011-01-01

Mammalian CLC proteins comprise both Cl− channels and Cl−/H+ antiporters that carry out fundamental physiological tasks by transporting Cl− across plasma membrane and intracellular compartments. The NO3− over Cl− preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl−/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3− over Cl− preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl−. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext. PMID:21921031

Binding proteins of somatomedins and their functions

International Nuclear Information System (INIS)

Kostelecka, Z.; Blahovec, J.

1998-01-01

In this paper the functions of binding proteins are discussed. One variable that provides insulin-like growth factors (IGFs) control at the extracellular level is the presence of high-affinity, soluble insulin-like growth factor proteins (IGFBPs). IGFBP-1 inhibits IGF effect on human osteosarcoma cells. Increased concentration of IGFBP-3 inhibits the proliferation of breast cancer cell line MCF 7 either directly or by competition for IGF receptors. Maybe IGFBPs work as anti-mitogens and IGFs are potential promotors of cancer growth

Extracellular histones play an inflammatory role in acid aspiration-induced acute respiratory distress syndrome.

Science.gov (United States)

Zhang, Yanlin; Wen, Zongmei; Guan, Li; Jiang, Ping; Gu, Tao; Zhao, Jinyuan; Lv, Xin; Wen, Tao

2015-01-01

Systemic inflammation is a key feature in acid aspiration-induced acute respiratory distress syndrome (ARDS), but the factors that trigger inflammation are unclear. The authors hypothesize that extracellular histones, a newly identified inflammatory mediator, play important roles in the pathogenesis of ARDS. The authors used a hydrochloric acid aspiration-induced ARDS model to investigate whether extracellular histones are pathogenic and whether targeting histones are protective. Exogenous histones and antihistone antibody were administered to mice. Heparin can bind to histones, so the authors studied whether heparin could protect from ARDS using cell and mouse models. Furthermore, the authors analyzed whether extracellular histones are clinically involved in ARDS patients caused by gastric aspiration. Extracellular histones in bronchoalveolar lavage fluid of acid-treated mice were significantly higher (1.832 ± 0.698) at 3 h after injury than in sham-treated group (0.63 ± 0.153; P = 0.0252, n = 5 per group). Elevated histones may originate from damaged lung cells and neutrophil infiltration. Exogenous histones aggravated lung injury, whereas antihistone antibody markedly attenuated the intensity of ARDS. Notably, heparin provided a similar protective effect against ARDS. Analysis of plasma from ARDS patients (n = 21) showed elevated histones were significantly correlated with the degree of ARDS and were higher in nonsurvivors (2.723 ± 0.2933, n = 7) than in survivors (1.725 ± 0.1787, P = 0.006, n = 14). Extracellular histones may play a contributory role toward ARDS by promoting tissue damage and systemic inflammation and may become a novel marker reflecting disease activity. Targeting histones by neutralizing antibody or heparin shows potent protective effects, suggesting a potentially therapeutic strategy.

PAM4 based symmetrical 112-Gbps long-reach TWDM-PON

Science.gov (United States)

Wu, Liyu; Gao, Fan; Zhang, Minming; Fu, Songnian; Deng, Lei; Choi, Michael; Chang, Donald; Lei, Gordon K. P.; Liu, Deming

2018-02-01

We experimentally demonstrate cost effective symmetrical 112-Gbps long-reach passive optical network (LR-PON) over 70-km standard signal mode fiber (SSMF), based on pulse amplitude modulation (PAM)-4. Four 10G-class directly modulated lasers (DMLs) at C-band are used for achieving 4 × 28-Gbps downstream transmission, while two 18G-class DMLs at O-band are used to realize 2 × 56-Gbps upstream transmission, without any optical amplification in optical distributed network (ODN). Both dispersion compensation fiber (DCF) for downstream signal and praseodymium-doped fiber amplifier (PDFA) for upstream signal are equipped at optical line terminal (OLT). Meanwhile, sparse Volterra filter (SVF) equalizer is proposed to mitigate the transmission impairments with substantial reduction of computation complexity. Finally, we can successfully provide a loss budget of 33 dB per downstream wavelength channel, indicating of 64 optical network units (ONUs) with more than 1.25 Gbps per ONU.

Extracellular small RNAs: what, where, why?

Science.gov (United States)

Hoy, Anna M.; Buck, Amy H.

2012-01-01

miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753

Secrecy Dimming Capacity in Multi-LED PAM-Based Visible Light Communications

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Byung Wook Kim

2017-01-01

Full Text Available Recently, mobile cloud computing (MCC has gained a lot of interest for researchers building the next-generation mobile applications. Because unauthorized access may cause serious problems, security and privacy with MCC have become significant issues. This paper addresses the secrecy dimming capacity of secure transmission in MCC over visible light communication (VLC channels. By obtaining the entropy-maximizing symbol probability of multiple light emitting diode- (LED- based pulse amplitude modulation (PAM, mathematical analysis of the secrecy dimming capacity of VLC was derived. Simulation results show that the secure transmission ability of multi-LED-based VLC is determined according to the number of activated LEDs and target dimming level. This can be a guideline for practical VLC-based mobile network designers intending to secure wireless transmission and to decide on the number of activated LEDs at target dimming level to operate.

Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

Science.gov (United States)

Guan, Xiaolu; Zhang, Yao; Yu, Mengmeng; Ren, Chaoqi; Gao, Yanni; Yun, Bingling; Liu, Yongzhen; Wang, Yongqiang; Qi, Xiaole; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Gao, Li; Li, Kai; Pan, Qing; Zhang, Baoshan; Wang, Xiaomei; Gao, Yulong

2018-01-01

Chicken Na + /H + exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry. IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are

Ascorbic acid: Nonradioactive extracellular space marker in canine heart

International Nuclear Information System (INIS)

Reil, G.H.; Frombach, R.; Kownatzki, R.; Quante, W.; Lichtlen, P.R.

1987-01-01

The distribution pattern of ascorbic acid and L-[ 14 C]ascorbic acid in myocardial tissue was compared with those of the classical radioactive extracellular space markers [ 3 H]-inulin, [ 3 H]sucrose, and Na 82 Br. A new polarographic techniques was developed for analogue registration of ascorbic acid concentration in coronary venous blood. The kinetic data of the markers were studied in an open-chest canine heart preparation during a constant tracer infusion of up to 9 min. Distribution volumes were calculated based on the mean transit time method of Zierler. The distribution volume of ascorbic acid as well as of L-[ 14 C]ascorbic acid in myocardial tissue agreed closely with those of [ 3 H]inulin and [ 3 H]sucrose as well as 82 Br. The obtained kinetic data confirmed that ascorbic acid exhibits the physicochemical properties of an extracellular space marker, though this compound was shown to leak slowly into myocardial cells. Favorable attributes of this indicator are its low molecular weight, high diffusibility in interstitial fluid, low binding affinity to macromolecules, and high transcapillary as well as low transplasmalemmal penetration rate. Therefore, this nonradioactive marker can be applied in a safe and simple fashion, and without untoward side effects in experimental animals as well as in patients

The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

Energy Technology Data Exchange (ETDEWEB)

Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. S.; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

2010-07-01

Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

Extracellular proteins: Novel key components of metal resistance in cyanobacteria?

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Joaquin eGiner-Lamia

2016-06-01

Full Text Available Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias towards the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

IGF binding proteins.

Science.gov (United States)

Bach, Leon A

2017-12-18

Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

An accessory to the 'Trinity': SR-As are essential pathogen sensors of extracellular dsRNA, mediating entry and leading to subsequent type I IFN responses.

Directory of Open Access Journals (Sweden)

Stephanie J DeWitte-Orr

2010-03-01

Full Text Available Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (dsRNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNbeta. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II(-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as 'carriers', facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.

Extracellular matrix of smooth muscle cells: interaction of collagen type V with heparan sulfate proteoglycan

International Nuclear Information System (INIS)

Gay, S.; Hoeoek, M.; Gay, R.E.; Magargal, W.W.; Reynertson, R.H.

1986-01-01

Alteration in the extracellular matrix produced by smooth muscle cells may play a role in the development of atherosclerotic lesions. Consequently the authors have initiated studies on the structural organization of the extracellular matrix produced by cultured smooth muscle cells. Immunohisotological examination of this matrix using well-characterized mono- and polyclonal antibodies showed a partial codistribution of heparan sulfate (HS) proteoglycans with a number of different matrix components including collagen types I, III, IV, V and VI, laminin and fibronectin. Subsequent binding studies between isolated matrix proteins and HS showed that the polysaccharide interacts strongly with type V collagen and to a lesser extent with fibronectin as well as collagen types III and VI. The interaction between type V and HS was readily inhibited by heparin and highly sulfated HS but not be dermatan sulfate, chondroitin sulfate or HS with a low sulfate content. Furthermore, [ 35 S]-HS proteoglycans isolated from cultured smooth muscle cells could be adsorbed on a column of sepharose conjugated with native type V collagen and eluted in a salt gradient. Hence, the interaction between type V and HS may play a major part in stabilizing the extracellular matrix of the vessel wall

Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism.

Science.gov (United States)

Edery, M; Rozakis-Adcock, M; Goujon, L; Finidori, J; Lévi-Meyrueis, C; Paly, J; Djiane, J; Postel-Vinay, M C; Kelly, P A

1993-01-01

A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism. The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor. All constructs were transiently expressed in COS-7 cells. Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively. Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities. In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand. These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients. Images PMID:8450064

Role of an extracellular loop in determining the stoichiometry of Na+–HCO3− cotransporters

Science.gov (United States)

Chen, Li-Ming; Liu, Ying; Boron, Walter F

2011-01-01

The Na+–HCO3− cotransporters (NBCs) of the solute carrier 4 family (SLC4) are critical for regulating pH in cells as well as in fluids such as blood and cerebrospinal fluid. Moreover, mutations and gene disruptions in NBC are linked to a wide range of pathologies. NBCe1 (SLC4A4) is electrogenic because it has an apparent Na+:HCO3− stoichiometry of 1:2 or 1:3, whereas NBCn1 (SLC4A7) is electroneutral because it has an apparent stoichiometry of 1:1. Because stoichiometry influences the effect of transport on membrane potential and vice versa, a central question is what structural features underlie electrogenicity versus electroneutrality. A previous study on rat NBCe1/n1 chimeras demonstrated that the structural elements determining the electrogenicity of NBCe1-A are located within the transmembrane domain, excluding the large third extracellular loop. In the present study we generated a series of chimeras of human NBCe1-A and human NBCn1-A. We found that replacing merely the predicted fourth extracellular loop (EL4) – containing 32 amino acid residues that include 7 prolines – of human NBCe1-A with EL4 of NBCn1-A creates an electroneutral NBC. The opposite switch converts an electroneutral construct to one with electrogenic properties. The introduction of an N-glycosylation site into EL4 confirms that at least a part of it is exposed to the extracellular fluid. We hypothesize that putative EL4 either contributes to the substrate-binding vestibule or indirectly influences substrate binding by interacting with one or more transmembrane segments, thereby controlling the nature of transport. PMID:21224233

Surface glycosylation profiles of urine extracellular vesicles.

Directory of Open Access Journals (Sweden)

Jared Q Gerlach

Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

Extracellular gadolinium contrast agents: Differences in stability

International Nuclear Information System (INIS)

Morcos, S.K.

2008-01-01

Extracellular gadolinium contrast agents (Gd-CA) are either linear or macrocyclic chelates available as ionic or non-ionic preparations. The molecular structure whether cyclic or linear and ionicity determines the stability of Gd-CA. Linear chelates are flexible open chains which do not offer a strong binding to Gd 3+ . In contrast, the macrocyclic chelates offer a strong binding to Gd 3+ by the virtue of being preorganized rigid rings of almost optimal size to cage the gadolinium atom. Non-ionic preparations are also less stable in comparison to the ionic ones as the binding between Gd 3+ with the negatively charged carboxyl groups is stronger in comparison to that with amides or alcohol in the non-ionic preparations. According to stability constants and kinetic measurements, the most stable Gd-CM is the ionic-macrocyclic chelate Gd-DOTA and the least stable agents are the non-ionic linear chelates gadodiamide and gadoversetamide. In vivo data confirmed the low stability of non-ionic linear chelates but no significant difference was observed amongst the macrocyclic agents whether ionic (Gd-DOTA) or non-ionic such as Gd-HP-DO3A and Gd-BT-DO3A. The stability of Gd-CA seems to be an important factor in the pathogenesis of the serious complication of nephrogenic systemic fibrosis. Gd-CA of low stability are likely to undergo transmetallation and release free Gd ions that deposit in tissue and attract circulating fibrocytes to initiate the process of fibrosis. No cases of NSF have been observed so far after the exclusive use of the stable macrocyclic Gd-CA

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

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Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

Molecular mechanisms in compatibility and mechanical properties of Polyacrylamide/Polyvinyl alcohol blends.

Science.gov (United States)

Wei, Qinghua; Wang, Yanen; Che, Yu; Yang, Mingming; Li, Xinpei; Zhang, Yingfeng

2017-01-01

The objectives of this study were to develop a computational model based on molecular dynamics technique to investigate the compatibility and mechanical properties of Polyacrylamide (PAM)/Polyvinyl alcohol (PVA) blends. Five simulation models of PAM/PVA with different composition ratios (4/0, 3/1, 2/2, 1/3, 0/4) were constructed and simulated by using molecular dynamics (MD) simulation. The interaction mechanisms of molecular chains in PAM/PVA blend system were elaborated from the aspects of the compatibility, mechanical properties, binding energy and pair correlation function, respectively. The computed values of solubility parameters for PAM and PVA indicate PAM has a good miscibility with PVA. The results of the static mechanical analysis, based on the equilibrium structures of blends with differing component ratios, shows us that the elastic coefficient, engineering modulus, and ductility are increased with the addition of PVA content, which is 4/0 PAM/PVAPVAPVAPVAPVA. Moreover, binding energy results indicate that a stronger interaction exists among PVA molecular chains comparing with PAM molecular chains, which is why the mechanical properties of blend system increasing with the addition of PVA content. Finally, the results of pair correlation functions (PCFs) between polar functional groups and its surrounding hydrogen atoms, indicated they interact with each other mainly by hydrogen bonds, and the strength of three types of polar functional groups has the order of O(-OH)>O(-C=O)>N(-NH 2 ). This further elaborates the root reason why the mechanical properties of blend system increase with the addition of PVA content. Copyright © 2016 Elsevier Ltd. All rights reserved.

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

DEFF Research Database (Denmark)

Erlendsson, Simon; Gotfryd, Kamil; Larsen, Flemming Hofmann

2017-01-01

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been...

A novel cell binding site in the coiled‐coil domain of laminin involved in capillary morphogenesis

DEFF Research Database (Denmark)

Sanz, Laura; García-Bermejo, Laura; Blanco, Francisco J

2003-01-01

Recently, we reported the isolation and characterization of an anti‐laminin antibody that modulates the extracellular matrix‐dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction....

What Happened to the IGF Binding Proteins?

Science.gov (United States)

Bach, Leon A

2018-02-01

Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

Directory of Open Access Journals (Sweden)

Jeffrey M Sanders

Full Text Available The epidermal growth factor receptor (EGFR is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of

Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

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Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

2014-04-01

Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

The YARHG domain: an extracellular domain in search of a function.

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Penny Coggill

Full Text Available We have identified a new bacterial protein domain that we hypothesise binds to peptidoglycan. This domain is called the YARHG domain after the most highly conserved sequence-segment. The domain is found in the extracellular space and is likely to be composed of four alpha-helices. The domain is found associated with protein kinase domains, suggesting it is associated with signalling in some bacteria. The domain is also found associated with three different families of peptidases. The large number of different domains that are found associated with YARHG suggests that it is a useful functional module that nature has recombined multiple times.

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

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Jiang, Bin; Suen, Rachel; Wertheim, Jason A; Ameer, Guillermo A

2016-12-12

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

The role of Cas8 in type I CRISPR interference.

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Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

2015-05-05

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

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Boriana K Tchernookova

Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

Integrin activation dynamics between the RGD-binding site and the headpiece hinge.

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Puklin-Faucher, Eileen; Vogel, Viola

2009-12-25

Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

Science.gov (United States)

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

International Nuclear Information System (INIS)

Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias; Malmberg, Karl-Johan; Troye-Blomberg, Marita; Ahlborg, Niklas; Karlsson, Anna; Johansson, Magnus; Lundberg, Mathias

2009-01-01

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.

Identification of a receptor for extracellular renalase.

Directory of Open Access Journals (Sweden)

Ling Wang

Full Text Available An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI in wild type (WT mice. Therefore, we sought to identity the receptor for extracellular renalase.RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

Validation of the German version of the patient activation measure 13 (PAM13-D) in an international multicentre study of primary care patients

NARCIS (Netherlands)

Brenk-Franz, K.; Hibbard, J.H.; Herrmann, W.J.; Freund, T.; Szecsenyi, J.; Djalali, S.; Steurer-Stey, C.; Sonnichsen, A.; Tiesler, F.; Storch, M.; Schneider, N.; Gensichen, J.

2013-01-01

The patients' active participation in their medical care is important for patients with chronic diseases. Measurements of patient activation are needed for studies and in clinical practice. This study aims to validate the Patient Activation Measure 13 (PAM13-D) in German-speaking primary care

Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function

International Nuclear Information System (INIS)

Marshman, Emma; Streuli, Charles H

2002-01-01

Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development in the mammary gland during puberty and pregnancy. IGFs interact with IGF-binding proteins and regulate their function. The present review focuses on the role of IGFs and IGF-binding proteins in the mammary gland and describes how modulation of their actions occurs by association with hormones, other growth factors and the extracellular matrix. The review will also highlight the involvement of the IGF axis in breast cancer

Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy*

Science.gov (United States)

Zandarashvili, Levani; Sahu, Debashish; Lee, Kwanbok; Lee, Yong Sun; Singh, Pomila; Rajarathnam, Krishna; Iwahara, Junji

2013-01-01

Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized 15N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear 1H-15N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ∼17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules. PMID:23447529

The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

DEFF Research Database (Denmark)

Behrendt, N; Ploug, M; Patthy, L

1991-01-01

with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

KAUST Repository

Suen, KinMan

2013-05-01

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc - induced through interaction with the phosphorylated receptor - releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells. © 2013 Nature America, Inc. All rights reserved.

Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

KAUST Repository

Suen, KinMan; Lin, Chichuan; George, Roger R.; Melo, Fernando A.; Biggs, Eleanor R.; Ahmed, Zamal; Drake, Melanie N.; Arur, Swathi; Arold, Stefan T.; Ladbury, John E S D

2013-01-01

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc - induced through interaction with the phosphorylated receptor - releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells. © 2013 Nature America, Inc. All rights reserved.

Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

International Nuclear Information System (INIS)

Ruffell, Brian; Johnson, Pauline

2005-01-01

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding

Cationic composition and acid-base state of the extracellular fluid, and specific buffer value of hemoglobin from the branchiopod crustacean Triops cancriformis.

Science.gov (United States)

Pirow, Ralph; Buchen, Ina; Richter, Marc; Allmer, Carsten; Nunes, Frank; Günsel, Andreas; Heikens, Wiebke; Lamkemeyer, Tobias; von Reumont, Björn M; Hetz, Stefan K

2009-04-01

Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein's native environment. This study determined the cationic composition and acid-base state of the animal's extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO(2) partial pressure of 1.36 kPa at pH 7.52 and 20 degrees C. The discrepancy between this high CO(2) partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L(-1) pH(-1). Hb covered 40-60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)(-1) pH(-1) suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.

The second extracellular loop of the adenosine A1 receptor mediates activity of allosteric enhancers.

Science.gov (United States)

Kennedy, Dylan P; McRobb, Fiona M; Leonhardt, Susan A; Purdy, Michael; Figler, Heidi; Marshall, Melissa A; Chordia, Mahendra; Figler, Robert; Linden, Joel; Abagyan, Ruben; Yeager, Mark

2014-02-01

Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

Mechanism of melphalan crosslink enhancement by misonidazole pretreatment

International Nuclear Information System (INIS)

Taylor, Y.C.; Sawyer, J.M.; Hsu, B.; Brown, J.M.

1984-01-01

Sensitization of Chinese hamster ovary cells to melphalan (L-PAM) toxicity by prior treatment with misonidazole is associated with increased levels of DNA crosslinks believed to be the critical lesion for bifunctional alkylating agent toxicity. Enhanced L-PAM crosslinking of DNA could occur by a variety of mechanisms in MISO-pretreated cells including: (1) increased transport or binding of L-PAM, (2) decreased repair of L-PAM monoadducts which would allow more time for their conversion to crosslinks, (3) decreased crosslink repair (unhooking of one arm), or (4) chemical modification of the DNA structure, presumably by bound MISO derivatives, such that crosslink formation is facilitated. Previous studies have eliminated mechanisms (1) and (3). Mechanism (4) was investigated by following MISO-pretreatments of whole cells with L-PAM treatments of the isolated DNA from these cells. Treatment of bare DNA with L-PAM modeled very well the crosslinking behavior in whole cells although it was somewhat more efficient. In the presence of double stranded DNA and absence of repair systems during and after the L-PAM exposure, it was determined that MISO-pretreatments did not increase the crosslinking efficiency of L-PAM

Focus on Extracellular Vesicles: Introducing the Next Small Big Thing

Directory of Open Access Journals (Sweden)

Hina Kalra

2016-02-01

Full Text Available Intercellular communication was long thought to be regulated exclusively through direct contact between cells or via release of soluble molecules that transmit the signal by binding to a suitable receptor on the target cell, and/or via uptake into that cell. With the discovery of small secreted vesicular structures that contain complex cargo, both in their lumen and the lipid membrane that surrounds them, a new frontier of signal transduction was discovered. These “extracellular vesicles” (EV were initially thought to be garbage bags through which the cell ejected its waste. Whilst this is a major function of one type of EV, i.e., apoptotic bodies, many EVs have intricate functions in intercellular communication and compound exchange; although their physiological roles are still ill-defined. Additionally, it is now becoming increasingly clear that EVs mediate disease progression and therefore studying EVs has ignited significant interests among researchers from various fields of life sciences. Consequently, the research effort into the pathogenic roles of EVs is significantly higher even though their protective roles are not well established. The “Focus on extracellular vesicles” series of reviews highlights the current state of the art regarding various topics in EV research, whilst this review serves as an introductory overview of EVs, their biogenesis and molecular composition.

Involvement of extracellular matrix constituents in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Lochter, Andre; Bissell, Mina J

1995-06-01

It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.

Science.gov (United States)

López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M

2017-10-13

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

RNA-binding IMPs promote cell adhesion and invadopodia formation

DEFF Research Database (Denmark)

Vikesaa, Jonas; Hansen, Thomas V O; Jønson, Lars

2006-01-01

Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and inva......Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading...... and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular......-mediated invadopodia formation. Taken together, our results indicate that RNA-binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation....