Self-assembly of nanorod motors into geometrically regular multimers and their propulsion by ultrasound.

PubMed

Ahmed, Suzanne; Gentekos, Dillon T; Fink, Craig A; Mallouk, Thomas E

2014-11-25

Segmented gold-ruthenium nanorods (300 ± 30 nm in diameter and 2.0 ± 0.2 μm in length) with thin Ni segments at one end assemble into few-particle, geometrically regular dimers, trimers, and higher multimers while levitated in water by ∼4 MHz ultrasound at the midpoint of a cylindrical acoustic cell. The assembly of the nanorods into multimers is controlled by interactions between the ferromagnetic Ni segments. These assemblies are propelled autonomously in fluids by excitation with ∼4 MHz ultrasound and exhibit several distinct modes of motion. Multimer assembly and disassembly are dynamic in the ultrasonic field. The relative numbers of monomers, dimers, trimers, and higher multimers are dependent upon the number density of particles in the fluid and their speed, which is in turn determined by the ultrasonic power applied. The magnetic binding energy of the multimers estimated from their speed-dependent equilibria is in agreement with the calculated strength of the magnetic dipole interactions. These autonomously propelled multimers can also be steered with an external magnetic field and remain intact after removal from the acoustic chamber for SEM imaging.

Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells.

PubMed

Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M; van der Burg, Sjoerd H; Walter, Steffen; Gouttefangeas, Cécile

2015-01-01

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry.

Cryopreservation of MHC Multimers: Recommendations for Quality Assurance in Detection of Antigen Specific T Cells

PubMed Central

Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M; van der Burg, Sjoerd H; Walter, Steffen; Gouttefangeas, Cécile

2015-01-01

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5–16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry PMID:25297339

Recombinant von Willebrand factor: preclinical development.

PubMed

Plaimauer, B; Schlokat, U; Turecek, P L; Mitterer, A; Mundt, W; Auer, W; Pichler, L; Gritsch, H; Schwarz, H P

2001-08-01

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

NASA Astrophysics Data System (ADS)

Iljina, Marija; Tosatto, Laura; Choi, Minee L.; Sang, Jason C.; Ye, Yu; Hughes, Craig D.; Bryant, Clare E.; Gandhi, Sonia; Klenerman, David

2016-09-01

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT.

PubMed

Shen, Chuanlai; Xu, Tao; Wu, You; Li, Xiaoe; Xia, Lingzhi; Wang, Wei; Shahzad, Khawar Ali; Zhang, Lei; Wan, Xin; Qiu, Jie

2017-11-27

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin 257-264 -specific CD8 + T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen 18-27 - and surface antigen 183-191 -specific CD8 + T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

ATZ11 recognizes not only Z-α1-antitrypsin-polymers and complexed forms of non-Z-α1-antitrypsin but also the von Willebrand factor.

PubMed

Goltz, Diane; Hittetiya, Kanishka; Yadegari, Hamideh; Driesen, Julia; Kirfel, Jutta; Neuhaus, Thomas; Steiner, Susanne; Esch, Christiane; Bedorf, Jörg; Hertfelder, Hans-Jörg; Fischer, Hans-Peter

2014-01-01

The ATZ11 antibody has been well established for the identification of α1-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue. Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens. ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes. ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

PubMed

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

2016-10-01

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide–MHC Class II Multimers

PubMed Central

Holland, Christopher J.; Dolton, Garry; Scurr, Martin; Ladell, Kristin; Schauenburg, Andrea J.; Miners, Kelly; Madura, Florian; Sewell, Andrew K.; Price, David A.

2015-01-01

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions. PMID:26553072

ATZ11 Recognizes Not Only Z-α1-Antitrypsin-Polymers and Complexed Forms of Non-Z-α1-Antitrypsin but Also the von Willebrand Factor

PubMed Central

Yadegari, Hamideh; Driesen, Julia; Kirfel, Jutta; Neuhaus, Thomas; Steiner, Susanne; Esch, Christiane; Bedorf, Jörg; Hertfelder, Hans-Jörg; Fischer, Hans-Peter

2014-01-01

Aims The ATZ11 antibody has been well established for the identification of α1-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue. Methods and Results Ultrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens. ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes. Conclusions ATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes. PMID:24646657

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

PubMed Central

Latif, Rauf; Michalek, Krzysztof; Morshed, Syed Ahmed; Davies, Terry F.

2010-01-01

Background The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction. Methodology/Principal Findings We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation. Conclusions/Significance These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3. PMID:20195479

Molecular dynamics studies of the 3D structure and planar ligand binding of a quadruplex dimer.

PubMed

Li, Ming-Hui; Luo, Quan; Xue, Xiang-Gui; Li, Ze-Sheng

2011-03-01

G-rich sequences can fold into a four-stranded structure called a G-quadruplex, and sequences with short loops are able to aggregate to form stable quadruplex multimers. Few studies have characterized the properties of this variety of quadruplex multimers. Using molecular modeling and molecular dynamics simulations, the present study investigated a dimeric G-quadruplex structure formed from a simple sequence of d(GGGTGGGTGGGTGGGT) (G1), and its interactions with a planar ligand of a perylene derivative (Tel03). A series of analytical methods, including free energy calculations and principal components analysis (PCA), was used. The results show that a dimer structure with stacked parallel monomer structures is maintained well during the entire simulation. Tel03 can bind to the dimer efficiently through end stacking, and the binding mode of the ligand stacked with the 3'-terminal thymine base is most favorable. PCA showed that the dominant motions in the free dimer occur on the loop regions, and the presence of the ligand reduces the flexibility of the loops. Our investigation will assist in understanding the geometric structure of stacked G-quadruplex multimers and may be helpful as a platform for rational drug design.

Purification and characterization of a heteromultimeric glycoprotein from Artocarpus heterophyllus latex with an inhibitory effect on human blood coagulation.

PubMed

Siritapetawee, Jaruwan; Thammasirirak, Sompong

2011-01-01

Plant latex has many health benefits and has been used in folk medicine. In this study, the biological effect of Artocarpus heterophyllus (jackfruit) latex on human blood coagulation was investigated. By a combination of heat precipitation and ion-exchange chromatography, a heat stable heteromultimeric glycoprotein (HSGPL1) was purified from jackfruit milky latex. The apparent molecular masses of the monomeric proteins on SDS/PAGE were 33, 31 and 29 kDa. The isoelectric points (pIs) of the monomers were 6.63, 6.63 and 6.93, respectively. Glycosylation and deglycosylation tests confirmed that each subunit of HSGPL1 formed the native multimer by sugar-based interaction. Moreover, the multimer of HSGPL1 also resisted 2-mercaptoethanol action. Peptide mass fingerprint analysis indicated that HSGPL1 was a complex protein related to Hsps/chaperones. HSGPL1 has an effect on intrinsic pathways of the human blood coagulation system by significantly prolonging the activated partial thrombin time (APTT). In contrast, it has no effect on the human extrinsic blood coagulation system using the prothrombin time (PT) test. The prolonged APTT resulted from the serine protease inhibitor property of HSGPL1, since it reduced activity of human blood coagulation factors XI(a) and α-XII(a).

DYSREGULATION OF MATERNAL SERUM ADIPONECTIN IN PRETERM LABOR

PubMed Central

Mazaki-Tovi, Shali; Romero, Roberto; Vaisbuch, Edi; Erez, Offer; Mittal, Pooja; Chaiworapongsa, Tinnakorn; Kim, Sun Kwon; Pacora, Percy; Yeo, Lami; Gotsch, Francesca; Dong, Zhong; Nhan-Chang, Chia-Ling; Jodicke, Cristiano; Yoon, Bo Hyun; Hassan, Sonia S.; Kusanovic, Juan Pedro

2013-01-01

Objective Intra-amniotic and systemic infection/inflammation have been causally linked to preterm parturition and fetal injury. An emerging theme is that adipose tissue can orchestrate a metabolic response to insults, but also an inflammatory response via the production of adipocytokines, and that these two phenomenon are interrelated. Adiponectin, an insulin-sensitizing, anti-inflammatory adipocytokine, circulates in multimeric complexes including low-molecular-weight (LMW) trimers, medium-molecular-weight (MMW) hexamers and high-molecular-weight (HMW) isoforms. Each of these complexes can exert differential biological effects. The aim of this study was to determine whether spontaneous preterm labor (PTL) with intact membranes and intra-amniotic infection/inflammation (IAI) is associated with changes in maternal serum circulating adiponectin multimers. Study design This cross-sectional study included patients in the following groups: 1) normal pregnant women (n=158); 2) patients with an episode of preterm labor and intact membranes without IAI who delivered at term (n=41); 3) preterm labor without IAI who delivered preterm (n=27); and 4) preterm labor with IAI who delivered preterm (n=36). Serum adiponectin multimers (total, HMW, MMW and LMW) concentrations were determined by ELISA. Non-parametric statistics were used for analyses. Results 1) Preterm labor leading to preterm delivery or an episode of preterm labor which does not lead to preterm delivery, was associated with a lower median maternal serum concentration of total and HMW adiponectin, a lower median HMW/total adiponectin ratio, and a higher median LMW/total adiponectin ratio than normal pregnancy; 2) among patients with preterm labor, those with IAI had the lowest median concentration of total and HMW adiponectin, as well as the lowest median HMW/total adiponectin ratio; 3) The changes in maternal adiponectin and adiponectin multimers remained significant after adjusting for confounding factors such as maternal age, BMI, gestational age at sampling, and parity. Conclusion 1) Preterm labor is characterized by a change in the profile of adiponectin multimers concentrations and their relative isoforms. These changes were observed in patients with an episode of preterm labor not leading to preterm delivery, in patients with intra-amniotic inflammation, or in those without evidence of intra-amniotic inflammation; 2) The changes in adiponectin multimer concentrations reported in preterm labor are different from those previously reported in spontaneous labor at term, suggesting that there is a fundamental difference between preterm labor and labor at term; 3) The findings reported herein, provide the first evidence for the participation of adiponectin multimer in preterm parturition. We propose that adiponectins and adipokines in general provide a mechanism to organize the metabolic demands generated by the process of preterm parturition regardless of the nature of the insult (intra-amniotic inflammation or not). PMID:19579094

Low-loss resonance modes in a gain-assisted plasmonic multimer

NASA Astrophysics Data System (ADS)

Pan, Gui-Ming; Yang, Da-Jie; Zhou, Li; Hao, Zhong-Hua

2018-03-01

We theoretically study the properties of optical losses in a plasmonic multimer and find modes with lower radiative losses due to the cancellation of the dipole moment. High order plasmonic resonances, including electric quadrupole and magnetic dipole resonances, can be achieved by the reduction of symmetry in a multimer. Meanwhile, the dipole moment can be significantly reduced in these high order modes, and consequently, the radiative losses decrease efficiently. The low-loss modes can lead to a lower gain threshold in the gain-assisted nanosystem. In particular, compared with the electric dipolar mode in a single nanoshell, the gain threshold of the electric quadrupolar and magnetic dipolar modes in a multimer can drop by 57.66% and 59.22%, respectively. On the other hand, the gain threshold can reflect the extent of the optical losses of the plasmonic mode in a nanosystem. These findings may have potential applications in the design of a nanolaser, plasmon waveguide and photo-thermal device.

Laboratory diagnosis of von Willebrand's disease.

PubMed

Rick, M E

1994-12-01

The diagnosis of von Willebrand's disease is becoming complex as more is understood about the disease. Clinical information and laboratory data are necessary for the diagnosis because of the overlap of normal and abnormal laboratory values. A complete evaluation including von Willebrand factor multimers, ristocetin-induced platelet aggregation, factor VIII activity level, and a template bleeding time is necessary to correctly classify the patient so that optimal treatment may be given.

Monomer-dimer control of the ColE1 P(cer) promoter.

PubMed

Chatwin, H M; Summers, D K

2001-11-01

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance. P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers. P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

PubMed Central

Soares, R.P.S.; Bydlowski, S.P.; Nascimento, N.M.; Thomaz, A.M.; Bastos, E.N.M.; Lopes, A.A.

2013-01-01

Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF. PMID:23558858

Dogs with heart diseases causing turbulent high-velocity blood flow have changes in platelet function and von Willebrand factor multimer distribution.

PubMed

Tarnow, Inge; Kristensen, Annemarie T; Olsen, Lisbeth H; Falk, Torkel; Haubro, Lotte; Pedersen, Lotte G; Pedersen, Henrik D

2005-01-01

The purpose of this prospective study was to investigate platelet function using in vitro tests based on both high and low shear rates and von Willebrand factor (vWf) multimeric composition in dogs with cardiac disease and turbulent high-velocity blood flow. Client-owned asymptomatic, untreated dogs were divided into 4 groups: 14 Cavalier King Charles Spaniels (Cavaliers) with mitral valve prolapse (MVP) and no or minimal mitral regurgitation (MR), 17 Cavaliers with MVP and moderate to severe MR, 14 control dogs, and 10 dogs with subaortic stenosis (SAS). Clinical examinations and echocardiography were performed in all dogs. PFA100 closure times (the ability of platelets to occlude a hole in a membrane at high shear rates), platelet activation markers (plasma thromboxane B2 concentration, platelet surface P-selectin expression), platelet aggregation (in whole blood and platelet-rich plasma with 3 different agonists), and vWf multimers were analyzed. Cavaliers with moderate to severe MR and dogs with SAS had longer closure times and a lower percentage of the largest vWf multimers than did controls. Maximal aggregation responses were unchanged in dogs with SAS but enhanced in Cavaliers with MVP (regardless of MR status) compared with control dogs. No significant difference in platelet activation markers was found among groups. The data suggest that a form of platelet dysfunction detected at high shear rates was present in dogs with MR and SAS, possibly associated with a qualitative vWf defect. Aggregation results suggest increased platelet reactivity in Cavaliers, but the platelets did not appear to circulate in a preactivated state in either disease.

Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial.

PubMed

Mannucci, Pier Mannuccio; Kempton, Christine; Millar, Carolyn; Romond, Edward; Shapiro, Amy; Birschmann, Ingvild; Ragni, Margaret V; Gill, Joan Cox; Yee, Thynn Thynn; Klamroth, Robert; Wong, Wing-Yen; Chapman, Miranda; Engl, Werner; Turecek, Peter L; Suiter, Tobias M; Ewenstein, Bruce M

2013-08-01

Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF-binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post-rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved.

Complexity in Acid–Base Titrations: Multimer Formation Between Phosphoric Acids and Imines

PubMed Central

Malm, Christian; Kim, Heejae; Wagner, Manfred

2017-01-01

Abstract Solutions of Brønsted acids with bases in aprotic solvents are not only common model systems to study the fundamentals of proton transfer pathways but are also highly relevant to Brønsted acid catalysis. Despite their importance the light nature of the proton makes characterization of acid–base aggregates challenging. Here, we track such acid–base interactions over a broad range of relative compositions between diphenyl phosphoric acid and the base quinaldine in dichloromethane, by using a combination of dielectric relaxation and NMR spectroscopy. In contrast to what one would expect for an acid–base titration, we find strong deviations from quantitative proton transfer from the acid to the base. Even for an excess of the base, multimers consisting of one base and at least two acid molecules are formed, in addition to the occurrence of proton transfer from the acid to the base and simultaneous formation of ion pairs. For equimolar mixtures such multimers constitute about one third of all intermolecular aggregates. Quantitative analysis of our results shows that the acid‐base association constant is only around six times larger than that for the acid binding to an acid‐base dimer, that is, to an already protonated base. Our findings have implications for the interpretation of previous studies of reactive intermediates in organocatalysis and provide a rationale for previously observed nonlinear effects in phosphoric acid catalysis. PMID:28597513

Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood.

PubMed

Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo

2015-05-20

Adoptive transfer of CMV-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in immunocompromised patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or months after G-CSF mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. Despite the tolerogenic effects associated with G-CSF mobilization, recent studies described that CMV-primed T cells generated from mobilized donors remain functional. MHC-multimers are potent tools that allow the rapid production of antigen-specific CTLs. Therefore, in the present study we have assessed the feasibility and efficacy of CMV-specific CTL manufacture from G-CSF mobilized apheresis using MHC-multimers. CMV-specific CTLs can be efficiently isolated from G-CSF mobilized samples with Streptamers and are able to express activation markers and produce cytokines in response to antigenic stimulation. However, this anti-viral functionality is moderately reduced when compared to non-mobilized products. The translation of Streptamer technology for the isolation of anti-viral CTLs from G-CSF mobilized PBMCs into clinical practice would widen the number of patients that could benefit from this therapeutic strategy, although our results need to be taken into consideration before the infusion of antigen-specific T cells obtained from G-CSF mobilized samples.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C

DOE PAGES

Gruschus, James M.; Jiang, Zhiping; Yap, Thai Leong; ...

2015-01-16

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (K d = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63–66 and 74–76, which is distinct from the region known tomore » enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. In conclusion, for the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.« less

Fact or artifact: the representativeness of ESI-MS for complex natural organic mixtures.

PubMed

Novotny, Nicole R; Capley, Erin N; Stenson, Alexandra C

2014-04-01

Because mass spectrometers provide their own dispersion and resolution of analytes, electrospray ionization mass spectrometry (ESI-MS) has become a workhorse for the characterization of complex mixtures from aerosols to crude oil. Unfortunately, ESI mass spectra commonly contain multimers, adducts and fragments. For the characterization of complex mixtures of unknown initial composition, this presents a significant concern. Mixed-multimer formation could potentially lead to results that bare no resemblance to the original mixture. Conversely, ESI-MS has continually reflected subtle differences between natural organic matter mixtures that are in agreement with prediction or theory. Knowing the real limitations of the technique is therefore critical to avoiding both over-interpretation and unwarranted skepticism. Here, data were collected on four mass spectrometers under a battery of conditions. Results indicate that formation of unrepresentative ions cannot entirely be ruled out, but non-covalent multimers do not appear to make a major contribution to typical natural organic matter spectra based on collision-induced dissociation results. Multimers also appear notably reduced when a cooling gas is present in the accumulation region of the mass spectrometer. For less complex mixtures, the choice of spray solvent can make a difference, but generally spectrum cleanliness (i.e. representativeness) comes at the price of increased selectivity. Copyright © 2014 John Wiley & Sons, Ltd.

Complexity in Acid-Base Titrations: Multimer Formation Between Phosphoric Acids and Imines.

PubMed

Malm, Christian; Kim, Heejae; Wagner, Manfred; Hunger, Johannes

2017-08-10

Solutions of Brønsted acids with bases in aprotic solvents are not only common model systems to study the fundamentals of proton transfer pathways but are also highly relevant to Brønsted acid catalysis. Despite their importance the light nature of the proton makes characterization of acid-base aggregates challenging. Here, we track such acid-base interactions over a broad range of relative compositions between diphenyl phosphoric acid and the base quinaldine in dichloromethane, by using a combination of dielectric relaxation and NMR spectroscopy. In contrast to what one would expect for an acid-base titration, we find strong deviations from quantitative proton transfer from the acid to the base. Even for an excess of the base, multimers consisting of one base and at least two acid molecules are formed, in addition to the occurrence of proton transfer from the acid to the base and simultaneous formation of ion pairs. For equimolar mixtures such multimers constitute about one third of all intermolecular aggregates. Quantitative analysis of our results shows that the acid-base association constant is only around six times larger than that for the acid binding to an acid-base dimer, that is, to an already protonated base. Our findings have implications for the interpretation of previous studies of reactive intermediates in organocatalysis and provide a rationale for previously observed nonlinear effects in phosphoric acid catalysis. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Platelet von Willebrand factor in Hermansky-Pudlak syndrome.

PubMed

McKeown, L P; Hansmann, K E; Wilson, O; Gahl, W; Gralnick, H R; Rosenfeld, K E; Rosenfeld, S J; Horne, M K; Rick, M E

1998-10-01

The Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive inherited disorder characterized by oculocutaneous albinism, tissue accumulation of ceroid pigment, and a mild to moderate bleeding diathesis attributed to storage-pool deficient (SPD) platlets. Patients have platelet aggregation and release abnormalities. In addition, low levels of plasma von Willebrand factor (vWF) antigen in some HPS patients have been associated with a greater bleeding tendency than would be predicted from either condition alone. Other HPS patients have severe bleeding despite normal levels of plasma vWF, suggesting that at least one additional factor is responsible for their bleeding diathesis. Because platelet vWF levels have been well correlated with clinical bleeding times in patients with von Willebrand's disease, we have measured the platelet vWF activity and antigen levels in 30 HPS patients and have attempted to correlate their clinical bleeding with these values. The platelet vWF activity levels in patients was significantly lower than that of normal subjects (P < 0.0001). The patients as a group also had slightly lower values of plasma vWF activity when compared with normals (P-0.03). In 11 of the HPS patients, the multimeric structure of plasma vWF showed a decrease in the high molecular weight multimers and an increase in the low molecular weight multimers. In correlating the platelet and plasma vWF values with the bleeding histories, we were not able to show a predictable relationship in the majority of the patients.

Fluorescence fluctuation spectroscopy for clinical applications

NASA Astrophysics Data System (ADS)

Olson, Eben

Fluorescence correlation spectroscopy (FCS) and the related techniques of brightness analysis have become standard tools in biological and biophysical research. By analyzing the statistics of fluorescence emitted from a restricted volume, a number of parameters including concentrations, diffusion coefficients and chemical reaction rates can be determined. The single-molecule sensitivity, spectral selectivity, small sample volume and non-perturbative measurement mechanism of FCS make it an excellent technique for the study of molecular interactions. However, its adoption outside of the research laboratory has been limited. Potential reasons for this include the cost and complexity of the required apparatus. In this work, the application of fluorescence fluctuation analysis to several clinical problems is considered. Optical designs for FCS instruments which reduce the cost and increase alignment tolerance are presented. Brightness analysis of heterogenous systems, with application to the characterization of protein aggregates and multimer distributions, is considered. Methods for FCS-based assays of two clinically relevant proteins, von Willebrand factor and haptoglobin, are presented as well.

Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami.

PubMed

Obser, T; Ledford-Kraemer, M; Oyen, F; Brehm, M A; Denis, C V; Marschalek, R; Montgomery, R R; Sadler, J E; Schneppenheim, S; Budde, U; Schneppenheim, R

2016-09-01

Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo. © 2016 International Society on Thrombosis and Haemostasis.

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

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

Huang, Yongjian; Bharill, Shashank; Karandur, Deepti

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if thismore » is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.« less

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

DOE PAGES

Huang, Yongjian; Bharill, Shashank; Karandur, Deepti; ...

2016-03-28

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if thismore » is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.« less

Adiponectin complexes composition in Japanese-Brazilians regarding their glucose tolerance status.

PubMed

Crispim, Felipe; Vendramini, Marcio F; Moisés, Regina S

2013-04-09

Adiponectin circulates in different multimer complexes comprised of low molecular weight trimeric form (LMW), hexamer of middle molecular weight (MMW) and high molecular weight multimers (HMW). In Japanese-Brazilians, a population with high prevalence of glucose metabolism disturbances, we examined the associations of total adiponectin and its multimers with diabetes mellitus. Two study groups were examined: 26 patients with diabetes mellitus (DM,14 women and 12 men, aged 55.3 ± 8.6 years) and 27 age-matched control subjects with normal glucose tolerance (NGT,12 women and 15 men, aged 54.0 ± 9.2 years). We found no significant differences in total [NGT: 6.90 ug/ml (4.38-13.43); DM: 5.38 ug/ml (3.76-8.56), p = 0.35], MMW [NGT:2.34 ug/ml (1.38-3.25); DM: 1.80 ug/ml (1.18-2.84), p = 0.48] or LMW adiponectin [NGT: 2.07 ug/ml (1.45-3.48), DM: 2.93 ug/ml (1.78-3.99), p = 0.32] between groups. In contrast, HMW adiponectin levels were significantly lower in patients with DM [TGN: 2.39 ug/ml (1.20-4.75); DM: 1.04 ug/ml (0.42-1.60), p = 0.001]. A logistic regression analysis was done to identify independent associations with diabetes mellitus. The results showed that HOMA-IR and HMW adiponectin in women were independently associated with diabetes mellitus. The current investigation demonstrates that in Japanese-Brazilians HMW adiponectin is selectively reduced in individuals with type 2 diabetes, while no differences were found in MMW and LMW adiponectin isoforms.

Adiponectin complexes composition in Japanese-Brazilians regarding their glucose tolerance status

PubMed Central

2013-01-01

Background Adiponectin circulates in different multimer complexes comprised of low molecular weight trimeric form (LMW), hexamer of middle molecular weight (MMW) and high molecular weight multimers (HMW). In Japanese-Brazilians, a population with high prevalence of glucose metabolism disturbances, we examined the associations of total adiponectin and its multimers with diabetes mellitus. Methods Two study groups were examined: 26 patients with diabetes mellitus (DM,14 women and 12 men, aged 55.3 ± 8.6 years) and 27 age-matched control subjects with normal glucose tolerance (NGT,12 women and 15 men, aged 54.0 ± 9.2 years). Results We found no significant differences in total [NGT: 6.90 ug/ml (4.38-13.43); DM: 5.38 ug/ml (3.76-8.56), p = 0.35], MMW [NGT:2.34 ug/ml (1.38-3.25); DM: 1.80 ug/ml (1.18-2.84), p = 0.48] or LMW adiponectin [NGT: 2.07 ug/ml (1.45-3.48), DM: 2.93 ug/ml (1.78-3.99), p = 0.32] between groups. In contrast, HMW adiponectin levels were significantly lower in patients with DM [TGN: 2.39 ug/ml (1.20-4.75); DM: 1.04 ug/ml (0.42-1.60), p = 0.001]. A logistic regression analysis was done to identify independent associations with diabetes mellitus. The results showed that HOMA-IR and HMW adiponectin in women were independently associated with diabetes mellitus. Conclusion The current investigation demonstrates that in Japanese-Brazilians HMW adiponectin is selectively reduced in individuals with type 2 diabetes, while no differences were found in MMW and LMW adiponectin isoforms. PMID:23570346

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction

PubMed Central

Müller, Jochen P.; Mielke, Salomé; Löf, Achim; Obser, Tobias; Beer, Christof; Bruetzel, Linda K.; Pippig, Diana A.; Vanderlinden, Willem; Lipfert, Jan; Schneppenheim, Reinhard; Benoit, Martin

2016-01-01

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF’s hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above ∼50 pN and provided ∼80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF’s effective length contributing to force sensing, they can be expected to tune VWF’s sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF’s function in hemostasis and thrombosis. PMID:26787887

Evidence that the rabbit proton-peptide co-transporter PepT1 is a multimer when expressed in Xenopus laevis oocytes.

PubMed

Panitsas, Konstantinos-E; Boyd, C A R; Meredith, David

2006-04-01

To test whether the rabbit proton-coupled peptide transporter PepT1 is a multimer, we have employed a combination of transport assays, luminometry and site-directed mutagenesis. A functional epitope-tagged PepT1 construct (PepT1-FLAG) was co-expressed in Xenopus laevis oocytes with a non-functional but normally trafficked mutant form of the same transporter (W294F-PepT1). The amount of PepT1-FLAG cRNA injected into the oocytes was kept constant, while the amount of W294F-PepT1 cRNA was increased over the mole fraction range of 0 to 1. The uptake of [(3)H]-D: -Phe-L: -Gln into the oocytes was measured at pH(out) 5.5, and the surface expression of PepT1-FLAG was quantified by luminometry. As the mole fraction of injected W294F-PepT1 increased, the uptake of D: -Phe-L: -Gln decreased. This occurred despite the surface expression of PepT1-FLAG remaining constant, and so we can conclude that PepT1 must be a multimer. Assuming that PepT1 acts as a homomultimer, the best fit for the modelling suggests that PepT1 could be a tetramer, with a minimum requirement of two functional subunits in each protein complex. Western blotting also showed the presence of higher-order complexes of PepT1-FLAG in oocyte membranes. It should be noted that we cannot formally exclude the possibility that PepT1 interacts with unidentified Xenopus protein(s). The finding that PepT1 is a multimer has important implications for the molecular modelling of this protein.

Purification of α-Synuclein from Human Brain Reveals an Instability of Endogenous Multimers as the Protein Approaches Purity

PubMed Central

2015-01-01

Despite two decades of research, the structure–function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson’s disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification. PMID:25490121

Left Ventricular Assist Device Design Reduces von Willebrand Factor Degradation: A Comparative Study Between the HeartMate II and the EVAHEART Left Ventricular Assist System.

PubMed

Bartoli, Carlo R; Kang, Jooeun; Zhang, David; Howard, Jessica; Acker, Michael; Atluri, Pavan; Motomura, Tadashi

2017-04-01

Supraphysiologic shear stress from continuous-flow left ventricular assist devices (LVADs) accelerates von Willebrand factor (vWF) degradation and predisposes patients to nonsurgical bleeding. It is unknown whether unique design characteristics of LVADs differentially affect vWF degradation. We tested the hypothesis that the centrifugal-flow EVAHEART (Evaheart, Houston, TX) left ventricular assist system (LVAS), which was designed to minimize shear stress (low operational revolutions per minute [rpm], larger flow gaps, low shear stress, flat H-Q curve), reduced vWF degradation versus the axial-flow HeartMate II (Thoratec, Pleasanton, CA) LVAD. Whole human blood was obtained from volunteer donors (n = 22). Blood was circulated for 12 hours in mock circulatory loops through a HeartMate II (n = 10; 11,400 rpm, 6.3 ± 0.8 L/min, 76 ± 2 mm Hg) or an EVAHEART LVAS (n = 12; 2,300 rpm, 5.7 ± 0.1 L/min, 80 ± 1 mm Hg). vWF degradation was characterized with electrophoresis and immunoblotting for large vWF multimers and 11 vWF degradation fragments. The HeartMate II eliminated large vWF multimers and significantly (p < 0.05) increased 10 of 11 vWF degradation fragments at 6 and 12 hours. The increase was approximately 2.0-fold at 6 hours and 2.2-fold at 12 hours. In contrast, the EVAHEART LVAS modestly reduced large vWF multimers and significantly increased 5 of 11 and 8 of 11 vWF degradation fragments at 6 and 12 hours, respectively. The increase was approximately 1.5-fold at 6 hours and 1.7-fold at 12 hours. The EVAHEART LVAS caused significantly less degradation (p < 0.01) than the HeartMate II of the 140 kDa vWF fragment (cleavage product of ADAMTS-13, the vWF protease). The EVAHEART LVAS caused significantly less vWF degradation than the HeartMate II in a mock circulatory loop with whole human blood. LVAD design features may minimize vWF degradation. These data may inform the design and operation of next-generation LVADs to minimize blood trauma. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Cellular localization and detergent dependent oligomerization of rice allene oxide synthase-1.

PubMed

Yoeun, Sereyvath; Kim, Jeong-Il; Han, Oksoo

2015-01-01

Allene oxide synthase-1 from Oryza sativa (OsAOS1) localizes to the chloroplast, but lacks a putative chloroplast targeting sequence typically found in dicot AOS. Here, kinetic parameters and the oligomerization state/subunit composition of OsAOS1 were characterized in vitro in the absence or presence of detergent micelles. The catalytic efficiency (k(cat)/K(m)) of OsAOS1 reached a maximum near the critical micelle concentration for polyoxyethylene 10 tridecyl ether. Native gel analysis showed that OsAOS1 exists as a multimer in the absence of detergent micelles. The multimeric form of OsAOS1 was stably cross-linked in the absence of detergents, while only monomeric OsAOS1 was detected in the presence of detergent micelles. Gel filtration analysis indicated that the oligomeric state of OsAOS1 depends strongly on the detergents and that the monomer becomes the predominant form in the presence of detergent micelles. These data suggest that the detergent-dependent oligomeric state of OsAOS1 is an important factor for the regulation of its catalytic efficiency.

CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

PubMed

Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo

2014-11-19

Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.

Imagens do céu ontem e hoje - um multimídia interativo de astronomia e uma nova exposição no MAST

NASA Astrophysics Data System (ADS)

Caretta, C. A.; Lima, F. P.; Requeijo, F.; Vieira, G. G.; Alves, F.; Valente, M. E. A.; de Almeida, R.; de Garcia, G. C.; Quixadá, A. C.

2003-08-01

"Imagens do Céu Ontem e Hoje" é o título de uma nova exposição que está sendo inaugurada no Museu de Astronomia e Ciências Afins (MCT), que inclui experimentos interativos, maquetes, réplicas e 8 terminais de computador com um multimídia interativo sobre Astronomia para consulta dos visitantes. O multimídia apresenta um conteúdo bastante extenso, que engloba quase todos os temas em Astronomia, consistindo numa fonte de divulgação e pesquisa para um público que vai das crianças até estudantes universitários. O conteúdo está distribuído em mais de 500 páginas de texto divididas em 4 módulos: "O Universo", "Espectroscopia", "Telescópios" e "Observando o Céu". Cada módulo é subdividido em 5 seções, em média, cada uma iniciada por uma animação que ilustra os temas a serem abordados na seção. Ao final da animação, uma lista de temas é apresentada sob o título "Saiba Mais". Para exemplificar, o módulo "O Universo" contém as seguintes seções: "O Universo visto pelo homem", "Conhecendo o Sistema Solar", "Indo além do Sistema Solar", "Nossa Galáxia, a Via-Láctea" e "Indo mais além, a imensidão do Universo". A seção "Conhecendo o Sistema Solar", por sua vez, tem os seguintes temas: "A origem do Sistema Solar", "O Sol", "Os planetas", "Satélites, asteróides, cometas e outros bichos..." e "O Sistema Solar em números". Cada texto é repleto de imagens, quadros, desenhos, esquemas, etc, além de passatempos ao final de cada seção, incluindo jogos interativos, quadrinhos e curiosidades, que auxiliam o aprendizado de forma divertida. Apresentamos neste trabalho as idéias gerais que permearam a produção da exposição, e uma viagem pelo multimídia para exemplificar sua estrutura e conteúdo. O multimídia será posteriormente disponibilizado para o público externo pela página eletrônica do MAst e/ou por intermédio de uma publicação comercial.

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair

PubMed Central

Nowarski, Roni; Wilner, Ofer I.; Cheshin, Ori; Shahar, Or D.; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S.; Goldberg, Michal; Willner, Itamar

2012-01-01

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. PMID:22645179

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

PubMed

Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

2012-07-12

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

Homotypic Interaction of Bunyamwera Virus Nucleocapsid Protein

PubMed Central

Leonard, Vincent H. J.; Kohl, Alain; Osborne, Jane C.; McLees, Angela; Elliott, Richard M.

2005-01-01

The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein. PMID:16189017

Expression of dengue-3 premembrane and envelope polyprotein in lettuce chloroplasts

PubMed Central

Kanagaraj, Anderson Paul; Verma, Dheeraj

2012-01-01

Dengue is an acute febrile viral disease with >100 million infections occurring each year and more than half of the world population is at risk. Global resurgence of dengue in many urban centers of the tropics is a major concern. Therefore, development of a successful vaccine is urgently needed that is economical and provide long-lasting protection from dengue virus infections. In this manuscript, we report expression of dengue-3 serotype polyprotein (prM/E) consisting of part of capsid, complete premembrane (prM) and truncated envelope (E) protein in an edible crop lettuce. The dengue sequence was controlled by endogenous Lactuca sativa psbA regulatory elements. PCR and Southern blot analysis confirmed transgene integration into the lettuce chloroplast genome via homologous recombination at the trnI/trnA intergenic spacer region. Western blot analysis showed expression of polyprotein prM/E in different forms as monomers (~65 kDa) or possibly heterodimers (~130 kDa) or multimers. Multimers were solubilized into monomers using guanidine hydrochloride. Transplastomic lettuce plants expressing dengue prM/E vaccine antigens grew normally and transgenes were inherited in the T1 progeny without any segregation. Transmission electron microscopy showed the presence of virus-like particles of ~20 nm diameter in chloroplast extracts of transplastomic lettuce expressing prM/E proteins, but not in untransformed plants. The prM/E antigens expressed in lettuce chloroplasts should offer a potential source for investigating an oral Dengue vaccine. PMID:21431782

Efficient trans-cleavage by the Schistosoma mansoni SMα1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus

PubMed Central

Vazquez-Tello, Alejandro; Castán, Pablo; Moreno, Renata; Smith, James M.; Berenguer, José; Cedergren, Robert

2002-01-01

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMα DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme–product dissociation step. The optimal temperature for trans-cleavage was 70°C. This result was confirmed when both the SMα1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMα1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMα1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo. PMID:11917021

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli.

PubMed

You, Chun; Percival Zhang, Y-H

2012-09-01

A simple and fast protocol for the preparation of a large-size mutant library for directed evolution in Escherichia coli was developed based on the DNA multimers generated by prolonged overlap extension polymerase chain reaction (POE-PCR). This protocol comprised the following: (i) a linear DNA mutant library was generated by error-prone PCR or shuffling, and a linear vector backbone was prepared by regular PCR; (ii) the DNA multimers were generated based on these two DNA templates by POE-PCR; and (iii) the one restriction enzyme-digested DNA multimers were ligated to circular plasmids, followed by transformation to E. coli. Because the ligation efficiency of one DNA fragment was several orders of magnitude higher than that of two DNA fragments for typical mutant library construction, it was very easy to generate a mutant library with a size of more than 10(7) protein mutants per 50 μl of the POE-PCR product. Via this method, four new fluorescent protein mutants were obtained based on monomeric cherry fluorescent protein. This new protocol was simple and fast because it did not require labor-intensive optimizations in restriction enzyme digestion and ligation, did not involve special plasmid design, and enabled constructing a large-size mutant library for directed enzyme evolution within 1 day. Copyright © 2012 Elsevier Inc. All rights reserved.

Adiponectin isoform patterns in ethnic-specific ADIPOQ mutation carriers: The IRAS Family Study

PubMed Central

Tabb, Keri L.; Gao, Chuan; Hicks, Pamela J.; Hawkins, Gregory A.; Rotter, Jerome I.; da Chen, Yii-Der I; Guo, Xiuqing; Norris, Jill M.; Lorenzo, Carlos; Freedman, Barry I.; Bowden, Donald W.; Palmer, Nicholette D.

2017-01-01

Objective Adiponectin is found in human serum in three groups of multimers (high, medium, and low molecular weight). Previously, we reported two ethnic-specific variants in ADIPOQ, G45R (Hispanic Americans) and R55C (African Americans). Although carriers of both variants had mean adiponectin levels ≤20% of those of non-carriers, they were not clinically different from non-carriers. To compare carriers of both variants and non-carriers, relative quantification of adiponectin isoforms to total adiponectin was performed on serum samples. Methods The multimeric patterns of serum adiponectin in G45R carriers (n=23), R55C carriers (n=3), and Hispanic and African American non-carriers (n=84 and 44, respectively) from the IRAS Family Study were explored using native western blotting and densitometry. Results Serum samples from carriers showed an absence of the high molecular weight (HMW) isoform and a marked reduction in the medium molecular weight isoform but an approximate two-fold increase in the amount of the low molecular weight isoform (LMW). Thus, individuals making only LMW adiponectin are metabolically normal. Conclusions The results contrast with the proposed biological importance of the HMW multimer. This suggests that the LMW isoform may functionally compensate for some of the loss/reduction of the higher-order multimers in carriers of the G45R and R55C mutations. PMID:28643464

Adiponectin and markers of metabolic syndrome in obese children and adolescents: impact of 8-mo regular physical exercise program.

PubMed

Nascimento, Henrique; Costa, Elísio; Rocha, Susana; Lucena, Clarice; Rocha-Pereira, Petronila; Rêgo, Carla; Mansilha, Helena Ferreira; Quintanilha, Alexandre; Aires, Luísa; Mota, Jorge; Santos-Silva, Alice; Belo, Luís

2014-08-01

Adiponectin circulates as low-, medium-, and high-molecular-weight multimers (LMW, MMW, and HMW) and influences lipid profile and insulin resistance (IR), HMW being considered as the most biologically active form. We aimed to study the relation between adiponectin and markers of metabolic syndrome (MS) in pediatric obesity, and the impact of physical exercise. The study consisted of a cross-sectional part and an 8-mo physical exercise program. Lipid profile, insulin, glucose, C-reactive protein (CRP), total adiponectin (TA), and homeostasis model assessment IR (HOMA-IR) were measured. Adiponectin multimers were studied in a prepubertal group. Obesity is associated with increased dyslipidemia, IR, and inflammation. TA is correlated inversely with adiposity, triglycerides, HOMA-IR, and CRP, and positively with high-density lipoprotein cholesterol (HDLc)/total cholesterol (TC) ratio. HMW mimicked TA associations. The intervention program led to a reduction of TC, low-density lipoprotein cholesterol (LDLc), insulin, HOMA-IR, and trunk percentage of fat, and an increase of HDLc/TC ratio, in the obese group. BMI improvements prevented adiponectin reduction and correlated with increments in HMW and MMW. Obesity-related increase in MS features might be linked to lower adiponectin. HMW and MMW were the multimers that most explained the MS features. The intervention program improved the lipid profile and IR, and prevented the reduction of adiponectin.

ADAMTS13 Gene Mutations in Children with Hemolytic Uremic Syndrome

PubMed Central

Choi, Hyoung Soo; Cheong, Hae Il; Kim, Nam Keun

2011-01-01

We investigated ADAMTS13 activity as well as the ADAMTS13 gene mutation in children with hemolytic uremic syndrome (HUS). Eighteen patients, including 6 diarrhea-negative (D-HUS) and 12 diarrhea-associated HUS (D+HUS) patients, were evaluated. The extent of von Willebrand factor (VWF) degradation was assayed by multimer analysis, and all exons of the ADAMTS13 gene were PCR-amplified using Taq DNA polymerase. The median and range for plasma activity of ADAMTS13 in 6 D-HUS and 12 D+HUS patients were 71.8% (22.8-94.1%) and 84.9% (37.9-119.9%), respectively, which were not statistically significantly different from the control group (86.4%, 34.2-112.3%) (p>0.05). Five ADAMTS13 gene mutations, including 2 novel mutations [1584+2T>A, 3941C>T (S1314L)] and 3 polymorphisms (Q448E, P475S, S903L), were found in 2 D-HUS and one D+HUS patients, which were not associated with deficiency of ADAMTS13 activity. Whether these mutations without reduced ADAMTS13 activity are innocent bystanders or predisposing factors in HUS remains unanswered. PMID:21488199

Recombinant MHC Tetramers for Isolation of Virus-Specific CD8+ Cells from Healthy Donors: Potential Approach for Cell Therapy of Posttransplant Cytomegalovirus Infection.

PubMed

Vdovin, A S; Filkin, S Y; Yefimova, P R; Sheetikov, S A; Kapranov, N M; Davydova, Y O; Egorov, E S; Khamaganova, E G; Drokov, M Y; Kuzmina, L A; Parovichnikova, E N; Efimov, G A; Savchenko, V G

2016-11-01

Patients undergoing allogeneic hematopoietic stem cell transplantation have a high risk of cytomegalovirus reactivation, which in the absence of T-cell immunity can result in the development of an acute inflammatory reaction and damage of internal organs. Transfusion of the virus-specific donor T-lymphocytes represents an alternative to a highly toxic and often ineffective antiviral therapy. Potentially promising cell therapy approach comprises transfusion of cytotoxic T-lymphocytes, specific to the viral antigens, immediately after their isolation from the donor's blood circulation without any in vitro expansion. Specific T-cells could be separated from potentially alloreactive lymphocytes using recombinant major histocompatibility complex (MHC) multimers, carrying synthetic viral peptides. Rapid transfusion of virus-specific T-cells to patients has several crucial advantages in comparison with methods based on the in vitro expansion of the cells. About 30% of hematopoietic stem cell donors and 46% of transplant recipients at the National Research Center for Hematology were carriers of the HLA-A*02 allele. Moreover, 94% of Russian donors have an immune response against the cytomegalovirus (CMV). Using recombinant HLA-A*02 multimers carrying an immunodominant cytomegalovirus peptide (NLV), we have shown that the majority of healthy donors have pronounced T-cell immunity against this antigen, whereas shortly after the transplantation the patients do not have specific T-lymphocytes. The donor cells have the immune phenotype of memory cells and can be activated and proliferate after stimulation with the specific antigen. Donor lymphocytes can be substantially enriched to significant purity by magnetic separation with recombinant MHC multimers and are not activated upon cocultivation with the antigen-presenting cells from HLA-incompatible donors without addition of the specific antigen. This study demonstrated that strong immune response to CMV of healthy donors and prevalence of HLA-A*02 allele in the Russian population make it possible to isolate a significant number of virus-specific cells using HLA-A*02-NLV multimers. After the transfusion, these cells should protect patients from CMV without development of allogeneic immune response.

A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation.

PubMed

Adam, Frédéric; Casari, Caterina; Prévost, Nicolas; Kauskot, Alexandre; Loubière, Cécile; Legendre, Paulette; Repérant, Christelle; Baruch, Dominique; Rosa, Jean-Philippe; Bryckaert, Marijke; de Groot, Philip G; Christophe, Olivier D; Lenting, Peter J; Denis, Cécile V

2016-05-23

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

Fibronectin alters the rate of formation and structure of the fibrin matrix.

PubMed

Ramanathan, Anand; Karuri, Nancy

2014-01-10

Plasma fibronectin is a vital component of the fibrin clot; however its role on clot structure is not clearly understood. The goal of this study was to examine the influence of fibronectin on the kinetics of formation, structural characteristics and composition of reconstituted fibrin clots or fibrin matrices. Fibrin matrices were formed by adding thrombin to 1, 2 or 4 mg/ml fibrinogen supplemented with 0-0.4 mg/ml fibronectin. The rate of fibrin matrix formation was then monitored by measuring light absorbance properties at different time points. Confocal microscopy of fluorescein conjugated fibrinogen was used to visualize the structural characteristics of fibrin matrices. The amount of fibronectin in fibrin matrices was determined through electrophoresis and immunoblotting of solubilized matrices. Fibronectin concentration positively correlated with the initial rate of fibrin matrix formation and with steady state light absorbance values of fibrin matrices. An increase in fibronectin concentration resulted in thinner and denser fibers in the fibrin matrices. Electrophoresis and immunoblotting showed that fibronectin was covalently and non-covalently bound to fibrin matrices and in the form of high molecular weight multimers. The formation of fibronectin multimers was attributed to cross-linking of fibronectin by trace amounts Factor XIIIa. These findings are novel because they link results from light absorbance studies to microcopy analyses and demonstrate an influence of fibronectin on fibrin matrix structural characteristics. This data is important in developing therapies that destabilize fibrin clots. Copyright © 2014. Published by Elsevier Inc.

Sonic hedgehog multimerization: a self-organizing event driven by post-translational modifications?

PubMed

Koleva, Mirella V; Rothery, Stephen; Spitaler, Martin; Neil, Mark A A; Magee, Anthony I

2015-01-01

Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh's method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane.

Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

PubMed

Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

2002-04-01

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay systems can be used for diagnosis and subtyping of vWD, but CBA is more sensitive than either of the two RCo methods. The analysis of vWF multimers in the different fractions obtained by affinity chromatography on heparin Sepharose showed that the activity measured both with RCo assay and CBA correlated with the degree of multimerization. Our results suggest that measurement of the functional activity of vWF by the RCo procedure can be replaced by the more reliable CBA, reflecting the physiological hemostatic activity of vWF. The CBA method appears not only to be more sensitive and easier to carry out than the RCo method is but also to have a higher reproducibility and allow better standardization.

Analysis of cellular and humoral immune responses against cytomegalovirus in patients with autoimmune Addison's disease.

PubMed

Edvardsen, Kine; Hellesen, Alexander; Husebye, Eystein S; Bratland, Eirik

2016-03-09

Autoimmune Addison's disease (AAD) is caused by multiple genetic and environmental factors. Variants of genes encoding immunologically important proteins such as the HLA molecules are strongly associated with AAD, but any environmental risk factors have yet to be defined. We hypothesized that primary or reactivating infections with cytomegalovirus (CMV) could represent an environmental risk factor in AAD, and that CMV specific CD8(+) T cell responses may be dysregulated, possibly leading to a suboptimal control of CMV. In particular, the objective was to assess the HLA-B8 restricted CD8(+) T cell response to CMV since this HLA class I variant is a genetic risk factor for AAD. To examine the CD8(+) T cell response in detail, we analyzed the HLA-A2 and HLA-B8 restricted responses in AAD patients and healthy controls seropositive for CMV antibodies using HLA multimer technology, IFN-γ ELISpot and a CD107a based degranulation assay. No differences between patients and controls were found in functions or frequencies of CMV-specific T cells, regardless if the analyses were performed ex vivo or after in vitro stimulation and expansion. However, individual patients showed signs of reactivating CMV infection correlating with poor CD8(+) T cell responses to the virus, and a concomitant upregulation of interferon regulated genes in peripheral blood cells. Several recently diagnosed AAD patients also showed serological signs of ongoing primary CMV infection. CMV infection does not appear to be a major environmental risk factor in AAD, but may represent a precipitating factor in individual patients.

Effect of disulfide bonding and multimerization on proteoglycan 4's cartilage boundary lubricating ability and adsorption.

PubMed

Abubacker, Saleem; Ponjevic, Dragana; Ham, Hyun O; Messersmith, Phillip B; Matyas, John R; Schmidt, Tannin A

2016-01-01

The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface. PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi-), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi- was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150, and 450 μg/mL). R/A and NR PRG4Multi- were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants. Separation into enriched populations of PRG4Multi+ and PRG4Multi- was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi- both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL, with PRG4Multi+ being more effective than PRG4Multi-. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi- did not. R/A PRG4Multi- lubricated similar to NR PRG4Multi-. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi- appeared to have less intensity. These results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.

Clinical use of a rapid collagen binding assay for von Willebrand factor cleaving protease in patients with thrombotic thrombocytopenic purpura.

PubMed

Rick, Margaret E; Moll, Stephan; Taylor, Mark A; Krizek, Dennis M; White, Gilbert C; Aronson, David L

2002-10-01

A simple collagen binding assay (CBA) for measuring activity of the von Willebrand factor cleaving protease in clinical samples is described, and results of fifty masked plasmapheresis samples rom patients with TTP/HUS and other diseases are presented. There was 97.5% concordance between the CBA and a multimer gel assay. The CBA identified low protease activity in 78% of patients who had a clinical syndrome consistent with TTP/HUS and in 2 of 10 sick controls, giving it a positive predictive value of 0.94. The heterogeneity regarding the presence or absence of vWF protease activity in patients with TTP/HUS was confirmed by finding a low negative predictive value of 0.50 with the CBA. The CBA detected inhibitors of the protease in 26 of 29 patients (90%) with TTP/HUS and low protease activity levels. The CBA is a useful clinical assay for examining von Willebrand factor protease activity and detecting inhibitors against the protease.

Quantitative T-cell repertoire analysis of peripheral blood mononuclear cells from lung cancer patients following long-term cancer peptide vaccination.

PubMed

Takeda, Kazuyoshi; Kitaura, Kazutaka; Suzuki, Ryuji; Owada, Yuki; Muto, Satoshi; Okabe, Naoyuki; Hasegawa, Takeo; Osugi, Jun; Hoshino, Mika; Tsunoda, Takuya; Okumura, Ko; Suzuki, Hiroyuki

2018-06-01

Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca 2+ influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

PubMed

Rius, Cristina; Attaf, Meriem; Tungatt, Katie; Bianchi, Valentina; Legut, Mateusz; Bovay, Amandine; Donia, Marco; Thor Straten, Per; Peakman, Mark; Svane, Inge Marie; Ott, Sascha; Connor, Tom; Szomolay, Barbara; Dolton, Garry; Sewell, Andrew K

2018-04-01

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations. Copyright © 2018 The Authors.

Pyrrole multimers and pyrrole-acetylene hydrogen bonded complexes studied in N2 and para-H2 matrixes using matrix isolation infrared spectroscopy and ab initio computations

NASA Astrophysics Data System (ADS)

Sarkar, Shubhra; Ramanathan, N.; Gopi, R.; Sundararajan, K.

2017-12-01

Hydrogen bonded interaction of pyrrole multimer and acetylene-pyrrole complexes were studied in N2 and p-H2 matrixes. DFT computations showed T-shaped geometry for the pyrrole dimer and cyclic complex for the trimer and tetramer were the most stable structures, stabilized by Nsbnd H⋯π interactions. The experimental vibrational wavenumbers observed in N2 and p-H2 matrixes for the pyrrole multimers were correlated with the computed wavenumbers. Computations performed at MP2/aug-cc-pVDZ level of theory showed that C2H2 and C4H5N forms 1:1 hydrogen-bonded complexes stabilized by Csbnd H⋯π interaction (Complex A), Nsbnd H⋯π interaction (Complex B) and π⋯π interaction (Complex C), where the former complex is the global minimum and latter two complexes were the first and second local minima, respectively. Experimentally, 1:1 C2H2sbnd C4H5N complexes A (global minimum) and B (first local minimum) were identified from the shifts in the Nsbnd H stretching, Nsbnd H bending, Csbnd H bending region of pyrrole and Csbnd H asymmetric stretching and bending region of C2H2 in N2 and p-H2 matrixes. Computations were also performed for the higher complexes and found two minima corresponding to the 1:2 C2H2sbnd C4H5N and three minima for the 2:1 C2H2sbnd C4H5N complexes. Experimentally the global minimum 1:2 and 2:1 C2H2sbnd C4H5N complexes were identified in N2 and p-H2 matrixes.

Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors

PubMed Central

Maschauer, Simone; Einsiedel, Jürgen; Reich, Dominik; Hübner, Harald; Gmeiner, Peter; Wester, Hans-Jürgen; Prante, Olaf; Notni, Johannes

2017-01-01

Neurotensin receptor 1 (NTS1) is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET). The metabolically-stabilized neurotensin (NT) derivative peptide Nlys8-Lys9-Pro10-Tyr11-Tle12-Leu13-OH was elongated at the N-terminus with 6-azido norleucine and coupled with the 1,4,7-triazacyclononane-1,4,7-tris[(2-carboxyethyl)methylenephosphinic acid] (TRAP) chelator TRAP(alkyne)3 in order to synthesize a NT trimer with subnanomolar affinity and high stability. The 68Ga-labeled peptide [68Ga]Ga-TRAP(NT4)3 was characterized in vitro using the NTS1-expressing human colorectal adenocarcinoma cell line HT29. It displayed fast and high internalization rates of >90%, but also fast efflux rates of 50% over 15 min. In vivo, [68Ga]Ga-TRAP(NT4)3 showed moderate HT29 tumor uptake values of 1.7 %ID/g at 60 min post-injection (p.i.), but also high uptake and retention in the kidneys and liver. A comparison of data for trimer/monomer pairs of NT ligands and other targeting vectors (peptides and peptoids targeting integrins αvβ3, α5β1, and αvβ6, the PSMA-ligand DUPA (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), and nitroimidazoles targeting hypoxia) revealed that multimers always exhibit higher target affinities and tumor uptake, but not necessarily improved tumor-to-tissue ratios. Thus, although in vitro data are not suitable for prediction of in vivo performance, multimers are potentially superior to monomers, particularly for applications where high tumor accumulation is crucial. PMID:28287433

Networking Foundations for Collaborative Computing at Internet Scope

DTIC Science & Technology

2006-01-01

network-supported synchronous multime- dia groupwork at Internet scope and for large user groups. Contributions entail an novel classification for...multimedia resources in interactive groupwork , generalized to the domain of CSCW from the “right to speak” [26]. A floor control protocol mediates access to

LMI Based Robust Blood Glucose Regulation in Type-1 Diabetes Patient with Daily Multi-meal Ingestion

NASA Astrophysics Data System (ADS)

Mandal, S.; Bhattacharjee, A.; Sutradhar, A.

2014-04-01

This paper illustrates the design of a robust output feedback H ∞ controller for the nonlinear glucose-insulin (GI) process in a type-1 diabetes patient to deliver insulin through intravenous infusion device. The H ∞ design specification have been realized using the concept of linear matrix inequality (LMI) and the LMI approach has been used to quadratically stabilize the GI process via output feedback H ∞ controller. The controller has been designed on the basis of full 19th order linearized state-space model generated from the modified Sorensen's nonlinear model of GI process. The resulting controller has been tested with the nonlinear patient model (the modified Sorensen's model) in presence of patient parameter variations and other uncertainty conditions. The performance of the controller was assessed in terms of its ability to track the normoglycemic set point of 81 mg/dl with a typical multi-meal disturbance throughout a day that yields robust performance and noise rejection.

Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.

PubMed

Post, K; Pitschke, M; Schäfer, O; Wille, H; Appel, T R; Kirsch, D; Mehlhorn, I; Serban, H; Prusiner, S B; Riesner, D

1998-11-01

The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.

A Separate Pool of Cardiac Phospholemman That Does Not Regulate or Associate with the Sodium Pump

PubMed Central

Wypijewski, Krzysztof J.; Howie, Jacqueline; Reilly, Louise; Tulloch, Lindsay B.; Aughton, Karen L.; McLatchie, Linda M.; Shattock, Michael J.; Calaghan, Sarah C.; Fuller, William

2013-01-01

Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser63 and one associated with the pump, both phosphorylated at Ser68 and unphosphorylated. Phosphorylation of PLM at Ser63 following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser63-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser63-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump. PMID:23532852

Hemorrhoids screening and treatment prior to LVAD: is it a necessity?

PubMed

Skouri, Hadi; Shurrab, Mohammed; Zahnan, Jad; Deeba, Samer; Sfeir, Pierre; Gharzuddin, Walid; Haj-Yahia, Saleem

2016-04-12

Continuous-flow left ventricle assist devices (CF-LVADs) has become an essential modality in the management of stage D heart failure (HF) with significant improvement in survival and quality of life. Due to the durability of such devices and long term support complications such as bleeding and aortic insufficiency has emerged. Bleeding accounts for more than 20 % with the majority being from the gastrointestinal tract. The increase of bleeding tendency are mainly attributed to the loss of large von Willebrand's Factor (vWF) multimers due to shear stress with the chronic intake of anticoagulants. We are reporting two cases of patients with Stage D HF and history of hemorrhoids presenting for LVAD implantation. Many efforts that decrease bleeding related to CF-LVADs will be discussed with focus on hemorrhoids.

Developing Modular and Adaptable Courseware Using TeachML.

ERIC Educational Resources Information Center

Wehner, Frank; Lorz, Alexander

This paper presents the use of an XML grammar for two complementary projects--CHAMELEON (Cooperative Hypermedia Adaptive MultimEdia Learning Objects) and EIT (Enabling Informal Teamwork). Areas of applications are modular courseware documents and the collaborative authoring process of didactical units. A number of requirements for a suitable…

Interfacing with a DMM.

ERIC Educational Resources Information Center

Beatty, Jim

1985-01-01

Suggests purchasing a digital multimer (DMM) with an IEEE-488 option to interface an instrument to a microcomputer, indicating that a DMM is well protected from overloads and is easy to connect. An example of its use in an experiment involving hydrolysis of tertiary butyl alcohol (with program listing) is given. (JN)

Model input and output files for the simulation of time of arrival of landfill leachate at the water table, Municipal Solid Waste Landfill Facility, U.S. Army Air Defense Artillery Center and Fort Bliss, El Paso County, Texas

USGS Publications Warehouse

Abeyta, Cynthia G.; Frenzel, Peter F.

1999-01-01

This report contains listings of model input and output files for the simulation of the time of arrival of landfill leachate at the water table from the Municipal Solid Waste Landfill Facility (MSWLF), about 10 miles northeast of downtown El Paso, Texas. This simulation was done by the U.S. Geological Survey in cooperation with the U.S. Department of the Army, U.S. Army Air Defense Artillery Center and Fort Bliss, El Paso, Texas. The U.S. Environmental Protection Agency-developed Hydrologic Evaluation of Landfill Performance (HELP) and Multimedia Exposure Assessment (MULTIMED) computer models were used to simulate the production of leachate by a landfill and transport of landfill leachate to the water table. Model input data files used with and output files generated by the HELP and MULTIMED models are provided in ASCII format on a 3.5-inch 1.44-megabyte IBM-PC compatible floppy disk.

Dynamic oligomeric conversions of the cytoplasmic RCK domains mediate MthK potassium channel activity.

PubMed

Kuo, Mario Meng-Chiang; Baker, Kent A; Wong, Lee; Choe, Senyon

2007-02-13

The crystal structure of the RCK-containing MthK provides a molecular framework for understanding the ligand gating mechanisms of K+ channels. Here we examined the macroscopic currents of MthK in enlarged Escherichia coli membrane by patch clamp and rapid perfusion techniques and showed that the channel undergoes desensitization in seconds after activation by Ca2+ or Cd2+. Additionally, MthK is inactivated by slightly acidic pH only from the cytoplasmic side. Examinations of isolated RCK domain by size-exclusion chromatography, static light scattering, analytical sedimentation, and stopped-flow spectroscopy show that Ca2+ rapidly converts isolated RCK monomers to multimers at alkaline pH. In contrast, the RCK domain at acidic pH remains firmly dimeric regardless of Ca2+ but restores predominantly to multimer or monomer at basic pH with or without Ca2+, respectively. These functional and biochemical analyses correlate the four functional states of the MthK channel with distinct oligomeric states of its RCK domains and indicate that the RCK domains undergo oligomeric conversions in modulating MthK activities.

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

PubMed

Horn, T; Chang, C A; Urdea, M S

1997-12-01

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology.

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

PubMed Central

Horn, T; Chang, C A; Urdea, M S

1997-01-01

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology. PMID:9365266

Histone H1 functions as a stimulatory factor in backup pathways of NHEJ

PubMed Central

Rosidi, Bustanur; Wang, Minli; Wu, Wenqi; Sharma, Aparna; Wang, Huichen; Iliakis, George

2008-01-01

DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ. PMID:18250087

Overview of a HLA-Ig based "Lego-like system" for T cell monitoring, modulation and expansion.

PubMed

Oelke, Mathias; Schneck, Jonathan P

2010-07-01

Recent advances in molecular medicine have shown that soluble MHC-multimers can be valuable tools for both analysis and modulation of antigen-specific immune responses in vitro and in vivo. In this review, we describe the use of dimeric human and mouse major histocompatibility complexes, MHC-Ig, as part of an artificial Antigen-Presenting Cell (aAPC). MHC-Ig-based aAPC and its derivatives represent an exciting new platform technology for measuring and manipulating immune responses in vitro as well as in vivo. This new technology has the potential to help overcome many of the obstacles associated with limitations in current antigen-specific approaches of immunotherapy for the treatment of cancer, infectious diseases and autoimmunity.

Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains.

PubMed

Bot, Simonetta; Andreuzzi, Eva; Capuano, Alessandra; Schiavinato, Alvise; Colombatti, Alfonso; Doliana, Roberto

2015-01-01

EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues. Copyright © 2014. Published by Elsevier B.V.

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation*

PubMed Central

Rishavy, Mark A.; Hallgren, Kevin W.; Wilson, Lee A.; Usubalieva, Aisulu; Runge, Kurt W.; Berkner, Kathleen L.

2013-01-01

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously. PMID:23918929

Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

PubMed

van Veen, Harrie A; Koiter, Jaco; Vogelezang, Carla J M; van Wessel, Noucha; van Dam, Tijtje; Velterop, Ingeborg; van Houdt, Kristina; Kupers, Luc; Horbach, Danielle; Salaheddine, Mourad; Nuijens, Jan H; Mannesse, Maurice L M

2012-12-31

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE. Copyright © 2012 Elsevier B.V. All rights reserved.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates*

PubMed Central

Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George; Weigand, Steven; Dunbrack, Roland L.; Skalka, Anna Marie

2013-01-01

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition. PMID:23322775

The ability of multimerized cyclophilin A to restrict retrovirus infection

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

Javanbakht, Hassan; Diaz-Griffero, Felipe; Yuan Wen

2007-10-10

In owl monkeys, the typical retroviral restriction factor of primates, TRIM5{alpha}, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 andmore » Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection.« less

Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo.

PubMed

Senis, Y A; Richardson, M; Tinlin, S; Maurice, D H; Giles, A R

1996-04-01

The pattern of distribution of von Willebrand factor (VWF) in relatively large sheets of rat aortic endothelial cells (EC) obtained by the Häutchen technique were analysed by immunocytochemistry and light microscopy. EC were examined pre and post administration of a procoagulant mixture of factor Xa (F.Xa) and phosphotidylcholine/phosphotidylserine (PCPS) vesicles which was demonstrated to result in the selective loss of high molecular weight multimers (HMWM) of plasma VWF in the rat. In placebo animals the pattern was heterogenous both in overall distribution and in individual cells which showed both a diffuse and granular pattern. Groups of intensely stained EC were oriented parallel to the longitudinal axis of the aorta and staining was particularly prominent around the orifices of the intercostal arteries, implicating shear-stress as a possible factor in VWF expression by EC. Changes in the pattern of distribution of staining were observed at various time points post-infusion of F.Xa/PCPS, suggesting the immediate release of VWF from EC stores followed by the recruitment of EC to synthesize and store VWF. These changes are consistent with the decrease in EC Weibel-Palade Body (WPB) content observed by EM in previously reported studies using this model.

Using mass spectrometry and small molecule reagents to detect distinctive structural features of different prion conformations (strains)

USDA-ARS?s Scientific Manuscript database

A prion (PrPSc) is a conformer of a normal cellular prion protein (PrPC). Although they are isosequential, PrPSc is an infectious protein able to convert PrPC into the prion conformation and thereby propagate an infection. PrPC is monomeric while PrPSc is a multimer. PrPSc can adopt more than one co...

Recombinant PrPSc shares structural features with brain-derived PrPSc suggesting that they have a similar architecture: Insights from limited proteolysis

USDA-ARS?s Scientific Manuscript database

An extensive body of experimental and spectroscopic evidence supports the hypothesis that PrPSc is a multimer of 4-rung ß-solenoids, and that individual PrPSc solenoids stack to form amyloid fibers. We recently used limited proteolysis to map the ß-strands and connecting loops that make up the PrPSc...

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

PubMed Central

Goodin, Jeremy L.; Nellis, David F.; Powell, Bradford S.; Vyas, Vinay V.; Enama, Jeffrey T.; Wang, Lena C.; Clark, Patrick K.; Giardina, Steven L.; Adamovicz, Jeffery. J.; Michiel, Dennis F.

2009-01-01

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-VC424S proteins were over-expressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2 mg per gram of cell paste of 95% pure, mono-disperse protein having ≤ 0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-VC424S were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible Alhydrogel® adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-VC424S monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 × 20 µg of F1-V, respectively, 100, 80, 80, and 70% of injected mice survived a subcutaneous lethal plague challenge with 108 LD50 Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection. PMID:17293124

Optical properties, excitation energy and primary charge transfer in photosystem II: theory meets experiment.

PubMed

Renger, Thomas; Schlodder, Eberhard

2011-01-01

In this review we discuss structure-function relationships of the core complex of photosystem II, as uncovered from analysis of optical spectra of the complex and its subunits. Based on descriptions of optical difference spectra including site directed mutagenesis we propose a revision of the multimer model of the symmetrically arranged reaction center pigments, described by an asymmetric exciton Hamiltonian. Evidence is provided for the location of the triplet state, the identity of the primary electron donor, the localization of the cation and the secondary electron transfer pathway in the reaction center. We also discuss the stationary and time-dependent optical properties of the CP43 and CP47 subunits and the excitation energy transfer and trapping-by-charge-transfer kinetics in the core complex. Copyright © 2011 Elsevier B.V. All rights reserved.

Circular trimers of gelatinase B/matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1

PubMed Central

Vandooren, Jennifer; Born, Benjamin; Solomonov, Inna; Zajac, Ewa; Saldova, Radka; Senske, Michael; Ugarte-Berzal, Estefanía; Martens, Erik; Van den Steen, Philippe E.; Van Damme, Jo; Garcia-Pardo, Angeles; Froeyen, Matheus; Deryugina, Elena I.; Quigley, James P.; Moestrup, Søren K.; Rudd, Pauline M.; Sagi, Irit; Opdenakker, Ghislain

2015-01-01

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers, and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical, and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast to a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, atomic force microscopy (AFM) and transmission electron microscopy (TEM), we generated a 3Dstructure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers versus monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1. PMID:25360794

Method for determining the composition and orientation of III-V {001} semiconductor surfaces

NASA Astrophysics Data System (ADS)

Sung, M. M.; Kim, C.; Rabalais, J. W.

1996-09-01

A method for determining the composition and orientation of III-V {001} semiconductor surfaces is presented and applications are described. The information is obtained from the techniques of time-of-flight scattering and recoiling spectrometry (TOF-SARS), using the composition from azimuth-specific elemental accessibilities (CASEA) method, and low energy electron diffraction (LEED). The azimuth-specific elemental accessibilities (ASEA) are measured experimentally and calculated from the number of accessible atoms in the unit cell and from three-dimensional trajectory simulations using the SARIC program. The in situ analyses identify the 1st-layer elemental species and determine the orientation of the reconstructed surface symmetry elements with respect to the bulk crystallographic directions. This is demonstrated for the III-V {001} compound semiconductor surfaces of GaAs and InAs in the (4 × 2) and (4 × 2) phases and InP in the (4 × 2) phase. The analyses confirm the missing-row-dimer (MRD) structure for GaAs and InAs in which the missing row direction is parallel to the direction of the 1st-layer multimers (dimers) and the missing-row-trimer-dimer (MRTD) structure for InP in which the missing row direction is perpendicular to the direction of the 1st-layer multimers (trimers).

Conformational and Thermal Stability Improvements for the Large-Scale Production of Yeast-Derived Rabbit Hemorrhagic Disease Virus-Like Particles as Multipurpose Vaccine

PubMed Central

Méndez, Lídice; González, Nemecio; Parra, Francisco; Martín-Alonso, José M.; Limonta, Miladys; Sánchez, Kosara; Cabrales, Ania; Estrada, Mario P.; Rodríguez-Mallón, Alina; Farnós, Omar

2013-01-01

Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans. PMID:23460801

Discontinuation of antithrombotic therapy for a year or more in patients with continuous-flow left ventricular assist devices.

PubMed

Pereira, Naveen L; Chen, Dong; Kushwaha, Sudhir S; Park, Soon J

2010-10-01

The recommended anticoagulation regimen during continuous-flow axial left ventricular assist device (LVAD) support is aspirin and warfarin with a targeted international normalized ratio of 2.0-3.0. We report two patients in whom recurrent gastrointestinal bleeding during LVAD support necessitated discontinuation of this anti-thrombotic regimen for a year or more. Despite this, neither patients developed thrombotic complications during 29 patient-months of follow-up. An acquired von Willebrand factor (VWF) abnormality reflected by the absence or decreased abundance of the highest molecular weight multimers was demonstrated in both patients. The gold standard test for platelet function, light transmission platelet aggregometry was measured in one patient and was normal, indicative that the predominant abnormality in the coagulation profile of these patients is an acquired VWF syndrome. Clinical trials are required to address the question whether it is safe to discontinue anticoagulation in LVAD patients with acquired VWF abnormalities.

Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer.

PubMed

Bernhard, Helga; Neudorfer, Julia; Gebhard, Kerstin; Conrad, Heinke; Hermann, Christine; Nährig, Jörg; Fend, Falko; Weber, Wolfgang; Busch, Dirk H; Peschel, Christian

2008-02-01

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

PubMed

Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

2009-02-01

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds

PubMed Central

Ballet, Steven; Marczak, Ewa D.; Feytens, Debby; Salvadori, Severo; Sasaki, Yusuke; Abell, Andrew D.; Lazarus, Lawrence H.; Balboni, Gianfranco; Tourwé, Dirk

2010-01-01

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced δ/μ antagonists, as determined by the functional [S35]GTPγS binding assay, the dimerization of potent Aia-based ‘parent’ ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds. PMID:20137938

SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

PubMed

Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

2015-03-01

To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

A soluble RecN homologue provides means for biochemical and genetic analysis of DNA double-strand break repair in Escherichia coli.

PubMed

Grove, Jane I; Wood, Stuart R; Briggs, Geoffrey S; Oldham, Neil J; Lloyd, Robert G

2009-12-03

RecN is a highly conserved, SMC-like protein in bacteria. It plays an important role in the repair of DNA double-strand breaks and is therefore a key factor in maintaining genome integrity. The insolubility of Escherichia coli RecN has limited efforts to unravel its function. We overcame this limitation by replacing the resident coding sequence with that of Haemophilus influenzae RecN. The heterologous construct expresses Haemophilus RecN from the SOS-inducible E. coli promoter. The hybrid gene is fully functional, promoting survival after I-SceI induced DNA breakage, gamma irradiation or exposure to mitomycin C as effectively as the native gene, indicating that the repair activity is conserved between these two species. H. influenzae RecN is quite soluble, even when expressed at high levels, and is readily purified. Its analysis by ionisation-mass spectrometry, gel filtration and glutaraldehyde crosslinking indicates that it is probably a dimer under physiological conditions, although a higher multimer cannot be excluded. The purified protein displays a weak ATPase activity that is essential for its DNA repair function in vivo. However, no DNA-binding activity was detected, which contrasts with RecN from Bacillus subtilis. RecN proteins from Aquifex aeolicus and Bacteriodes fragilis also proved soluble. Neither binds DNA, but the Aquifex RecN has weak ATPase activity. Our findings support studies indicating that RecN, and the SOS response in general, behave differently in E. coli and B. subtilis. The hybrid recN reported provides new opportunities to study the genetics and biochemistry of how RecN operates in E. coli.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network*

PubMed Central

Miszta, Adam; Pelkmans, Leonie; Lindhout, Theo; Krishnamoorthy, Ganeshram; de Groot, Philip G.; Hemker, Coenraad H.; Heemskerk, Johan W. M.; Kelchtermans, Hilde; de Laat, Bas

2014-01-01

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. PMID:25381443

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

PubMed Central

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-01-01

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. PMID:27335124

A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis

PubMed Central

Noor, Nudrat; Bitoun, Emmanuelle; Tumian, Afidalina; Imbeault, Michael; Chapman, J Ross; Aricescu, A Radu

2017-01-01

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. PMID:29072575

Life-threatening subdural hematoma after aortic valve replacement in a patient with Heyde syndrome: a case report.

PubMed

Uchida, Tetsuro; Hamasaki, Azumi; Ohba, Eiichi; Yamashita, Atsushi; Hayashi, Jun; Sadahiro, Mitsuaki

2017-08-08

Heyde syndrome is known as a triad of calcific aortic stenosis, anemia due to gastrointestinal bleeding from angiodysplasia, and acquired type 2A von Willebrand disease. This acquired hemorrhagic disorder is characterized by the loss of the large von Willebrand factor multimers due to the shear stress across the diseased aortic valve. The most frequently observed type of bleeding in these patients is mucosal or skin bleeding, such as epistaxis, followed by gastrointestinal bleeding. On the other hand, intracranial hemorrhage complicating Heyde syndrome is extremely rare. A 77-year-old woman presented to our hospital with severe aortic stenosis and severe anemia due to gastrointestinal bleeding and was diagnosed with Heyde syndrome. Although aortic valve replacement was performed without recurrent gastrointestinal bleeding, postoperative life-threatening acute subdural hematoma occurred with a marked midline shift. Despite prompt surgical evacuation of the hematoma, she did not recover consciousness and she died 1 month after the operation. Postoperative subdural hematoma is rare, but it should be kept in mind as a devastating hemorrhagic complication, especially in patients with Heyde syndrome.

ADAMTS13 activity as a novel risk factor for incident type 2 diabetes mellitus: a population-based cohort study.

PubMed

de Vries, Paul S; van Herpt, Thijs T W; Ligthart, Symen; Hofman, Albert; Ikram, M Arfan; van Hoek, Mandy; Sijbrands, Eric J G; Franco, Oscar H; de Maat, Moniek P M; Leebeek, Frank W G; Dehghan, Abbas

2017-02-01

ADAMTS13 is a protease that breaks down von Willebrand factor (VWF) multimers into smaller, less active particles. VWF has been associated with an increased risk of incident type 2 diabetes mellitus. Here, we determine whether ADAMTS13 activity and VWF antigen are associated with incident diabetes. This study included 5176 participants from the Rotterdam Study, a prospective population-based cohort study. Participants were free of diabetes at baseline and followed up for more than 20 years. Cox proportional hazards models were used to examine the association of ADAMTS13 activity and VWF antigen with incident diabetes. ADAMTS13 activity was associated with an increased risk of incident diabetes (HR 1.17 [95% CI 1.08, 1.27]) after adjustment for known risk factors and VWF antigen levels. Although ADAMTS13 activity was positively associated with fasting glucose and insulin, the association with incident diabetes did not change when we adjusted for these covariates. ADAMTS13 activity was also associated with incident prediabetes (defined on the basis of both fasting and non-fasting blood glucose) after adjustment for known risk factors (HR 1.11 [95% CI 1.03, 1.19]), while the VWF antigen level was not. VWF antigen was associated with incident diabetes, but this association was attenuated after adjustment for known risk factors. ADAMTS13 activity appears to be an independent risk factor for incident prediabetes and type 2 diabetes. As the association between ADAMTS13 and diabetes did not appear to be explained by its cleavage of VWF, ADAMTS13 may have an independent role in the development of diabetes.

Rhabdomyosarcome paratesticulaire (RMSP) multimétastatique : à propos d’un cas

PubMed Central

Bennani, Hassan; Ziouziou, Imad; El Ghanmi, Jihad; Karmouni, Tarik; El Khader, Khalid; Koutani, Abdellatif; Andaloussi, Ahmed Iben Attya

2014-01-01

Résumé Nous rapportons, dans le présent article, le cas clinique d’un RMSP découvert à un stade tardif chez un adolescent, dans le but de confirmer l’évolution fatale de cette pathologie au potentiel métastatique « affreux ». Nous discutons aussi les causes du retard diagnostique, l’implication du sous-type histologique comme facteur pronostique et la place de la chimiothérapie dans le traitement des formes évoluées de la maladie. PMID:25295143

Pulse EPR distance measurements to study multimers and multimerisation

NASA Astrophysics Data System (ADS)

Ackermann, Katrin; Bode, Bela E.

2018-06-01

Pulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.

The ClusPro web server for protein-protein docking

PubMed Central

Kozakov, Dima; Hall, David R.; Xia, Bing; Porter, Kathryn A.; Padhorny, Dzmitry; Yueh, Christine; Beglov, Dmitri; Vajda, Sandor

2017-01-01

The ClusPro server (https://cluspro.org) is a widely used tool for protein-protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank format. However, ClusPro also offers a number of advanced options to modify the search that include the removal of unstructured protein regions, applying attraction or repulsion, accounting for pairwise distance restraints, constructing homo-multimers, considering small angle X-ray scattering (SAXS) data, and finding heparin binding sites. Six different energy functions can be used depending on the type of proteins. Docking with each energy parameter set results in ten models defined by centers of highly populated clusters of low energy docked structures. This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results. Although the server is heavily used, runs are generally completed in < 4 hours. PMID:28079879

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

PubMed Central

Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

2016-01-01

Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

Novel role for glutathione S-transferase pi. Regulator of protein S-Glutathionylation following oxidative and nitrosative stress.

PubMed

Townsend, Danyelle M; Manevich, Yefim; He, Lin; Hutchens, Steven; Pazoles, Christopher J; Tew, Kenneth D

2009-01-02

Glutathione S-transferase Pi (GSTpi) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTpi potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTpi is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTpi, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTpi multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTpi and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs.

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry.

PubMed

Borrenberghs, Doortje; Dirix, Lieve; De Wit, Flore; Rocha, Susana; Blokken, Jolien; De Houwer, Stéphanie; Gijsbers, Rik; Christ, Frauke; Hofkens, Johan; Hendrix, Jelle; Debyser, Zeger

2016-11-10

Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.

Characterization of three types of human alpha s1-casein mRNA transcripts.

PubMed Central

Johnsen, L B; Rasmussen, L K; Petersen, T E; Berglund, L

1995-01-01

Here we report the molecular cloning and sequencing of three types of human alpha s1-casein transcripts and present evidence indicating that exon skipping is responsible for deleted mRNA transcripts. The largest transcript comprised 981 bp encoding a signal peptide of 15 amino acids followed by the mature alpha s1-casein sequence of 170 amino acids. Human alpha s1-casein has been reported to exist naturally as a multimer in complex with kappa-casein in mature human milk, thereby being unique among alpha s1-caseins [Rasmussen, Due and Petersen (1995) Comp. Biochem. Physiol., in the press]. The present demonstration of three cysteines in the mature protein provides a molecular explanation of the interactions in this complex. Tissue-specific expression of human alpha s1-casein was indicated by Northern-blot analysis. In addition, two cryptic exons were localized in the bovine alpha s1-casein gene. Images Figure 3 PMID:7619062

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches

PubMed Central

Hyeon, Jae Wook; Choi, Jiwon; Kim, Su Yeon; Govindaraj, Rajiv Gandhi; Jam Hwang, Kyu; Lee, Yeong Seon; An, Seong Soo A.; Lee, Myung Koo; Joung, Jong Young; No, Kyoung Tai; Lee, Jeongmin

2015-01-01

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrPC) to the pathogenic form, PrPSc. Compounds that inhibit this process by blocking conversion to the PrPSc could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrPC interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrPSc levels and one of these compounds also showed a high binding affinity for PrPC. These results provide a promising starting point for the development of anti-prion compounds. PMID:26449325

Inhibition of ADAMTS-13 by Doxycycline Reduces von Willebrand Factor Degradation During Supraphysiological Shear Stress: Therapeutic Implications for Left Ventricular Assist Device-Associated Bleeding.

PubMed

Bartoli, Carlo R; Kang, Jooeun; Restle, David J; Zhang, David M; Shabahang, Cameron; Acker, Michael A; Atluri, Pavan

2015-11-01

The aim of this study was to investigate a potential therapy for left ventricular assist device (LVAD)-associated bleeding. Nonsurgical bleeding is the most frequent complication of LVAD support. Recent evidence has demonstrated that supraphysiological shear stress from continuous-flow LVADs accelerates von Willebrand factor (vWF) metabolism by the action of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) (the vWF protease). An acquired vWF deficiency causes bleeding. This suggests that ADAMTS-13 is a clinical target to reduce vWF degradation. We tested the hypothesis that inhibition of ADAMTS-13 with doxycycline, an inexpensive, clinically approved drug, reduces vWF degradation during shear stress. Whole blood was collected from human donors (n = 15), and purified, recombinant ADAMTS-13 protein was obtained. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the dose relationship between doxycycline and ADAMTS-13 activity prior to shear stress (n = 10). To determine the effect of shear stress, plasma and recombinant ADAMTS-13 were exposed to LVAD-like supraphysiological shear stress (approximately 175 dyne/cm(2)). vWF multimers and degradation fragments were characterized with electrophoresis and immunoblotting (n = 10). Förster resonance energy transfer was used to quantify plasma ADAMTS-13 activity (n = 10). An ELISA was used to quantify vWF:collagen binding activity. Platelet aggregometry was performed with adenosine 5'-diphosphate, collagen, and ristocetin (vWF-platelet pathway) agonism (n = 10). Doxycycline significantly decreased plasma ADAMTS-13 activity (p = 0.01) and the activity of recombinant human ADAMTS-13 protein by 21%. After plasma was exposed to shear stress, the same pattern of vWF degradation was observed as previously reported for LVAD patients, and vWF:collagen binding activity decreased significantly (p = 0.002). Doxycycline significantly decreased ADAMTS-13 activity (p = 0.04) and the activity of recombinant ADAMTS-13 by 18%, protected large vWF multimers from degradation, and significantly decreased the levels of the 5 smallest vWF fragments by 12 ± 2% (p < 0.05). As a result, vWF:collagen binding activity was significantly restored (p = 0.004). ADAMTS-13 inhibition with doxycycline did not hyperactivate platelets. Inhibition of ADAMTS-13 by doxycycline decreased vWF degradation and improved vWF function during supraphysiological shear stress without hyperactivating platelets. ADAMTS-13 is a clinical target to reduce vWF degradation, improve vWF function, and potentially reduce bleeding during LVAD support. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra.

PubMed

Chu, Teng-Ping J; Yuann, Jeu-Ming P

2011-05-01

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% β-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin☆

PubMed Central

Faraj, Santiago E.; Venturutti, Leandro; Roman, Ernesto A.; Marino-Buslje, Cristina B.; Mignone, Astor; Tosatto, Silvio C.E.; Delfino, José M.; Santos, Javier

2013-01-01

The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42–80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56–210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81–210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56–81, the moiety putatively responsible for promoting protein–protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or β-strand structure. In this context, we explored the conformation and stability of hFXN56–210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90–210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81–210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function. PMID:23951553

Autoinducer 2 activity in Escherichia coli culture supernatants can be actively reduced despite maintenance of an active synthase, LuxS.

PubMed

Hardie, Kim R; Cooksley, Clare; Green, Andrew D; Winzer, Klaus

2003-03-01

Production of the signalling molecule (autoinducer-2) synthesized by LuxS has been proposed to be pivotal to a universal mechanism of inter-species bacterial cell-cell communication (quorum sensing); however recently the function of LuxS has been noted to be integral to central metabolism since it contributes to the activated methyl cycle. This paper shows that when Helicobacter pylori LuxS is overproduced in Escherichia coli, it forms cross-linkable multimers. These multimers persist at comparable levels after 24 h of growth if glucose is omitted from the growth medium; however, the levels of extracellular autoinducer-2 decline (Glucose Retention of AI-2 Levels: GRAIL). Glycerol, maltose, galactose, ribose and L-arabinose could substitute for glucose, but lactose, D-arabinose, acetate, citrate and pyruvate could not. Mutations in (i). metabolic pathways (glycolytic enzymes eno, pgk, pgm; galactose epimerase; the Pta-AckA pathway), (ii). sugar transport (pts components, rbs operon, mgl, trg), and (iii). regulators involved in conventional catabolic repression (crp, cya), cAMP-independent catabolite repression (creC, fruR, rpoS,) the stringent response (relA, spoT) and the global carbon storage regulator (csrA) did not prevent GRAIL. Although the basis of GRAIL remains uncertain, it is clear that the mechanism is distinct from conventional catabolite repression. Moreover, GRAIL is not due to inactivation of the enzymic activity of LuxS, since in E. coli, LuxS contained within stationary-phase cells grown in the absence of glucose maintains its activity in vitro.

Formation of multimeric antibodies for self-delivery of active monomers.

PubMed

Dekel, Yaron; Machluf, Yossy; Gefen, Tal; Eidelshtein, Gennady; Kotlyar, Alexander; Bram, Yaron; Shahar, Ehud; Reslane, Farah; Aizenshtein, Elina; Pitcovski, Jacob

2017-11-01

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

PubMed Central

De Meyer, Simon F.; Vanhoorelbeke, Karen; Chuah, Marinee K.; Pareyn, Inge; Gillijns, Veerle; Hebbel, Robert P.; Collen, Désiré; Deckmyn, Hans; VandenDriessche, Thierry

2006-01-01

Von Willebrand disease (VWD) is an inherited bleeding disorder, caused by quantitative (type 1 and 3) or qualitative (type 2) defects in von Willebrand factor (VWF). Gene therapy is an appealing strategy for treatment of VWD because it is caused by a single gene defect and because VWF is secreted into the circulation, obviating the need for targeting specific organs or tissues. However, development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA (8.4 kb [kilobase]) and the inherent complexity of the VWF protein that requires extensive posttranslational processing. In this study, a gene-based approach for VWD was developed using lentiviral transduction of blood-outgrowth endothelial cells (BOECs) to express functional VWF. A lentiviral vector encoding complete human VWF was used to transduce BOECs isolated from type 3 VWD dogs resulting in high-transduction efficiencies (95.6% ± 2.2%). Transduced VWD BOECs efficiently expressed functional vector-encoded VWF (4.6 ± 0.4 U/24 hour per 106 cells), with normal binding to GPIbα and collagen and synthesis of a broad range of multimers resulting in phenotypic correction of these cells. These results indicate for the first time that gene therapy of type 3 VWD is feasible and that BOECs are attractive target cells for this purpose. PMID:16478886

VP08R from Infectious Spleen and Kidney Necrosis Virus Is a Novel Component of the Virus-Mock Basement Membrane

PubMed Central

Xu, Xiaopeng; Yan, Muting; Wang, Rui; Lin, Ting; Tang, Junliang; Li, Chaozheng; Weng, Shaoping

2014-01-01

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement membrane (BM)-like structure, termed virus-mock basement membrane (VMBM), on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R is located on the plasma membrane of infected cells and shows an expression profile similar to that of VP23R. Coimmunoprecipitation showed that VP08R interacts with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in the maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV; genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct virus-mock basement membranes (VMBMs) on the surface of infected cells to provide attaching sites for LECs. Although VMBMs lack the collagen IV network, which is an essential structural part of true BMs, VMBMs still show an intact structure. An ISKNV-encoded VP08R protein can self-assemble into a multimer and bind both VP23R and nidogen-1 to maintain the integrity and stability of VMBMs. On the basis of these facts, we redrew the putative schematic illustration of the VMBM structure. Our study suggests that the virus adopts a strategy to remodel the cellular matrix and may provide an important reference to elucidate BM functions and the mechanisms of lymphangiogenesis. PMID:24599992

Supramolecular curcumin-barium prodrugs for formulating with ceramic particles.

PubMed

Kamalasanan, Kaladhar; Anupriya; Deepa, M K; Sharma, Chandra P

2014-10-01

A simple and stable curcumin-ceramic combined formulation was developed with an aim to improve curcumin stability and release profile in the presence of reactive ceramic particles for potential dental and orthopedic applications. For that, curcumin was complexed with barium (Ba(2+)) to prepare curcumin-barium (BaCur) complex. Upon removal of the unbound curcumin and Ba(2+) by dialysis, a water-soluble BaCur complex was obtained. The complex was showing [M+1](+) peak at 10,000-20,000 with multiple fractionation peaks of MALDI-TOF-MS studies, showed that the complex was a supramolecular multimer. The (1)H NMR and FTIR studies revealed that, divalent Ba(2+) interacted predominantly through di-phenolic groups of curcumin to form an end-to-end complex resulted in supramolecular multimer. The overall crystallinity of the BaCur was lower than curcumin as per XRD analysis. The complexation of Ba(2+) to curcumin did not degrade curcumin as per HPLC studies. The fluorescence spectrum was blue shifted upon Ba(2+) complexation with curcumin. Monodisperse nanoparticles with size less than 200dnm was formed, out of the supramolecular complex upon dialysis, as per DLS, and upon loading into pluronic micelles the size was remaining in similar order of magnitude as per DLS and AFM studies. Stability of the curcumin was improved greater than 50% after complexation with Ba(2+) as per UV/Vis spectroscopy. Loading of the supramloecular nanoparticles into pluronic micelles had further improved the stability of curcumin to approx. 70% in water. These BaCur supramolecule nanoparticles can be considered as a new class of prodrugs with improved solubility and stability. Subsequently, ceramic nanoparticles with varying chemical composition were prepared for changing the material surface reactivity in terms of the increase in, degradability, surface pH and protein adsorption. Further, these ceramic particles were combined with curcumin prodrug formulations and optimized the curcumin release properties in the combined formulations. Our proof concept study shows that, the conversion of curcumin to a metal-organic supramolecular prodrug improved the solubility, stability and release profile of curcumin. The prodrug approach with the micellisation strategy appears to be more appropriate to deliver intact curcumin in the presence of ceramic particles of varying surface reactivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional diversification of B MADS-box homeotic regulators of flower development: Adaptive evolution in protein-protein interaction domains after major gene duplication events.

PubMed

Hernández-Hernández, Tania; Martínez-Castilla, León Patricio; Alvarez-Buylla, Elena R

2007-02-01

B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus propose a mechanism of functional diversification and persistence of gene duplicates by the appearance of novel multimerization capabilities after duplications. Multimer formation in different combinations of regulatory proteins can be a mechanistic basis for the origin of novel regulatory functions and a gene regulatory mechanism for the appearance of morphological innovations.

Dominant von Willebrand disease type 2A groups I and II due to missense mutations in the A2 domain of the von Willebrand factor gene: diagnosis and management.

PubMed

Michiels, Jan Jacques; van Vliet, Huub H D M

2009-01-01

Pertinent findings in patients with von Willebrand disease (VWD) type 2A include prolonged bleeding time (BT), consistently low von Willebrand factor (VWF):ristocetin cofactor activity (RCo)/antigen concentration (Ag) and VWF:collagen binding (CB)/Ag ratios, absence of high, and (depending on severity) intermediate and large VWF multimers, the presence of pronounced triplet structure of individual bands and increased VWF degradation products due to increased proteolysis caused by mutations in the A2 domain of VWF. Two categories of VWD type 2A can be distinguished: group I with severe and group II with mild VWD. A minority of VWD type 2A have mild VWD characterized by near normal to prolonged BT, normal factor VIII coagulant activity and VWF:Ag, low VWF:RCo and VWF:CB, a normal ristocetin-induced platelet aggregation and complete but transient correction of BT and functional VWF parameters to normal levels for only a few hours due to short half-lives for VWF:RCo and CWF:CB. Such transient complete responses to desmopressin (DDAVP) lasting only a few hours may facilitate treatment and prophylaxis of minor bleedings, but may not be able to prevent bleeding during minor and major surgery. Most VWD type 2A patients have pronounced VWD with very low VWF:RCo, prolonged BT, PFA-100 closure times >250 s, and response to DDAVP is only transient, minor, poor or absent, with no correction of the BT despite some increase in VWF:RCo, thus being candidates for factor VIII-VWF concentrate substitution for the acute and prophylactic treatment of bleeding symptoms. Copyright (c) 2009 S. Karger AG, Basel.

Presentation of the Multimédia Game "Geolover" Concept, to Educational Enchancement of the Geolocical Heritage of the Following Regions: "Ilha do Fogo" (Cabo-Verde), Seridó (Brasil), Sabugal (Portugal) and Açores (Portugal)

NASA Astrophysics Data System (ADS)

Cabral, João; Gomes, Ana; Alfama, Vera; Oliveira, Sirlene; Pinharandas, Carlos; Fonseca, Pedro; Campos, José; Nobre, José

2013-04-01

"Geolover" - Presentation of the multimédia game concept, to educational enchancement of the geolocical heritage of the following regions: : "Ilha do Fogo" (Cabo-Verde), Seridó (Brasil), Sabugal and Açores (Portugal). "Geolover" is a multitouch game, played by four players simultaneously, identified by 4 mascots and using as sceneries, the four regions landscapes, aimed to the young people with ages between 8 and 12 years old. The main objective is value the geological heritage of the Ilha do Fogo (Cabo Verde), Seridó in State of Rio Grande do Norte (Brasil) , Sabugal in Beira Alta province (Portugal) and Arquipélago dos Açores (Portuguese autonomous region). These regions have a great geological heritage like volcanology, plutonic rocks, sedimentar formations, metamorphic, paleontologic, mineralogic, geomorphologic, hydric and mining resources. Such heritage is being used in the different regions has base of studies to senior scientists and were used to great scientific researches. The diversified and distinguished cultural heritage of these four regions is referenced and it's a value to the union of the students from these three continents, with the Portuguese language as communication tool. The variety of the geological wealth and cultural of these regions, results in the common objective of their valuing like Geoparks. His creation on these three regions is a strategy with a great relevance to the socio-economic development. With the creation of this game, we promote the union of these 3 countries from these three continents, the universal values of the heritage richness that are offered by our planet.

Selective protein extraction from Chlorobium tepidum chlorosomes using detergents. Evidence that CsmA forms multimers and binds bacteriochlorophyll a.

PubMed

Bryant, Donald A; Vassilieva, Elena V; Frigaard, Niels-Ulrik; Li, Hui

2002-12-03

Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins. Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters. When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes. Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed. These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes. Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.

Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF.

PubMed

Yago, Tadayuki; Lou, Jizhong; Wu, Tao; Yang, Jun; Miner, Jonathan J; Coburn, Leslie; López, José A; Cruz, Miguel A; Dong, Jing-Fei; McIntire, Larry V; McEver, Rodger P; Zhu, Cheng

2008-09-01

Arterial blood flow enhances glycoprotein Ibalpha (GPIbalpha) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbalpha/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbalpha dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbalpha-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbalpha on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.

Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy.

PubMed

Yang, Yantao; Qu, Na; Tan, Jie; Rushdi, Muaz N; Krueger, Christopher J; Chen, Antony K

2018-06-11

During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (Gag ZiL ) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and Gag ZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while Gag ZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not Gag ZiL , was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses. Copyright © 2018 the Author(s). Published by PNAS.

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.

PubMed

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-08-15

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. © 2016 Li, Miao, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The aldo-keto reductase superfamily homepage.

PubMed

Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

2003-02-01

The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

The intrinsically disordered C-terminal region of Arabidopsis thaliana TCP8 transcription factor acts both as a transactivation and self-assembly domain.

PubMed

Valsecchi, Isabel; Guittard-Crilat, Emilie; Maldiney, Régis; Habricot, Yvette; Lignon, Sabrina; Lebrun, Régine; Miginiac, Emile; Ruelland, Eric; Jeannette, Emmanuelle; Lebreton, Sandrine

2013-09-01

TCPs are plant specific transcription factors with non-canonical basic helix-loop-helix domains. While Arabidopsis thaliana has 24 TCPs involved in cell proliferation and differentiation, their mode of action has not been fully elucidated. Using bioinformatic tools, we demonstrate that TCP transcription factors belong to the intrinsically disordered proteins (IDP) family and that disorder is higher in class I TCPs than in class II TCPs. In particular, using bioinformatic and biochemical approaches, we have characterized TCP8, a class I TCP. TCP8 exhibits three intrinsically disordered regions (IDR) made of more than 50 consecutive residues, in which phosphorylable Ser residues are mainly clustered. Phosphorylation of Ser-211 that belongs to the central IDR was confirmed by mass spectrometry. Yeast two-hybrid assays also showed that the C-terminal IDR corresponds to a transactivation domain. Moreover, biochemical experiments demonstrated that TCP8 tends to oligomerize in dimers, trimers and higher-order multimers. Bimolecular fluorescence complementation (BiFC) experiments carried out on a truncated form of TCP8 lacking the C-terminal IDR indicated that it is effectively required for the pronounced self-assembly of TCP8. These data were reinforced by the prediction of a coiled coil domain in this IDR. The C-terminal IDR acts thus as an oligomerization domain and also a transactivation domain. Moreover, many Molecular Recognition Features (MoRFs) were predicted, indicating that TCP8 could interact with several partners to fulfill a fine regulation of transcription in response to various stimuli.

Clinical usefulness of a functional assay for the von Willebrand factor cleaving protease (ADAMTS 13) and its inhibitor in a patient with thrombotic thrombocytopenic purpura.

PubMed

Rick, M E; Austin, H; Leitman, S F; Krizek, D M; Aronson, D L

2004-02-01

Decreased von Willebrand factor cleaving protease activity (VWFCP, ADAMTS 13) leads to persistence of unusually large multimers of von Willebrand factor that bind to platelets, causing platelet aggregates, microangiopathic hemolysis, and thrombocytopenia in patients with thrombotic thrombocytopenic purpura (TTP). The clinical value of measuring ADAMTS 13 and its inhibitor is not fully defined; the case reported here illustrates the usefulness of the assay to help confirm the clinical diagnosis in a patient with other potential causes for thrombotic microangiopathy; the assay also helped in making treatment decisions. A patient with systemic lupus erythematosis (SLE) presented with fever and abdominal pain, thrombocytopenia, and anemia. Thrombotic microangiopathy was diagnosed by the appearance of schistocytes, decreasing platelet count, and evidence of hemolysis. ADAMTS 13 was decreased and an inhibitor was demonstrated in the patient's initial blood sample within 24 hr of admission. Plasma exchange was initiated, and serial assays showed increased ADAMTS 13 activity and decreased inhibitor after each plasma exchange; there was a rebound in inhibitor and a decrease in ADAMTS 13 activity prior to the next exchange that lessened over time. Increasing levels of protease activity correlated with clinical and laboratory improvement. Measurement of ADAMTS 13 activity and its inhibitor aided in the diagnosis of this complicated case of a patient with other potential causes for microangiopathic hemolysis. Subsequent levels correlated with the clinical course, and disappearance of the inhibitor indicated that long-term plasma exchange or other immunosuppressive treatment was not needed.

Von Willebrand factor regulation of blood vessel formation.

PubMed

Randi, Anna M; Smith, Koval E; Castaman, Giancarlo

2018-06-04

Several important physiological processes, from permeability to inflammation to haemostasis, take place at the vessel wall and are regulated by endothelial cells (EC). Thus, proteins that have been identified as regulators of one process are increasingly found to be involved in other vascular functions. Such is the case for Von Willebrand Factor (VWF), a large glycoprotein best known for its critical role in haemostasis. In vitro and in vivo studies have shown that lack of VWF causes enhanced vascularisation, both constitutively and following ischemia. This evidence is supported by studies on blood outgrowth endothelial cells (BOEC) from patients with lack of VWF synthesis (type 3 von Willebrand disease [VWD]). The molecular pathways are likely to involve VWF binding partners, such as integrin αvβ3, and components of Weibel Palade bodies (WPB), such as Angiopoietin-2 and Galectin-3, whose storage is regulated by VWF; these converge on the master regulator of angiogenesis and endothelial homeostasis, vascular endothelial growth factor (VEGF) signalling. Recent studies suggest that the roles of VWF may be tissue-specific. The ability of VWF to regulate angiogenesis has clinical implications for a subset of VWD patients with severe, intractable gastrointestinal bleeding due to vascular malformations. In this article, we review the evidence showing that VWF is involved in blood vessel formation, discuss the role of VWF high molecular weight multimers in regulating angiogenesis, and the value of studies on BOEC in developing a precision medicine approach to validate novel treatments for angiodysplasia in congenital VWD and acquired von Willebrand syndrome. Copyright © 2018 American Society of Hematology.

On the Effect of Sphere-Overlap on Super Coarse-Grained Models of Protein Assemblies

NASA Astrophysics Data System (ADS)

Degiacomi, Matteo T.

2018-05-01

Ion mobility mass spectrometry (IM/MS) can provide structural information on intact protein complexes. Such data, including connectivity and collision cross sections (CCS) of assemblies' subunits, can in turn be used as a guide to produce representative super coarse-grained models. These models are constituted by ensembles of overlapping spheres, each representing a protein subunit. A model is considered plausible if the CCS and sphere-overlap levels of its subunits fall within predetermined confidence intervals. While the first is determined by experimental error, the latter is based on a statistical analysis on a range of protein dimers. Here, we first propose a new expression to describe the overlap between two spheres. Then we analyze the effect of specific overlap cutoff choices on the precision and accuracy of super coarse-grained models. Finally, we propose a method to determine overlap cutoff levels on a per-case scenario, based on collected CCS data, and show that it can be applied to the characterization of the assembly topology of symmetrical homo-multimers. [Figure not available: see fulltext.

Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system

DOE PAGES

Scharadin, Tiffany M.; He, Wei; Yiannakou, Yianni; ...

2017-06-06

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. Themore » resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. Lastly, we anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.« less

Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system

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

Scharadin, Tiffany M.; He, Wei; Yiannakou, Yianni

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. Themore » resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. Lastly, we anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.« less

Reduced ADAMTS13 activity is associated with thrombotic risk in systemic lupus erythematosus.

PubMed

Martin-Rodriguez, S; Reverter, J C; Tàssies, D; Espinosa, G; Heras, M; Pino, M; Escolar, G; Diaz-Ricart, M

2015-10-01

Severe deficiency of ADAMTS13 activity leads to von Willebrand factor (VWF) ultralarge multimers with high affinity for platelets, causing thrombotic thrombocytopenic purpura. Other pathological conditions with moderate ADAMTS13 activity exhibit a thrombotic risk. We examined the ADAMTS13 activity in systemic lupus erythematosus (SLE) and its value as a thrombotic biomarker. ADAMTS13 activity, VWF antigen and multimeric structure, and vascular cell adhesion molecule 1 (VCAM-1) were measured in plasma samples from 50 SLE patients and 50 healthy donors. Disease activity (systemic lupus erythematosus disease activity index; SLEDAI) and organ damage (systemic lupus international collaborating clinics) scores, thrombotic events, antiphospholipid syndrome (APS) and antiphospholipid antibodies (aPLs) were registered. SLE patients showed decreased ADAMTS13 activity and high VWF levels compared with controls (66 ± 27% vs. 101 ± 8%, P < 0.01, and 325 ± 151% vs. 81 ± 14%, P < 0.001). VCAM-1 levels were higher in SLE patients (P < 0.05). Considering three groups of SLE patients depending on ADAMTS13 activity (>60%, 60-40% and <40%), comparative analysis showed significant association between ADAMTS13 activity and SLEDAI (P < 0.05), presence of aPLs (P < 0.001), APS (P < 0.01) and thrombotic events (P < 0.01). Reduced ADAMTS13 activity together with increased VWF levels were especially notable in patients with active disease and with aPLs. ADAMTS13 activity, in combination with other laboratory parameters, could constitute a potential prognostic biomarker of thrombotic risk in SLE. © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

PubMed

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

2014-12-01

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization and Higher-Order Structure Assessment of an Interchain Cysteine-Based ADC: Impact of Drug Loading and Distribution on the Mechanism of Aggregation.

PubMed

Guo, Jianxin; Kumar, Sandeep; Chipley, Mark; Marcq, Olivier; Gupta, Devansh; Jin, Zhaowei; Tomar, Dheeraj S; Swabowski, Cecily; Smith, Jacquelynn; Starkey, Jason A; Singh, Satish K

2016-03-16

The impact of drug loading and distribution on higher order structure and physical stability of an interchain cysteine-based antibody drug conjugate (ADC) has been studied. An IgG1 mAb was conjugated with a cytotoxic auristatin payload following the reduction of interchain disulfides. The 2-D LC-MS analysis shows that there is a preference for certain isomers within the various drug to antibody ratios (DARs). The physical stability of the unconjugated monoclonal antibody, the ADC, and isolated conjugated species with specific DAR, were compared using calorimetric, thermal, chemical denaturation and molecular modeling techniques, as well as techniques to assess hydrophobicity. The DAR was determined to have a significant impact on the biophysical properties and stability of the ADC. The CH2 domain was significantly perturbed in the DAR6 species, which was attributable to quaternary structural changes as assessed by molecular modeling. At accelerated storage temperatures, the DAR6 rapidly forms higher molecular mass species, whereas the DAR2 and the unconjugated mAb were largely stable. Chemical denaturation study indicates that DAR6 may form multimers while DAR2 and DAR4 primarily exist in monomeric forms in solution at ambient conditions. The physical state differences were correlated with a dramatic increase in the hydrophobicity and a reduction in the surface tension of the DAR6 compared to lower DAR species. Molecular modeling of the various DAR species and their conformers demonstrates that the auristatin-based linker payload directly contributes to the hydrophobicity of the ADC molecule. Higher order structural characterization provides insight into the impact of conjugation on the conformational and colloidal factors that determine the physical stability of cysteine-based ADCs, with implications for process and formulation development.

Computer Simulation Studies of Sputtering and Multimer Formation from Clean and Oxygen Reacted Surfaces of Titanium, Vanadium and Niobium.

DTIC Science & Technology

1983-12-01

100 - - 75 IP - - - Ti TFB 64 - - 92 100 100 - - - Ti TF(A+B) 82 - - 84 50 100 - - - C(2x2) Ti ATOP - - - 98 50 - - - V ATOP 56 - - 81 67 iP - 100 100...Nb ATOP 100 100 - 74 40 - X X - Ti BR 40 - - 92 100 100 IP IP - Ti TRA 33 - - 80 50 - - - Ti TFB 86 X - 73 100 - 50 - - Ti TR(A+B) 60 x - 77 75 - 50...NOTE: A tack (-) indicates that the species was not formed, while "X" indicates that none were formed by frag- mentation. IP indicates that only

Cyclo[n]pyrroles and methods thereto

DOEpatents

Sessler, Jonathan L.; Seidel, Daniel; Bolze, Frederic R.; Koehler, Thomas

2006-01-10

The present invention provides an oxidative coupling procedure that allows efficient synthesis of novel cyclo[n]pyrrole macrocycles. Therefore, the present invention provides cyclo[n]pyrroles where n is 6, 7, 8, 9, 10, 11, or 12, and derivatives, multimers, isomers, and ion and neutral molecule complexes thereof as new compositions of matter. A protonated form of cyclo[n]pyrrole displays a gap of up to 700 nm between strong Soret and Q-like absorption bands in the electronic spectrum, demonstrating no significant ground state absorption in the visible portion of the electronic spectrum. Uses of cyclo[n]pyrroles as separation media, nonlinear optical materials, information storage media and infrared filters are provided.

Concept of a self-associated multimer structure of coal

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

Gagarin, S.G.; Krichko, A.A.

1984-01-01

The paper examines the role of donor-acceptor reaction in the binding of the individual components forming the structure of the organic coal mass, and analyses the manifestations of this reaction during liquefaction. The authors put forward the concept of self-associated polymers in the coal structure, in accordance with which the organic coal mass has spatial and energetic distribution of the donor and acceptor sectors of structure. It is the specific reaction between these which produces the necessary stability to the polymer system under normal conditions. The authors propose a mechanism for the action of solvents and various additives in themore » liquefaction of coal.« less

Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)*

PubMed Central

Hershko, Klilah; Simhadri, Vijaya L.; Blaisdell, Adam; Hunt, Ryan C.; Newell, Jordan; Tseng, Sandra C.; Hershko, Alon Y.; Choi, Jae Won; Sauna, Zuben E.; Wu, Andrew; Bram, Richard J.; Komar, Anton A.; Kimchi-Sarfaty, Chava

2012-01-01

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients. PMID:23144461

Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

PubMed

Hershko, Klilah; Simhadri, Vijaya L; Blaisdell, Adam; Hunt, Ryan C; Newell, Jordan; Tseng, Sandra C; Hershko, Alon Y; Choi, Jae Won; Sauna, Zuben E; Wu, Andrew; Bram, Richard J; Komar, Anton A; Kimchi-Sarfaty, Chava

2012-12-28

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

Geohydrology of the unsaturated zone and simulated time of arrival of landfill leachate at the water table, municipal solid waste landfill facility, US Army Air Defense Artillery Center and Fort Bliss, El Paso County, Texas

USGS Publications Warehouse

Frenzel, Peter F.; Abeyta, Cynthia G.

1999-01-01

The U.S. Air Defense Artillery Center and Fort Bliss Municipal Solid Waste Landfill Facility (MSWLF) is located about 10 miles northeast of downtown El Paso, Texas. The landfill is built on the Hueco Bolson, a deposit that yields water to five public-supply wells within 1.1 miles of the landfill boundary on all sides. The bolson deposits consist of lenses and mixtures of sand, clay, silt, gravel, and caliche. The unsaturated zone at the landfill is about 300 feet thick. The Hydrologic Evaluation of Landfill Performance (HELP) and the Multimedia Exposure Assessment Model for Evaluating the Land Disposal of Wastes (MULTIMED) computer models were used to simulate the time of first arrival of landfill leachate at the water table. Site-specific data were collected for model input. At five sites on the landfill cover, hydraulic conductivity was measured by an in situ method; in addition, laboratory values were obtained for porosity, moisture content at field capacity, and moisture content at wilting point. Twenty-seven sediment samples were collected from two adjacent boreholes drilled near the southwest corner of the landfill. Of these, 23 samples were assumed to represent the unsaturated zone beneath the landfill. The core samples were analyzed in the laboratory for various characteristics required for the HELP and MULTIMED models: initial moisture content, dry bulk density, porosity, saturated hydraulic conductivity, moisture retention percentages at various suction values, total organic carbon, and pH. Parameters were calculated for the van Genuchten and Brooks-Corey equations that relate hydraulic conductivity to saturation. A reported recharge value of 0.008 inch per year was estimated on the basis of soil- water chloride concentration. The HELP model was implemented using input values that were based mostly on site-specific data or assumed in a conservative manner. Exceptions were the default values used for waste characteristics. Flow through the landfill was assumed to be at steady state. The HELP-estimated landfill leakage rate was 101.6 millimeters per year, approximately 500 times the estimated recharge rate for the area near the landfill. The MULTIMED model was implemented using input values that were based mainly on site-specific data and some conservatively assumed values. Landfill leakage was assumed to begin when the landfill was established and to continue at a steady-state rate of 101.6 millimeters per year as estimated by the HELP model. By using an assumed solute concentration in the leachate of 1 milligram per liter and assuming no delay or decay of solute, the solute serves as a tracer to indicate the first arrival of landfill leachate. The simulated first arrival of leachate at the water table was 204 to 210 years after the establishment of the landfill.

Thrombotic thrombocytopenic purpura presenting with pathologic fracture: a case report.

PubMed

Berber, Ilhami; Erkurt, Mehmet Ali; Kuku, Irfan; Kaya, Emin; Unlu, Serkan; Ertem, Kadir; Nizam, Ilknur

2014-08-01

Thrombotic thrombocytopenic purpura is an acute syndrome with abnormalities in multiple organ systems, which becomes manifest with microangiopathic hemolytic anemia and thrombocytopenia. The hereditary or acquired deficiency of ADAMTS-13 activity leads to an excess of high molecular weight von Willebrand factor multimers in plasma, leading to platelet aggregation and diffuse intravascular thrombus formation, resulting in thrombotic thrombocytopenic purpura. Thrombotic lesions occurring in TTP leads to ischemia and convulsion. Depending on the properties of the bony tissue, fractures are divided into three groups as traumatic, pathological, and stress fractures. A pathologic fracture is a broken bone caused by disease leading to weakness of the bone. This process is most commonly due to osteoporosis, but may also be due to other pathologies such as cancer, infections, inherited bone disorders, or a bone cyst. We herein report a case with a pathologic fracture due to convulsion secondary to thrombotic thrombocytopenic pupura. Thrombotic lesions occurring in TTP may lead to ischemia and convulsion, as in our patient and pathological fractures presented in our case report may occur as a result of severe muscle contractions associated with convulsive activity. Thrombotic thrombocytopenic pupura is a disease that involves many organ systems and thus may have a very wide spectrum of clinical presentations. Copyright © 2014. Published by Elsevier Ltd.

Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in the Yersinia enterocolitica Cytoplasmic Membrane.

PubMed

Srivastava, Disha; Moumene, Amal; Flores-Kim, Josué; Darwin, Andrew J

2017-09-12

The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers. IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers. Copyright © 2017 Srivastava et al.

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens.

PubMed

MacLachlan, Bruce J; Greenshields-Watson, Alexander; Mason, Georgina H; Schauenburg, Andrea J; Bianchi, Valentina; Rizkallah, Pierre J; Sewell, Andrew K; Fuller, Anna; Cole, David K

2017-02-06

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Parsimonious Charge Deconvolution for Native Mass Spectrometry

PubMed Central

2018-01-01

Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new “parsimonious” charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies. PMID:29376659

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state.

PubMed

Findeisen, Felix; Tolia, Alexandra; Arant, Ryan; Kim, Eun Young; Isacoff, Ehud; Minor, Daniel L

2011-01-01

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.

Platelet glycoprotein Ibα forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF

PubMed Central

Yago, Tadayuki; Lou, Jizhong; Wu, Tao; Yang, Jun; Miner, Jonathan J.; Coburn, Leslie; López, José A.; Cruz, Miguel A.; Dong, Jing-Fei; McIntire, Larry V.; McEver, Rodger P.; Zhu, Cheng

2008-01-01

Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged (“catch”) and then shortened (“slip”) bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif–13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding. PMID:18725999

Synaptic control of local translation: the plot thickens with new characters.

PubMed

Thomas, María Gabriela; Pascual, Malena Lucía; Maschi, Darío; Luchelli, Luciana; Boccaccio, Graciela Lidia

2014-06-01

The production of proteins from mRNAs localized at the synapse ultimately controls the strength of synaptic transmission, thereby affecting behavior and cognitive functions. The regulated transcription, processing, and transport of mRNAs provide dynamic control of the dendritic transcriptome, which includes thousands of messengers encoding multiple cellular functions. Translation is locally modulated by synaptic activity through a complex network of RNA-binding proteins (RBPs) and various types of non-coding RNAs (ncRNAs) including BC-RNAs, microRNAs, piwi-interacting RNAs, and small interference RNAs. The RBPs FMRP and CPEB play a well-established role in synaptic translation, and additional regulatory factors are emerging. The mRNA repressors Smaug, Nanos, and Pumilio define a novel pathway for local translational control that affects dendritic branching and spines in both flies and mammals. Recent findings support a role for processing bodies and related synaptic mRNA-silencing foci (SyAS-foci) in the modulation of synaptic plasticity and memory formation. The SyAS-foci respond to different stimuli with changes in their integrity thus enabling regulated mRNA release followed by translation. CPEB, Pumilio, TDP-43, and FUS/TLS form multimers through low-complexity regions related to prion domains or polyQ expansions. The oligomerization of these repressor RBPs is mechanistically linked to the aggregation of abnormal proteins commonly associated with neurodegeneration. Here, we summarize the current knowledge on how specificity in mRNA translation is achieved through the concerted action of multiple pathways that involve regulatory ncRNAs and RBPs, the modification of translation factors, and mRNA-silencing foci dynamics.

Prospective study of hemostatic alterations in children with acute lymphoblastic leukemia.

PubMed

Giordano, Paola; Molinari, Angelo Claudio; Del Vecchio, Giovanni Carlo; Saracco, Paola; Russo, Giovanna; Altomare, Maria; Perutelli, Paolo; Crescenzio, Nicoletta; Santoro, Nicola; Marchetti, Marina; De Mattia, Domenico; Falanga, Anna

2010-05-01

In a group of newly diagnosed acute lymphocytic leukemia (ALL) children we evaluated a number of hemostatic and inflammatory markers at diagnosis and at different time points during chemotherapy for the remission induction to identify alterations in the plasma levels of prothrombotic markers before and during the course of chemotherapy. The following plasma markers were evaluated: thrombin-antithrombin complex (TAT), D-Dimer, plasminogen activator inhibitor 1 (PAI-1), antithrombin, fibrinogen, von Willebrand factor (VWF) antigen and high molecular weight VWF (HMW-VWF) multimers, P-selectin, tumor necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6). Plasma samples were collected at the following time points: at T0 (baseline) and T1 (+24 days of therapy), T2 (+36 days therapy), and T3 (+64 days therapy). The results show that, at diagnosis, ALL children presented with laboratory signs of increased thrombin generation and fibrin formation (i.e. high TAT and D-dimer levels), fibrinolysis inhibition (i.e. high PAI-1 level), endothelial activation (i.e., high HMW-VWF and soluble P-selectin levels) and inflammation (i.e. high TNF-alpha and IL-6 levels). After starting induction therapy, the thrombin generation markers and inflammatory cytokines significantly decreased. To the opposite, PAI-1 and P-selectin significantly increased, suggesting an insult by chemotherapy on the vascular endothelium. These effects were more evident during steroid administration. Symptomatic venous thromboembolism (VTE) episodes developed in two cases during induction therapy, which did not allow the evaluation of the predictive value for VTE of laboratory markers. (c) 2010 Wiley-Liss, Inc.

Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans.

PubMed

Devi, Seram Nganbiton; Kiehler, Brittany; Haggett, Lindsey; Fujita, Masaya

2015-08-01

Entry into sporulation in Bacillus subtilis is governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur in trans. To test this hypothesis, we generated a series of B. subtilis strains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in a trans fashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressed in vivo. Taken together, these in vitro and in vivo results reinforce the evidence that KinA autophosphorylation is able to occur in a trans fashion. Autophosphorylation of histidine kinases is known to occur by either the cis (one subunit of kinase phosphorylating itself within the multimer) or the trans (one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided direct in vivo and in vitro evidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur in trans within the homotetramer complex. While the physiological and mechanistic significance of the trans autophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Monomeric rhodopsin is the minimal functional unit required for arrestin binding.

PubMed

Tsukamoto, Hisao; Sinha, Abhinav; DeWitt, Mark; Farrens, David L

2010-06-11

We have tested whether arrestin binding requires the G-protein-coupled receptor be a dimer or a multimer. To do this, we encapsulated single-rhodopsin molecules into nanoscale phospholipid particles (so-called nanodiscs) and measured their ability to bind arrestin. Our data clearly show that both visual arrestin and beta-arrestin 1 can bind to monomeric rhodopsin and stabilize the active metarhodopsin II form. Interestingly, we find that the monomeric rhodopsin in nanodiscs has a higher affinity for wild-type arrestin binding than does oligomeric rhodopsin in liposomes or nanodiscs, as assessed by stabilization of metarhodopsin II. Together, these results establish that rhodopsin self-association is not required to enable arrestin binding. Copyright 2010 Elsevier Ltd. All rights reserved.

Transcriptome Analysis of Mycobacteria-Specific CD4+ T Cells Identified by Activation-Induced Expression of CD154.

PubMed

Kunnath-Velayudhan, Shajo; Goldberg, Michael F; Saini, Neeraj K; Johndrow, Christopher T; Ng, Tony W; Johnson, Alison J; Xu, Jiayong; Chan, John; Jacobs, William R; Porcelli, Steven A

2017-10-01

Analysis of Ag-specific CD4 + T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4 + T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4 + T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4 + T cells from bacillus Calmette-Guérin-vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154 + cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4 + CD154 + cells was distinct from that of CD154 - cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4 + T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4 + T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts. Copyright © 2017 by The American Association of Immunologists, Inc.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Localization of electrons and excitations

NASA Astrophysics Data System (ADS)

Larsson, Sven

2006-07-01

Electrons, electron holes, or excitations in finite or infinite 'multimer systems' may be localized or delocalized. In the theory of Hush, localization depends on the ratio Δ/ λ ( Δ/2 = coupling; λ = reorganization energy). The latter theory has been extended to the infinite system [S. Larsson, A. Klimkāns, Mol. Cryst. Liq. Cryst. 355 (2000) 217]. The metal/insulator transition often takes place abruptly as a function of Δ/ λ. It is argued that localization in a system with un-filled bands cannot be determined on the basis of Mott-Hubbard U alone, but depends on the number of accessible valence states, reorganization energy λ and coupling Δ (=2t). In fact U = 0 does not necessarily imply delocalization. The analysis here shows that there are many different situations for an insulator to metal transition. Charge transfer in doped NiO is characterized by Ni 2+ - Ni 3+ exchange while charge transfer in pure NiO is characterized by a disproportionation 2Ni 2+ → Ni + + Ni 3+. In spite of the great differences between these two cases, U has been applied without discrimination to both. The relevant localization parameters appear to be Δ and λ in the first case, with only two oxidation states, and U, Δ and λ in the second case with three oxidation states. The analysis is extended to insulator-metal transitions, giant magnetic resistance (GMR) and high Tc superconductivity (SC). λ and Δ can be determined quite accurately in quantum mechanical calculations involving only one and two monomers, respectively.

Von Willebrand Factor Deposition and ADAMTS-13 Consumption in Allograft Tissue of Thrombotic Microangiopathy-like Disorder After Living Donor Liver Transplantation: A Case Report.

PubMed

Nakanuma, S; Miyashita, T; Hayashi, H; Ohbatake, Y; Takamura, H; Okazaki, M; Yamaguchi, T; Sakai, S; Makino, I; Oyama, K; Tajima, H; Ninomiya, I; Fushida, S; Ohta, T

2017-09-01

Thrombotic microangiopathy (TMA) pathogenesis after living donor liver transplantation (LDLT) is thought to be caused by release of unusually large von Willebrand factor multimers (UL-vWFMs) resulting from sinusoidal endothelial cell damage and induction of platelet adhesion and aggregation. A decrease in a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13 (ADAMTS-13) that cleave UL-vWFMs might cause excessive UL-vWFMs activity and result in platelet thrombus formation. However, this phenomenon has not undergone a full pathologic assessment. A 60-year-old man was diagnosed with hepatitis C-related end-stage cirrhosis. His son was the donor, and he underwent LDLT. On postoperative day 44, his laboratory findings met most TMA diagnostic criteria, and he was diagnosed with TMA-like disorder (TMALD). Localization of CD42b as a platelet marker, vWF, and ADAMTS-13 in allograft tissue of this patient were evaluated using immunohistochemistry. CD42b expression was observed as platelet aggregates attached to hepatocytes or within the hepatocyte cytoplasm, a morphology called extravasated platelet aggregation (EPA). vWF expression was observed mainly as deposited compact clusters, and ADAMTS-13 expression resembled distinct dots throughout the liver tissue. These findings suggest that EPA indicated sinusoidal endothelial cell damage followed by detachment, and vWF deposition resulted from UL-vWFM oversynthesis. ADAMTS-13 might be consumed in the allograft tissue to cleave UL-vWFMs, but ADAMTS-13 levels might be insufficient to cleave all the deposited UL-vWFMs. We present the case of an LDLT recipient diagnosed with TMALD using blood tests, which showed the presence of TMA pathogenesis in the allograft. Copyright © 2017 Elsevier Inc. All rights reserved.

Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor.

PubMed

Chen, Zengsheng; Mondal, Nandan K; Ding, Jun; Koenig, Steven C; Slaughter, Mark S; Wu, Zhongjun J

2016-07-01

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices. Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Paradoxical Effect of Non-Physiological Shear Stress on Platelets and von Willebrand factor

PubMed Central

Chen, Zengsheng; Mondal, Nandan K; Ding, Jun; Koenig, Steven C.; Slaughter, Mark S.; Wu, Zhongjun J.

2016-01-01

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that non-physiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25Pa, 125Pa) with an exposure time of 0.5 sec., generated by using a novel blood shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWM) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices. PMID:26582038

Scoring functions for protein-protein interactions.

PubMed

Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

2013-12-01

The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhancement of Resonant Energy Transfer Due to an Evanescent Wave from the Metal.

PubMed

Poudel, Amrit; Chen, Xin; Ratner, Mark A

2016-03-17

The high density of evanescent modes in the vicinity of a metal leads to enhancement of the near-field Förster resonant energy transfer (FRET) rate. We present a classical approach to calculate the FRET rate based on the dyadic Green's function of an arbitrary dielectric environment and consider the nonlocal limit of material permittivity in the case of the metallic half-space and thin film. In a dimer system, we find that the FRET rate is enhanced due to shared evanescent photon modes bridging a donor and an acceptor. Furthermore, a general expression for the FRET rate for multimer systems is derived. The presence of a dielectric environment and the path interference effect enhance the transfer rate, depending on the combination of distance and geometry.

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

PubMed

Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

2015-04-03

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

Immunological Prediction of Cytomegalovirus (CMV) Replication Risk in Solid Organ Transplantation Recipients: Approaches for Regulating the Targeted Anti-CMV Prevention Strategies

PubMed Central

2017-01-01

The current cytomegalovirus (CMV) prevention strategies in solid organ transplantation (SOT) recipients have contributed towards overcoming the detrimental effects caused by CMV lytic infection, and improving the long-term success rate of graft survival. Although the quantification of CMV in peripheral blood is the standard method, and an excellent end-point for diagnosing CMV replication and modulating the anti-CMV prevention strategies in SOT recipients, a novel biomarker mimicking the CMV control mechanism is required. CMV-specific immune monitoring can be employed as a basic tool predicting CMV infection or disease after SOT, since uncontrolled CMV replication mostly originates from the impairment of immune responses against CMV under immunosuppressive conditions in SOT recipients. Several studies conducted during the past few decades have indicated the possibility of measuring the CMV-specific cell-mediated immune response in clinical situations. Among several analytical assays, the most advancing standardized tool is the QuantiFERON®-CMV assay. The T-Track® CMV kit that uses the standardized enzyme-linked immunospot assay is also widely employed. In addition to these assays, immunophenotyping and intracellular cytokine analysis using flow cytometry (with fluorescence-labeled monoclonal antibodies or peptide-major histocompatibility complex multimers) needs to be adequately standardized and validated for potential clinical applications. PMID:29027383

Experimental and Theoretical Investigation of Sodiated Multimers of Steroid Epimers with Ion Mobility-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Chouinard, Christopher D.; Cruzeiro, Vinícius Wilian D.; Roitberg, Adrian E.; Yost, Richard A.

2017-02-01

Ion mobility-mass spectrometry (IM-MS) has recently seen increased use in the analysis of small molecules, especially in the field of metabolomics, for increased breadth of information and improved separation of isomers. In this study, steroid epimers androsterone and trans-androsterone were analyzed with IM-MS to investigate differences in their relative mobilities. Although sodiated monomers exhibited very similar collision cross-sections (CCS), baseline separation was observed for the sodiated dimer species (RS = 1.81), with measured CCS of 242.6 and 256.3 Å2, respectively. Theoretical modeling was performed to determine the most energetically stable structures of solution-phase and gas-phase monomer and dimer structures. It was revealed that these epimers differ in their preferred dimer binding mode in solution phase: androsterone adopts a R=O - Na+ - OH—R' configuration, whereas trans-androsterone adopts a R=O - Na+ - O=R' configuration. This difference contributes to a significant structural variation, and subsequent CCS calculations based on these structures relaxed in the gas phase were in agreement with experimentally measured values (ΔCCS 5%). Additionally, these calculations accurately predicted the relative difference in mobility between the epimers. This study illustrates the power of combining experimental and theoretical results to better elucidate gas-phase structures.

Spontaneous CD4+ and CD8+ T‐cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients

PubMed Central

Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard

2017-01-01

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune‐privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T‐cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg‐specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8+ and CD4+ T‐cell responses against MAGE‐A family antigens were present in 44% (20/45) of patients’ samples assayed by ex vivo IFN‐γ ELISPOT. The presence of MAGE‐specific CD8+ T cells was further determined following short‐term in vitro expansion through the use of pMHC‐I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE‐specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg‐specific T‐cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T‐cell pool following treatment. Spontaneous T‐cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. PMID:28555838

Human RAD50 makes a functional DNA-binding complex.

PubMed

Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

2015-06-01

The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Von Willebrand's disease with spontaneous platelet aggregation induced by an abnormal plasma von Willebrand factor.

PubMed Central

Grainick, H R; Williams, S B; McKeown, L P; Rick, M E; Maisonneuve, P; Jenneau, C; Sultan, Y

1985-01-01

We have investigated and characterized the abnormalities in four unrelated patients with von Willebrand's disease (vWd) who have (a) enhanced ristocetin-induced platelet aggregation (RIPA) at low ristocetin concentrations, (b) absence of the largest plasma von Willebrand factor (vWf) multimers, and (c) thrombocytopenia. The platelet-rich plasma of these patients aggregates spontaneously without the addition of any agonists. When isolated normal platelets are resuspended in patient plasma spontaneous aggregation occurs; however, the patients' plasmas did not induce platelet aggregation of normal washed formalinized platelets. When the patients' platelets are suspended in normal plasma, spontaneous aggregation is not observed. The spontaneous platelet aggregation (SPA) is associated with dense granule secretion as measured by ATP release and alpha granule release as measured by beta-thromboglobulin and platelet factor 4 release. The SPA is totally inhibited by 5 mM EDTA, prostaglandin I2, and dibutryl cyclic AMP, while it is only partially inhibited by 1 mM EDTA, acetylsalicylic acid, or apyrase. A monoclonal antibody directed against glycoprotein Ib (GPIb) and/or a monoclonal antibody against the glycoprotein IIb/IIIa (GPIIb/IIIa) complex totally inhibits the SPA. The vWf was isolated from the plasma of one of these patients. The purified vWf induced platelet aggregation of normal platelets resuspended in either normal or severe vWd plasma, but the vWf did not induce platelet aggregation of normal platelets resuspended in afibrinognemic plasma. Sialic acid and galactose quantification of the patient's vWf revealed approximately a 50% reduction compared with normal vWf. These studies indicate that a form of vWd exists, which is characterized by SPA that is induced by the abnormal plasma vWf. The SPA is dependent on the presence of plasma fibrinogen, and the availability of the GPIb and the GPIIb/IIIa complex. In this variant form of vWd the abnormal vWf causes enhanced RIPA, SPA, and thrombocytopenia. Images PMID:2932469

PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

PubMed Central

Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

2018-01-01

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative control-transduced groups. In conclusion, a novel HLA-A2-restricted and PLAC1-specific TCR was identified. The present study demonstrated PLAC1 to be a potential target for breast cancer treatment; and the usage of PLAC1-specific TCR-engineered T cells may be a novel strategy for PLAC1-positive breast cancer treatment. PMID:29556312

Dose response relationship in anti-stress gene regulatory networks.

PubMed

Zhang, Qiang; Andersen, Melvin E

2007-03-02

To maintain a stable intracellular environment, cells utilize complex and specialized defense systems against a variety of external perturbations, such as electrophilic stress, heat shock, and hypoxia, etc. Irrespective of the type of stress, many adaptive mechanisms contributing to cellular homeostasis appear to operate through gene regulatory networks that are organized into negative feedback loops. In general, the degree of deviation of the controlled variables, such as electrophiles, misfolded proteins, and O2, is first detected by specialized sensor molecules, then the signal is transduced to specific transcription factors. Transcription factors can regulate the expression of a suite of anti-stress genes, many of which encode enzymes functioning to counteract the perturbed variables. The objective of this study was to explore, using control theory and computational approaches, the theoretical basis that underlies the steady-state dose response relationship between cellular stressors and intracellular biochemical species (controlled variables, transcription factors, and gene products) in these gene regulatory networks. Our work indicated that the shape of dose response curves (linear, superlinear, or sublinear) depends on changes in the specific values of local response coefficients (gains) distributed in the feedback loop. Multimerization of anti-stress enzymes and transcription factors into homodimers, homotrimers, or even higher-order multimers, play a significant role in maintaining robust homeostasis. Moreover, our simulation noted that dose response curves for the controlled variables can transition sequentially through four distinct phases as stressor level increases: initial superlinear with lesser control, superlinear more highly controlled, linear uncontrolled, and sublinear catastrophic. Each phase relies on specific gain-changing events that come into play as stressor level increases. The low-dose region is intrinsically nonlinear, and depending on the level of local gains, presence of gain-changing events, and degree of feedforward gene activation, this region can appear as superlinear, sublinear, or even J-shaped. The general dose response transition proposed here was further examined in a complex anti-electrophilic stress pathway, which involves multiple genes, enzymes, and metabolic reactions. This work would help biologists and especially toxicologists to better assess and predict the cellular impact brought about by biological stressors.

Ras Dimer Formation as a New Signaling Mechanism and Potential Cancer Therapeutic Target

PubMed Central

Chen, Mo; Peters, Alec; Huang, Tao; Nan, Xiaolin

2016-01-01

The K-, N-, and HRas small GTPases are key regulators of cell physiology and are frequently mutated in human cancers. Despite intensive research, previous efforts to target hyperactive Ras based on known mechanisms of Ras signaling have been met with little success. Several studies have provided compelling evidence for the existence and biological relevance of Ras dimers, establishing a new mechanism for regulating Ras activity in cells additionally to GTP-loading and membrane localization. Existing data also start to reveal how Ras proteins dimerize on the membrane. We propose a dimer model to describe Ras-mediated effector activation, which contrasts existing models of Ras signaling as a monomer or as a 5-8 membered multimer. We also discuss potential implications of this model in both basic and translational Ras biology. PMID:26423697

TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice.

PubMed

Oda, Sayaka; Numaga-Tomita, Takuro; Kitajima, Naoyuki; Toyama, Takashi; Harada, Eri; Shimauchi, Tsukasa; Nishimura, Akiyuki; Ishikawa, Tatsuya; Kumagai, Yoshito; Birnbaumer, Lutz; Nishida, Motohiro

2017-08-08

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.

Remission of recurrent gastrointestinal bleeding after septal reduction therapy in patients with hypertrophic obstructive cardiomyopathy-associated acquired von Willebrand syndrome.

PubMed

Blackshear, J L; Stark, M E; Agnew, R C; Moussa, I D; Safford, R E; Shapiro, B P; Waldo, O A; Chen, D

2015-02-01

Gastrointestinal hemorrhage is considered to be a severe complication of von Willebrand disease. The optimal therapy for acquired von Willebrand syndrome and severe gastrointestinal bleeding with hypertrophic cardiomyopathy is undefined. Seventy-seven patients (median age, 67 years; interquartile range [IQR], 56-75 years; 49% women) with hypertrophic cardiomyopathy underwent von Willebrand factor multimer testing and acquisition of bleeding history. Bleeding was detected in 27 (36%) (median age, 74 years; IQR 66-76 years; 74% women), 20 with gastrointestinal bleeding, including 11 women with transfusion dependence. In these 11 women, the median duration of transfusion dependency was 36 months (IQR 18-44 months), and the median number of transfusions required was 25 (IQR 20-38). Two patients had undergone bowel resection for bleeding, one of them twice. Seven patients showed angiodysplasia, and the remainder had no endoscopic lesion. Bleeding recurred after bowel surgery or endoscopic intervention and medical therapy for hypertrophic cardiomyopathy in 10 of 11 patients. Two patients had septal myectomy, and six patients underwent alcohol septal ablation. With the exception of one patient in whom a significant gradient persisted after septal ablation, after the periprocedural period, patients after septal reduction therapy remained free of recurrent bleeding and need for transfusions. Acquired von Willebrand syndrome is common in hypertrophic cardiomyopathy. Gastrointestinal bleeding often recurs after endoscopic therapy, but may be relieved by structural cardiac repair. © 2014 International Society on Thrombosis and Haemostasis.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway. PMID:21595909

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

PubMed

Hauptmann, Valeska; Menzel, Matthias; Weichert, Nicola; Reimers, Kerstin; Spohn, Uwe; Conrad, Udo

2015-02-19

Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

Immunological Prediction of Cytomegalovirus (CMV) Replication Risk in Solid Organ Transplantation Recipients: Approaches for Regulating the Targeted Anti-CMV Prevention Strategies.

PubMed

Han, Sang Hoon

2017-09-01

The current cytomegalovirus (CMV) prevention strategies in solid organ transplantation (SOT) recipients have contributed towards overcoming the detrimental effects caused by CMV lytic infection, and improving the long-term success rate of graft survival. Although the quantification of CMV in peripheral blood is the standard method, and an excellent end-point for diagnosing CMV replication and modulating the anti-CMV prevention strategies in SOT recipients, a novel biomarker mimicking the CMV control mechanism is required. CMV-specific immune monitoring can be employed as a basic tool predicting CMV infection or disease after SOT, since uncontrolled CMV replication mostly originates from the impairment of immune responses against CMV under immunosuppressive conditions in SOT recipients. Several studies conducted during the past few decades have indicated the possibility of measuring the CMV-specific cell-mediated immune response in clinical situations. Among several analytical assays, the most advancing standardized tool is the QuantiFERON®-CMV assay. The T-Track® CMV kit that uses the standardized enzyme-linked immunospot assay is also widely employed. In addition to these assays, immunophenotyping and intracellular cytokine analysis using flow cytometry (with fluorescence-labeled monoclonal antibodies or peptide-major histocompatibility complex multimers) needs to be adequately standardized and validated for potential clinical applications. Copyright © 2017 by The Korean Society of Infectious Diseases and Korean Society for Chemotherapy.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

PubMed

García-Sanz, Nuria; Fernández-Carvajal, Asia; Morenilla-Palao, Cruz; Planells-Cases, Rosa; Fajardo-Sánchez, Emmanuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

2004-06-09

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

Spontaneous CD4+ and CD8+ T-cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients.

PubMed

Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard; Moss, Paul

2017-07-01

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune-privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T-cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg-specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8 + and CD4 + T-cell responses against MAGE-A family antigens were present in 44% (20/45) of patients' samples assayed by ex vivo IFN-γ ELISPOT. The presence of MAGE-specific CD8 + T cells was further determined following short-term in vitro expansion through the use of pMHC-I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE-specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg-specific T-cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T-cell pool following treatment. Spontaneous T-cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering of the function of diamond-like carbon binding peptides through structural design.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Singh, Vivek K; Mikkilä, Joona; Kostiainen, Mauri A; Koskinen, Jari; Linder, Markus B

2015-02-09

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

Wafer scale formation of monocrystalline silicon-based Mie resonators via silicon-on-insulator dewetting.

PubMed

Abbarchi, Marco; Naffouti, Meher; Vial, Benjamin; Benkouider, Abdelmalek; Lermusiaux, Laurent; Favre, Luc; Ronda, Antoine; Bidault, Sébastien; Berbezier, Isabelle; Bonod, Nicolas

2014-11-25

Subwavelength-sized dielectric Mie resonators have recently emerged as a promising photonic platform, as they combine the advantages of dielectric microstructures and metallic nanoparticles supporting surface plasmon polaritons. Here, we report the capabilities of a dewetting-based process, independent of the sample size, to fabricate Si-based resonators over large scales starting from commercial silicon-on-insulator (SOI) substrates. Spontaneous dewetting is shown to allow the production of monocrystalline Mie-resonators that feature two resonant modes in the visible spectrum, as observed in confocal scattering spectroscopy. Homogeneous scattering responses and improved spatial ordering of the Si-based resonators are observed when dewetting is assisted by electron beam lithography. Finally, exploiting different thermal agglomeration regimes, we highlight the versatility of this technique, which, when assisted by focused ion beam nanopatterning, produces monocrystalline nanocrystals with ad hoc size, position, and organization in complex multimers.

Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

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

Kuroyanagi, Kayo; Kang, Min-Sook; Goto, Tsuyoshi

Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-{alpha} and PPAR{gamma}. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPAR{gamma} antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPAR{gamma} activations. The results indicate that auraptene activates PPAR{gamma} in adipocytes to control adipocytekines such as adiponectin and MCP-1 andmore » suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.« less

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Maternal and Cord Blood Adiponectin Multimeric Forms in Gestational Diabetes Mellitus

PubMed Central

Ballesteros, Mónica; Simón, Inmaculada; Vendrell, Joan; Ceperuelo-Mallafré, Victoria; Miralles, Ramon M.; Albaiges, Gerard; Tinahones, Francisco; Megia, Ana

2011-01-01

OBJECTIVE To analyze the relationship between maternal adiponectin (mAdiponectin) and cord blood adiponectin (cbAdiponectin) multimeric forms (high molecular weight [HMW], medium molecular weight [MMW], and low molecular weight [LMW]) in a cohort of gestational diabetes mellitus (GDM) and normal glucose–tolerant (NGT) pregnant women. RESEARCH DESIGN AND METHODS A total of 212 women with a singleton pregnancy, 132 with NGT and 80 with GDM, and their offspring were studied. Maternal blood was obtained in the early third trimester and cord blood was obtained at delivery. Total adiponectin and the multimeric forms of adiponectin were determined in cord blood and maternal serum. Spearman rank correlation and stepwise linear correlation analysis were used to assess the relationship between cbAdiponectin levels and clinical and analytical parameters. RESULTS No differences in cbAdiponectin concentration or its multimeric forms were observed in the offspring of diabetic mothers compared with NGT mothers. The HMW-to-total adiponectin ratio was higher in cord blood than in maternal serum, whereas the MMW- and LMW-to-total adiponectin ratio was lower. Cord blood total and HMW adiponectin levels were positively correlated with birth weight and the ponderal index (PI), whereas cord blood MMW adiponectin was negatively correlated with the PI. In addition, cbAdiponectin and its multimeric forms were correlated with mAdiponectin concentrations. In the multivariate analysis, maternal multimeric forms of adiponectin emerged as independent predictors of cbAdiponectin, its multimers, and their distribution. CONCLUSIONS cbAdiponectin concentrations are independently related to mAdiponectin levels and unrelated to the diagnosis of GDM. Maternal multimeric forms of adiponectin are independent predictors of the concentrations of cbAdiponectin and its multimeric forms at delivery. PMID:21911780

NAD Kinases: Metabolic Targets Controlling Redox Co-enzymes and Reducing Power Partitioning in Plant Stress and Development

PubMed Central

Li, Bin-Bin; Wang, Xiang; Tai, Li; Ma, Tian-Tian; Shalmani, Abdullah; Liu, Wen-Ting; Li, Wen-Qiang; Chen, Kun-Ming

2018-01-01

NAD(H) and NADP(H) are essential co-enzymes which dominantly control a number of fundamental biological processes by acting as reducing power and maintaining the intracellular redox balance of all life kingdoms. As the only enzymes that catalyze NAD(H) and ATP to synthesize NADP(H), NAD Kinases (NADKs) participate in many essential metabolic reactions, redox sensitive regulation, photosynthetic performance and also reactive oxygen species (ROS) homeostasis of cells and therefore, play crucial roles in both development and stress responses of plants. NADKs are highly conserved enzymes in amino acid sequences but have multiple subcellular localization and diverse functions. They may function as monomers, dimers or multimers in cells but the enzymatic properties in plants are not well elucidated yet. The activity of plant NADK is regulated by calcium/calmodulin and plays crucial roles in photosynthesis and redox co-enzyme control. NADK genes are expressed in almost all tissues and developmental stages of plants with specificity for different members. Their transcripts can be greatly stimulated by a number of environmental factors such as pathogenic attack, irritant applications and abiotic stress treatments. Using transgenic approaches, several studies have shown that NADKs are involved in chlorophyll synthesis, photosynthetic efficiency, oxidative stress protection, hormone metabolism and signaling regulation, and therefore contribute to the growth regulation and stress tolerance of plants. In this review, the enzymatic properties and functional mechanisms of plant NADKs are thoroughly investigated based on literature and databases. The results obtained here are greatly advantageous for further exploration of NADK function in plants. PMID:29662499

Technological advances in diagnostic testing for von Willebrand disease: new approaches and challenges.

PubMed

Hayward, C P M; Moffat, K A; Graf, L

2014-06-01

Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays. © 2014 John Wiley & Sons Ltd.

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA

PubMed Central

Fusco, William G.; Choudhary, Neelima R.; Stewart, Shelley M.; Alam, S. Munir; Sempowski, Gregory D.; Elkins, Christopher

2015-01-01

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrAI) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine. PMID:25897604

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

PubMed

Fusco, William G; Choudhary, Neelima R; Stewart, Shelley M; Alam, S Munir; Sempowski, Gregory D; Elkins, Christopher; Leduc, Isabelle

2015-04-01

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13

PubMed Central

Bonnefoy, Arnaud; Daenens, Kim; Feys, Hendrik B.; De Vos, Rita; Vandervoort, Petra; Vermylen, Jos; Lawler, Jack; Hoylaerts, Marc F.

2006-01-01

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 ± 377 seconds) and arterioles (858 ± 289 seconds) than in WT vessels (559 ± 241 seconds, P < .001; 443 ± 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively. PMID:16204318

[Variety of thrombotic thrombocytopenic purpura clinical course in Polish family members with ADAMTS 13 gene mutation].

PubMed

Hyla-Klekot, Lidia; Kucharska, Grazyna; Słonka, Karina

2013-03-01

The congenital form of thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrom) is a result of genetically conditioned dysfunction of protease ADAMTS 13 enzyme which is responsible for von Wiellebrand factor multimer disintegration. The disease is inherited autosomally and recessively. The decrease of ADAMTS 13 activity results in intravascular clotting process activation with rapid lowering of platelet count, haemolytic anaemia, and occurence of schistocytes. Clinically, the disease is characterized by a range of symptoms such as severe jaundice in neonatal period, embolicthrombotic incidents of nervous system and progressive dysfunction of kidneys and other organs. Delaying diagnosis and hence administering of freshly frozen plasma leads to death. Molecular diagnosis allows for identification of genetical profile of the patient, and showing lowered enzyme activity is a basis for regular prophylactic plasma administration which is the protease donor. In our study we present members of a Polish family identified with ADAMTS 13 mutation. 52 old male with heterozygotic mutation of exon 29 (4143_4144insA) and in exon 19 (c2281G>A; Gly761Ser), experienced a few episodes of ischaemic stroke with ongoing neurological deficiency and developed chronic kidney disease. His 16-year old daughter with double homozygotic mutation in exon 29 (4143_4144insA) after severe episode of TTP at the age of 4 has been receiving plasma every 2 weeks for 12 years, which prevented her from other disorders. Target treatment introduced to clinical practice by means of ADAMTS 13 obtained by genetic recombination technology raises hopes.

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

PubMed

Ma, Xianyue; Cline, Kenneth

2013-03-01

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

PubMed Central

Thiel, U; Pirson, S; Müller-Spahn, C; Conrad, H; Busch, D H; Bernhard, H; Burdach, S; Richter, G H S

2011-01-01

Background: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8+ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. Methods: Following repetitive CHM1319 (VIMPCSWWV) and EZH2666 (YMCSFLFNL) peptide-driven stimulations with HLA-A*0201+ dendritic cells (DC), allo-restricted HLA-A*0201− CD8+ T cells were stained with HLA-A*0201/peptide multimers, sorted and expanded by limiting dilution. Results: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A*0201 and killed HLA-A*0201+ ET lines expressing the antigen while HLA-A*0201– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2−/−γC−/− mice. Within this context, we identified the CHM1319 peptide as a new candidate target antigen for ET immunotherapy. Conclusion: These results clearly identify the ET-derived antigens, EZH2666 and CHM1319, as suitable targets for protective allo-restricted human CD8+ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation. PMID:21407224

Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface

NASA Astrophysics Data System (ADS)

Lu, Xiaolong; Shi, Ruixin; Hao, Changchun; Chen, Huan; Zhang, Lei; Li, Junhua; Xu, Guoqing; Sun, Runguang

2016-09-01

The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics. The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine (DPPE) by analyzing the data of surface pressure-area (π-A) isotherms and surface pressure-time (π-T) curves. Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 mN/m. However, the changes of DPPE are larger than DPPC, indicating stronger interaction of lysozyme with DPPE than DPPC. The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 mN/m. Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers, which leads to self-aggregation and self-assembly, forming irregular multimers and conical multimeric. Through analysis, we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central Universities of China (Grant No. GK201603026), and the National University Science and Technology Innovation Project of China (Grant No. 201610718013).

Efficient creation of dipolar coupled nitrogen-vacancy spin qubits in diamond

NASA Astrophysics Data System (ADS)

Jakobi, I.; Momenzadeh, S. A.; Fávaro de Oliveira, F.; Michl, J.; Ziem, F.; Schreck, M.; Neumann, P.; Denisenko, A.; Wrachtrup, J.

2016-09-01

Coherently coupled pairs or multimers of nitrogen-vacancy defect electron spins in diamond have many promising applications especially in quantum information processing (QIP) but also in nanoscale sensing applications. Scalable registers of spin qubits are essential to the progress of QIP. Ion implantation is the only known technique able to produce defect pairs close enough to allow spin coupling via dipolar interaction. Although several competing methods have been proposed to increase the resulting resolution of ion implantation, the reliable creation of working registers is still to be demonstrated. The current limitation are residual radiation-induced defects, resulting in degraded qubit performance as trade-off for positioning accuracy. Here we present an optimized estimation of nanomask implantation parameters that are most likely to produce interacting qubits under standard conditions. We apply our findings to a well-established technique, namely masks written in electron-beam lithography, to create coupled defect pairs with a reasonable probability. Furthermore, we investigate the scaling behavior and necessary improvements to efficiently engineer interacting spin architectures.

IgX antibodies in the urodele amphibian Ambystoma mexicanum.

PubMed

Schaerlinger, Bérénice; Frippiat, Jean-Pol

2008-01-01

Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.

An RNA-Binding Multimer Specifies Nematode Sperm Fate.

PubMed

Aoki, Scott T; Porter, Douglas F; Prasad, Aman; Wickens, Marvin; Bingman, Craig A; Kimble, Judith

2018-06-26

FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

PubMed Central

Ploier, Birgit; Caro, Lydia N.; Morizumi, Takefumi; Pandey, Kalpana; Pearring, Jillian N.; Goren, Michael A.; Finnemann, Silvia C.; Graumann, Johannes; Arshavsky, Vadim Y.; Dittman, Jeremy S.; Ernst, Oliver P.; Menon, Anant K.

2016-01-01

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5. PMID:27694816

Innate Immunity in Lobsters: Partial Purification and Characterization of a Panulirus cygnus Anti-A Lectin.

PubMed

Flower, Robert L P

2012-01-01

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was a multimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125 mmol/L sucrose: 500 mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates.

Tailoring galvanic replacement reaction for the preparation of Pt/Ag bimetallic hollow nanostructures with controlled number of voids.

PubMed

Zhang, Weiqing; Yang, Jizheng; Lu, Xianmao

2012-08-28

Here we report the synthesis of Pt/Ag bimetallic nanostructures with controlled number of void spaces via a tailored galvanic replacement reaction (GRR). Ag nanocubes (NCs) were employed as the template to react with Pt ions in the presence of HCl. The use of HCl in the GRR caused rapid precipitation of AgCl, which grew on the surface of Ag NCs and acted as a removable secondary template for the deposition of Pt. The number of nucleation sites for AgCl was tailored by controlling the amount of HCl added to the Ag NCs or by introducing PVP to the reaction. This strategy led to the formation of Pt/Ag hollow nanoboxes, dimers, multimers, or popcorn-shaped nanostructures consisting of one, two, or multiple hollow domains. Due to the presence of large void space and porous walls, these nanostructures exhibited high surface area and improved catalytic activity for methanol oxidation reaction.

Protein Attachment on Nanodiamonds.

PubMed

Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

2015-07-16

A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging.

PubMed

Zhang, Liang; Thurber, Greg M

2016-02-01

Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type 1 diabetes. The glucagon-like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower-clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, down-regulation of the GLP-1 receptor and non-specific background uptake result in a higher target-to-background ratio for fast-clearing agents.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

PubMed Central

Zhang, Liang; Thurber, Greg M.

2016-01-01

Purpose Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type-1 diabetes. The glucagon like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Procedures Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Results Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Conclusions Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, downregulation of the GLP-1 receptor and non-specific background uptake result in a higher TBR for fast-clearing agents. PMID:26194012

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

Gewirth, Andrew A.; Kenis, Paul J.; Nuzzo, Ralph G.

In this research, we prosecuted a comprehensive plan of research directed at developing new catalysts and new understandings relevant to the operation of low temperature hydrogen-oxygen fuel cells. The focal point of this work was one centered on the Oxygen Reduction Reaction (ORR), the electrochemical process that most fundamentally limits the technological utility of these environmentally benign energy conversion devices. Over the period of grant support, we developed new ORR catalysts, based on Cu dimers and multimers. In this area, we developed substantial new insight into design rules required to establish better ORR materials, inspired by the three-Cu active sitemore » in laccase which has the highest ORR onset potential of any material known. We also developed new methods of characterization for the ORR on conventional (metal-based) catalysts. Finally, we developed a new platform to study the rate of proton transfer relevant to proton coupled electron transfer (PCET) reactions, of which the ORR is an exemplar. Other aspects of work involved theory and prototype catalyst testing.« less

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

NASA Astrophysics Data System (ADS)

Ploier, Birgit; Caro, Lydia N.; Morizumi, Takefumi; Pandey, Kalpana; Pearring, Jillian N.; Goren, Michael A.; Finnemann, Silvia C.; Graumann, Johannes; Arshavsky, Vadim Y.; Dittman, Jeremy S.; Ernst, Oliver P.; Menon, Anant K.

2016-10-01

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations--F45L, V209M and F220C--yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

Correlation Between Chain Architecture and Hydration Water Structure in Polysaccharides

NASA Astrophysics Data System (ADS)

Grossutti, Michael; Dutcher, John

The physical properties of confined water can differ dramatically from those of bulk water. Hydration water associated with polysaccharides provides a particularly important example of confined water, with differences in polysaccharide structure providing different spatially confined environments for water adsorption. We have used attenuated total reflection infrared (ATR-IR) spectroscopy to investigate the structure of hydration water in films of three different polysaccharides under controlled relative humidity (RH) conditions. We compare the results obtained for films of highly branched, monodisperse phytoglycogen nanoparticles to those obtained for two unbranched polysaccharides, hyaluronic acid (HA) and chitosan. We find similarities between water structuring in the two linear polysaccharides, and significant differences for phytoglycogen. In particular, the phytoglycogen nanoparticles exhibited high network water connectivity, and a large increase in the fraction of multimer water clusters with increasing RH, whereas the water structure for HA and chitosan was found to be insensitive to changes in RH. These measurements provide unique insight into the relationship between the chain architecture and hydration of polysaccharides.

On the structure and functions of gelatinase B/matrix metalloproteinase-9 in neuroinflammation.

PubMed

Vandooren, Jennifer; Van Damme, Jo; Opdenakker, Ghislain

2014-01-01

The blood-brain barrier (BBB) is a specific structure that is composed of two basement membranes (BMs) and that contributes to the control of neuroinflammation. As long as the BBB is intact, extravasated leukocytes may accumulate between two BMs, generating vascular cuffs. Specific matrix metalloproteinases, MMP-2 and MMP-9, have been shown to cleave BBB beta-dystroglycan and to disintegrate thereby the parenchymal BM, resulting in encephalomyelitis. This knowledge has been added to the molecular basis of the REGA model to understand the pathogenesis of multiple sclerosis, and it gives further ground for the use of MMP inhibitors for the treatment of acute neuroinflammation. MMP-9 is associated with central nervous system inflammation and occurs in various forms: monomers and multimers. None of the various neurological and neuropathologic functions of MMP-9 have been associated with either molecular structure or molecular form, and therefore, in-depth structure-function studies are needed before medical intervention with MMP-9-specific inhibitors is initiated.

PML/RARA oxidation and arsenic binding initiate the antileukemia response of As2O3.

PubMed

Jeanne, Marion; Lallemand-Breitenbach, Valérie; Ferhi, Omar; Koken, Marcel; Le Bras, Morgane; Duffort, Stéphanie; Peres, Laurent; Berthier, Caroline; Soilihi, Hassane; Raught, Brian; de Thé, Hugues

2010-07-13

As(2)O(3) cures acute promyelocytic leukemia (APL) by initiating PML/RARA oncoprotein degradation, through sumoylation of its PML moiety. However, how As(2)O(3) initiates PML sumoylation has remained largely unexplained. As(2)O(3) binds vicinal cysteines and increases reactive oxygen species (ROS) production. We demonstrate that upon As(2)O(3) exposure, PML undergoes ROS-initiated intermolecular disulfide formation and binds arsenic directly. Disulfide-linked PML or PML/RARA multimers form nuclear matrix-associated nuclear bodies (NBs), become sumoylated and are degraded. Hematopoietic progenitors transformed by an As(2)O(3)-binding PML/RARA mutant exhibit defective As(2)O(3) response. Conversely, nonarsenical oxidants elicit PML/RARA multimerization, NB-association, degradation, and leukemia response in vivo, but do not affect PLZF/RARA-driven APLs. Thus, PML oxidation regulates NB-biogenesis, while oxidation-enforced PML/RARA multimerization and direct arsenic-binding cooperate to enforce APL's exquisite As(2)O(3) sensitivity. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Mechanical circulatory support is associated with loss of platelet receptors glycoprotein Ibα and glycoprotein VI.

PubMed

Lukito, P; Wong, A; Jing, J; Arthur, J F; Marasco, S F; Murphy, D A; Bergin, P J; Shaw, J A; Collecutt, M; Andrews, R K; Gardiner, E E; Davis, A K

2016-11-01

Essentials Relationship of acquired von Willebrand disease (VWD) and platelet dysfunction is explored. Patients with ventricular assist devices and on extracorporeal membrane oxygenation are investigated. Acquired VWD and platelet receptor shedding is demonstrated in the majority of patients. Loss of platelet adhesion receptors glycoprotein (GP) Ibα and GPVI may increase bleeding risk. Background Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. Objectives To investigate whether loss of platelet receptors occurs in vivo, and the relationship with acquired von Willebrand syndrome (AVWS). Methods Platelet counts, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 21 continuous flow VAD (CF-VAD), 20 ECMO, 12 heart failure and seven aortic stenosis patients. Levels of platelet receptors were measured by flow cytometry or ELISA. Results The loss of high molecular weight VWF multimers was observed in 18 of 19 CF-VAD and 14 of 20 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Conclusions These data link AVWS and increased platelet receptor shedding in patients with CF-VADs or ECMO for the first time. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CF-VAD and ECMO patients may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions. © 2016 International Society on Thrombosis and Haemostasis.

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

PubMed Central

Lieberman, Joshua A.; Frost, Nicholas A.; Hoppert, Michael; Fernandes, Paula J.; Vogt, Stefanie L.; Raivio, Tracy L.; Blanpied, Thomas A.

2012-01-01

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis. PMID:22247509

Oral administration of γ-aminobutyric acid and γ-oryzanol prevents stress-induced hypoadiponectinemia.

PubMed

Ohara, Kazuyuki; Kiyotani, Yuka; Uchida, Asako; Nagasaka, Reiko; Maehara, Hiroyuki; Kanemoto, Shigeharu; Hori, Masatoshi; Ushio, Hideki

2011-06-15

Metabolic syndrome is a cluster of risk factors including insulin resistance and type 2 diabetes and is found to associate partly with chronic stress at work in human. Adiponectin circulates in mammal blood mainly as a low molecular weight (LMW) trimer, hexamer, and a high molecular weight (HMW) multimers. Low circulating levels of adiponectin are related to metabolic syndrome. We have then investigated the influence of immobilization stress on plasma adiponectin concentrations in mice. Relative LMW and HMW adiponectin levels were markedly reduced by immobilization stress (0.66±0.07 and 0.59±0.06 after 102 h, respectively), significantly different from the control values (p<0.01 and 0.05, respectively). γ-Aminobutyric acid (GABA) and γ-oryzanol abundantly contained in germinated brown rice have some physiological functions. We further investigated the effect of GABA, γ-oryzanol, GABA plus γ-oryzanol on adiponectin levels in mice subjected to immobilization stress. GABA and γ-oryzanol significantly increased the relative LMW and HMW adiponectin levels under immobilization stress (1.10±0.11 and 0.99±0.19 after 102 h, respectively, for GABA; 1.08±0.17 and 1.15±0.17 after 102 h, respectively, for γ-oryzanol). Additionally, the co-administration of GABA and γ-oryzanol also increased both relative LMW and HMW adiponectin levels (1.02±0.07 and 0.99±0.10 after 102 h, respectively) and was effective in an earlier phase from 30 to 54 h. The results indicate that the co-administration of GABA and γ-oryzanol might be effective in preventing stress-induced hypoadiponectinemia in mice and be also a promising tool for improving metabolic syndrome aggravated by chronic stress. Copyright © 2011 Elsevier GmbH. All rights reserved.

Families of short interspersed elements in the genome of the oomycete plant pathogen, Phytophthora infestans.

PubMed

Whisson, Stephen C; Avrova, Anna O; Lavrova, Olga; Pritchard, Leighton

2005-04-01

The first known families of tRNA-related short interspersed elements (SINEs) in the oomycetes were identified by exploiting the genomic DNA sequence resources for the potato late blight pathogen, Phytophthora infestans. Fifteen families of tRNA-related SINEs, as well as predicted tRNAs, and other possible RNA polymerase III-transcribed sequences were identified. The size of individual elements ranges from 101 to 392 bp, representing sequences present from low (1) to highly abundant (over 2000) copy number in the P. infestans genome, based on quantitative PCR analysis. Putative short direct repeat sequences (6-14 bp) flanking the elements were also identified for eight of the SINEs. Predicted SINEs were named in a series prefixed infSINE (for infestans-SINE). Two SINEs were apparently present as multimers of tRNA-related units; four copies of a related unit for infSINEr, and two unrelated units for infSINEz. Two SINEs, infSINEh and infSINEi, were typically located within 400 bp of each other. These were also the only two elements identified as being actively transcribed in the mycelial stage of P. infestans by RT-PCR. It is possible that infSINEh and infSINEi represent active retrotransposons in P. infestans. Based on the quantitative PCR estimates of copy number for all of the elements identified, tRNA-related SINEs were estimated to comprise 0.3% of the 250 Mb P. infestans genome. InfSINE-related sequences were found to occur in species throughout the genus Phytophthora. However, seven elements were shown to be exclusive to P. infestans.

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.

PubMed

Kim, Chang-Yub; Webster, Cecelia; Roberts, Justin K M; Moon, Jin Ho; Alipio Lyon, Emily Z; Kim, Heungbok; Yu, Minmin; Hung, Li-Wei; Terwilliger, Thomas C

2009-12-01

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.

Characterization and two-dimensional crystallization of membrane component AlkB of the medium-chain alkane hydroxylase system from Pseudomonas putida GPo1.

PubMed

Alonso, Hernan; Roujeinikova, Anna

2012-11-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C(12)E(8)]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-D-maltopyranoside (DM), n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.

Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

PubMed

Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

1996-02-15

Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U).

Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

PubMed Central

Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

1996-01-01

Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U). PMID:8604302

Preliminary Work in Obtaining Site-Directed Mutants of Hen Egg White Lysozyme

NASA Technical Reports Server (NTRS)

Holmes, Leonard D.

1996-01-01

Protein crystal growth studies are recognized as a critical endeavor in the field of molecular biotechnology. The scientific applications of this field include the understanding of how enzymes function and the accumulation of accurate information of atomic structures, a key factor in the process of rational drug design. NASA has committed substantial investment and resources to the field of protein crystal growth and has conducted many microgravity protein crystal growth experiments aboard shuttle flights. Crystals grown in space tend to be larger, denser and have a more perfect habit and geometry. These improved properties gained in the microgravity environment of space result largely from the reduction of solutal convection, and the elimination of sedimentation at the growing crystal surface. Shuttle experiments have yielded many large, high quality crystals that are suitable for high resolution X-ray diffraction analysis. Examples of biologically important macromolecules which have been successfully crystallized during shuttle missions include: lysozyme, isocitrate lyase, gamma-interferon, insulin, human serum albumin and canavalin. Numerous other examples are also available. In addition to obtaining high quality crystals, investigators are also interested in learning the mechanisms by which the growth events take place. Crystallization experiments indicate that for the enzyme HEWL, measured growth rates do not follow mathematical models for 2D nucleation and dislocation-led growth of tetragonal protein crystals. As has been suggested by the laboratory of Marc L. Pusey, a possible explanation for the disagreement between observation and data is that HEWL tetraconal crystals form by aggregated units of lysozyme in supersaturated solutions. Surface measurement data was shown to fit very well with a model using an octamer unit cell as the growth unit. According to this model, the aggregation pathway and subsequent crystal growth is described by: monomer < ------ > dimer < ------- > tetramer < ------ > octamer < ------ > higher order. It is believed that multimer aggregation of lysozyme occurs by interaction at specific binding sites on the surface of the protein crystals. If the presence of discrete binding sites and the aggregation hypothesis is true, then it follows that the alteration of the binding site(s) should have significant effect on the measurements obtained during growth experiments. Site-directed mutagenesis allows the specific alteration of proteins by replacement, deletion or addition of specific amino acid residues. This report outlines the approach for this strategy and the progress made thus far toward that end.

RNA Nanotechnology: Engineering, Assembly and Applications in Detection, Gene Delivery and Therapy

PubMed Central

Guo, Peixuan

2010-01-01

Biological macromolecules including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA molecules can be designed and manipulated with a level of simplicity characteristic of DNA while possessing versatility in structure and function similar to that of proteins. RNA molecules typically contain a large variety of single stranded loops suitable for inter- and intra-molecular interaction. These loops can serve as mounting dovetails obviating the need for external linking dowels in fabrication and assembly. The self-assembly of nanoparticles from RNA involves cooperative interaction of individual RNA molecules that spontaneously assemble in a predefined manner to form a larger two- or three-dimensional structure. Within the realm of self-assembly there are two main categories, namely template and non-template. Template assembly involves interaction of RNA molecules under the influence of specific external sequence, forces, or spatial constraints such as RNA transcription, hybridization, replication, annealing, molding, or replicas. In contrast, non-template assembly involves formation of a larger structure by individual components without the influence of external forces. Examples of non-template assembly are ligation, chemical conjugation, covalent linkage, and loop/loop interaction of RNA, especially the formation of RNA multimeric complexes. The best characterized RNA multiplier and the first to be described in RNA nanotechnological application is the motor pRNA of bacteriophage phi29 which form dimers, trimers, and hexamers, via hand-in-hand interaction. phi29 pRNA can be redesigned to form a variety of structures and shapes including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. 3D RNA array formation requires a defined nucleotide number for twisting and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH. Both the therapeutic siRNA or ribozyme and a receptor-binding RNA aptamer or other ligands have been engineered into individual pRNAs. Individual chimeric RNA building blocks harboring siRNA or other therapeutic molecules have been fabricated subsequently into a trimer through hand-in-hand interaction of the engineered right and left interlocking RNA loops. The incubation of these particles containing the receptor-binding aptamer or other ligands results in the binding and co-entry of trivalent therapeutic particles into cells. Such particles were subsequently shown to modulate the apoptosis of cancer cells in both cell cultures and animal trials. The use of such antigen-free 20–40 nm particles holds promise for the repeated long-term treatment of chronic diseases. Other potentially useful RNA molecules that form multimers include HIV RNA that contain kissing loop to form dimers, tecto-RNA that forms a “jigsaw puzzle,” and the Drosophila bicoid mRNA that forms multimers via “hand-by-arm” interactions. Applications of RNA molecules involving replication, molding, embossing, and other related techniques, have recently been described that allow the utilization of a variety of materials to enhance diversity and resolution of nanomaterials. It should eventually be possible to adapt RNA to facilitate construction of ordered, patterned, or pre-programmed arrays or superstructures. Given the potential for 3D fabrication, the chance to produce reversible self-assembly, and the ability of self-repair, editing and replication, RNA self-assembly will play an increasingly significant role in integrated biological nanofabrication. A random 100-nucleotide RNA library may exist in 1.6 × 1060 varieties with multifarious structure to serve as a vital system for efficient fabrication, with a complexity and diversity far exceeding that of any current nanoscale system. This review covers the basic concepts of RNA structure and function, certain methods for the study of RNA structure, the approaches for engineering or fabricating RNA into nanoparticles or arrays, and special features of RNA molecules that form multimers. The most recent development in exploration of RNA nanoparticles for pathogen detection, drug/gene delivery, and therapeutic application is also introduced in this review. PMID:16430131

Relationship between ADAMTS13 activity, von Willebrand factor antigen levels and platelet function in the early and late phases after TIA or ischaemic stroke.

PubMed

McCabe, Dominick J H; Murphy, Stephen J X; Starke, Richard; Harrison, Paul; Brown, Martin M; Sidhu, Paul S; Mackie, Ian J; Scully, Marie; Machin, Samuel J

2015-01-15

Reduced ADAMTS13 activity is seen in thrombotic thrombocytopenic purpura (TTP), and may lead to accumulation of prothrombotic ultra-large von Willebrand factor (ULVWF) multimers in vivo. ADAMTS13 activity and its relationship with VWF antigen (VWF:Ag) levels and platelet function in 'non-TTP related' TIA or ischaemic stroke has not been comprehensively studied. In this prospective pilot observational analytical case-control study, ADAMTS13 activity and VWF:Ag levels were quantified in platelet poor plasma in 53 patients in the early phase (≤ 4 weeks) and 34 of these patients in the late phase (≥ 3 months) after TIA or ischaemic stroke on aspirin. Data were compared with those from 22 controls not on aspirin. The impact of ADAMTS13 on platelet function in whole blood was quantified by measuring Collagen-ADP (C-ADP) and Collagen-Epinephrine closure times on a platelet function analyser (PFA-100(®)). Median ADAMTS13 activity was significantly reduced in the early phase (71.96% vs. 95.5%, P <0.01) but not in the late phase after TIA or stroke compared with controls (86.3% vs. 95.5%, P=0.19). There was a significant inverse relationship between ADAMTS13 activity and VWF:Ag levels in the early phase (r=-0.31; P=0.024), but not in the late phase after TIA or stroke (P=0.74). There was a positive correlation between ADAMTS13 activity and C-ADP closure times in early phase patients only, likely mediated via VWF:Ag levels. ADAMTS13 activity is reduced and VWF:Ag expression is increased within 4 weeks of TIA or ischaemic stroke onset, and can promote enhanced platelet adhesion and aggregation in response to stimulation with collagen and ADP via VWF-mediated pathways. These data improve our understanding of the dynamic haemostatic and thrombotic profiles of ischaemic cerebrovascular disease (CVD) patients, and are important in view of the potential future role that ADAMTS13 may have to play as an anti-thrombotic agent in CVD. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced plastic deformations of nanofibrillated cellulose film by adsorbed moisture and protein-mediated interactions.

PubMed

Malho, Jani-Markus; Ouellet-Plamondon, Claudiane; Rüggeberg, Markus; Laaksonen, Päivi; Ikkala, Olli; Burgert, Ingo; Linder, Markus B

2015-01-12

Biological composites are typically based on an adhesive matrix that interlocks rigid reinforcing elements in fiber composite or brick-and-mortar assemblies. In nature, the adhesive matrix is often made up of proteins, which are also interesting model systems, as they are unique among polymers in that we know how to engineer their structures with atomic detail and to select protein elements for specific interactions with other components. Here we studied how fusion proteins that consist of cellulose binding proteins linked to proteins that show a natural tendency to form multimer complexes act as an adhesive matrix in combination with nanofibrillated cellulose. We found that the fusion proteins are retained with the cellulose and that the proteins mainly affect the plastic yield behavior of the cellulose material as a function of water content. Interestingly, the proteins increased the moisture absorption of the composite, but the well-known plastifying effect of water was clearly decreased. The work helps to understand the functional basis of nanocellulose composites as materials and aims toward building model systems for molecular biomimetic materials.

Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae.

PubMed

Liu, Lifang; Martínez, José L; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

2014-01-01

Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (α2β2). In this work we evaluated the expression of different combinations of α and β peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group. © 2013 Published by International Metabolic Engineering Society on behalf of International Metabolic Engineering Society.

Na+ influx via Orai1 inhibits intracellular ATP-induced mTORC2 signaling to disrupt CD4 T cell gene expression and differentiation.

PubMed

Miao, Yong; Bhushan, Jaya; Dani, Adish; Vig, Monika

2017-05-11

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napa hyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napa hyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP] i . Depletion of [ATP] i inhibited mTORC2 dependent NFκB activation in Napa hyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napa hyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function.

Peculiarities of RFLP of highly repetitive DNA in crow genomes

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

Chelomina, G.N.; Kryukov, A.P.; Ivanov, S.V.

1995-02-01

We present a study of the structural organization of highly repetitive DNA in genomes of hooded crow Corvus cornix L., carrion crow C. corone L., and jungle crow C. macrorhynchos Wagl. RFLP and blot-hybridization with {sup 32}P-labeled Msp I fragment from hooded crow nDNA suggest the interspecific structural conservatism of the most repetitive DNA. The family of repeats we studied had tandem organization and the same (210 bp) period of reiteration for a set of restriction enzymes. However, in parallel to the general similarity of restriction patterns, there are species-specific peculiarities. The repetitive family revealed (Alu I, BsuR I, andmore » Msp I fragments) has quantitative RFLP of nDNA and interspecific differences in the extent of the multimer {open_quotes}ladder{close_quotes} pattern of Msp I fragments. The latter is more pronounced in nDNA of carrion crow than in that of phylogenetically distant jungle crow and closely related hooded crow. This suggests a recent amplification event for highly organized homological repeats in crow genomes. 10 refs., 2 figs.« less

Oxidative stress/damage induces multimerization and interaction of Fanconi anemia proteins.

PubMed

Park, Su-Jung; Ciccone, Samantha L M; Beck, Brian D; Hwang, Byounghoon; Freie, Brian; Clapp, D Wade; Lee, Suk-Hee

2004-07-16

Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function.

Novel Approach to Prepare {sup 99m}Tc-Based Multivalent RGD Peptides

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

Shuang Liu

2012-10-24

This project presents a novel approach to prepare the {sup 99m}Tc-bridged multivalent RGD (arginine-glycine-aspartate) peptides. This project will focus on fundamentals of {sup 99m}Tc radiochemistry. The main objective of this project is to demonstrate the proof-of-principle for the proposed radiotracers. Once a kit formulation is developed for preparation of the {sup 99m}Tc-bridged multivalent RGD peptides, various tumor-bearing animal models will be used to evaluate their potential for SPECT (single photon-emission computed tomography) imaging of cancer. We have demonstrated that (1) multimerization of cyclic RGD peptides enhances the integrin {alpha}{sub v}{beta}{sub 3} bonding affinity and radiotracer tumor uptake; (2) addition ofmore » G{sub 3} or PEG{sub 4} linkers makes it possible for two RGD motifs in 3P-RGD{sub 2} and 3G-RGD{sub 2} to achieve simultaneous integrin {alpha}{sub v}{beta}{sub 3} binding; and (3) multimers are actually bivalent (not multivalent), the presence of extra RGD motifs can enhance the tumor retention time of the radiotracer.« less

Spectroscopic properties of reaction center pigments in photosystem II core complexes: revision of the multimer model.

PubMed

Raszewski, Grzegorz; Diner, Bruce A; Schlodder, Eberhard; Renger, Thomas

2008-07-01

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., P(+)Pheo(-),P(+)Q(A)(-),(3)P) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His(198), the axial ligand of the special-pair chlorophyll P(D1), and D1-Thr(179), an amino-acid residue nearest to the accessory chlorophyll Chl(D1), on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl(D1). Compared to isolated reaction centers, the site energy of Chl(D1) is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl(D1) rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by approximately 10 nm than the low-energy exciton state of the two special-pair chlorophylls P(D1) and P(D2) which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl(D1) and P(D1), reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are considerable contributions from all pigments to the equilibrated excited state P*. The contribution of Chl(D1) is twice that of P(D1) at ambient temperature, making it likely that the primary charge separation will be initiated by Chl(D1) under these conditions. The calculations of absorbance difference spectra provide independent evidence that after primary electron transfer the hole stabilizes at P(D1), and that the physiologically dangerous charge recombination triplets, which may form under light stress, equilibrate between Chl(D1) and P(D1).

Aqueous Silicate Polymers: An Alternative to `Supercritical' Fluids as Transport Agents in Subduction Zones

NASA Astrophysics Data System (ADS)

Mannig, C. E.

2005-12-01

The chemistry of subduction-zone fluids is complicated by melt-vapor miscibility and the existence of critical end-points in rock-H2O systems. It is commonly assumed that fluids in subduction zones attain properties intermediate in composition between hydrous silicate liquid and H2O, and that such fluids possess enhanced material transport capabilities. However, the relevance of supercritical, intermediate fluids to subduction zones presents four problems. (1) Albite-H2O is typically used as an analogue system, but the favorable position of its critical curve is not representative; critical curves for polymineralic subduction-zone lithologies lie at substantially higher P. (2) Even if albite-H2O is relevant, jadeite may interfere because of its different solubility and the positive clapeyron slope of its solidus, which points to liquid-structure changes that could cause reappearance of the liquid+vapor field. (3) Critical curves are features of very H2O-rich compositions; low-porosity, H2O-poor natural systems will coexist with intermediate fluids only over a narrow PT interval. (4) Intermediate fluids are expected only over short length scales because their migration will likely result in compositional shifts via reaction and mineral precipitation in the mantle wedge. Although supercritical, intermediate fluids are probably relatively unimportant in subduction zones, they reflect a chemical process that may hold the key to understanding high- P mass transfer. Miscibility in melt-vapor systems is a consequence of polymerization of dissolved components, primarily Si ± Al, Na and Ca. This behavior yields, e.g., aqueous Si-Si, Si-Al, Si-Na-Al, and Si-Ca oxide dimers and other multimers of varying stoichiometry (silicate polymers), even in subcritical, dilute, H2O-rich vapor. Silicate polymers in subcritical aqueous solutions have been inferred from high- P mineral-solubility experiments. The abundance of these species at high P shows that the chemistry of aqueous fluids in subduction-zones differs fundamentally from the more familiar ionic solutions of the upper crust. This has important consequences for minor element transport. Measurements of Fe, phosphorous and Ti solubility reveal that dissolved concentrations rise with increased aqueous albite content at fixed P and T, with maximum enhancements exceeding 10X at melt saturation. Subcritical silicate polymerization thus permits transport of low solubility components via their substitution into sites on aqueous multimers constructed of "polymer formers" such as Na, Al, and Si, even in dilute solutions. The partitioning of elements between the bulk fluid, the polymer network, and the rock matrix likely controls the overall compositional evolution of subduction-zone fluids. Because they form over a wider PT and bulk X range, subcritical silicate polymers in dilute solutions are likely responsible for more mass transfer in subduction zones than intermediate, supercritical fluids.

Copper speciation in sulfidic solutions at low sulfur activity: Further evidence for cluster complexes?

NASA Astrophysics Data System (ADS)

Thompson, Richard A.; Helz, George R.

1994-07-01

The solubility of two as0-buffering assemblages in the Cu-S system have been studied: chalcocite-djurleite (Cc-Dj) and anilite-covellite (An-Cv). Ion activity products, [Cu +]HS -] 1/2[H +] - 1/2 (25°C, I = 0) at equilibrium, derived from solubility measurements in penicillamine solutions, are 10 -17.01 ± 0.05 (Cc-Dj) and 10 -17.14 ± 0.10 (An-Cv), from which ΔG° f = -82.11 kJ/mol for Cc and -74.77 kJ/mol for An. In the An-Cv assemblage, aCu2S = 0.55 (2 σ = 0.2) vs. 1.00 in the Cc-containing assemblage. The difference in aCu2S between the two assemblages is used in a novel way to estimate stoichiometry of Cu-HS complexes. The solubility of both assemblages (0.7-0.01 M NaHS, pH 7-12.5, 25°C) can be fit with a model incorporating the same two chemical species, one containing an odd number of Cu atoms (Cu(HS) 2-3, CU 3S 4H 2-3, or a higher multimer) and the other containing an even number of Cu atoms (Cu 2S(HS) 22-, Cu 4S 4H 22-, etc.). The trimer-tetramer model fits the combined data for the two assemblages distinctly better than the monomer-dimer model, but this result is very sensitive to uncertainty in aCu2S. Along with EXAFS results, the weight of the evidence favors small cluster complexes (2-5 Cu atoms), but is inconclusive at the present level of resolution. Multimers can be rationalized because condensation of metal-centered monomers to clusters provides a means for soft acid/base elements to maintain favored coordination geometries at low ligand to metal ratios. Based on the fitting methods developed here, previous covellite solubility data from this laboratory are reinterpreted in terms of Cu 2S 2(HS) 33-, Cu 2S 3)(S 4) 2-, and Cu(S 9)S 10) 3-; the last of these could also be represented by the trimer, Cu 3(S 7) 33-, which is homologous with a known complex. With the measured equilibrium constants, the speciation of Cu in the sulfidic zone of the Black Sea is calculated. Covellite is the stable Cu-S mineral, but the sulfidic water column is vastly supersaturated with respect to it. Most of the sulfidic water column is modestly (2.5-5.5 times) supersaturated with respect to Cc, hinting that this mineral metastably controls ΣCu. The slight supersaturation suggests that Cc occurs as 10-100 nm particles.

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

PubMed Central

Alonso, Hernan

2012-01-01

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C12E8]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-d-maltopyranoside (DM), n-dodecyl-β-d-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism. PMID:22941083

Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

PubMed

Kwun, Jean; Farris, Alton B; Song, Hyunjin; Mahle, William T; Burlingham, William J; Knechtle, Stuart J

2015-12-01

Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

PubMed

Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2017-10-01

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Surfactants reduce platelet-bubble and platelet-platelet binding induced by in vitro air embolism.

PubMed

Eckmann, David M; Armstead, Stephen C; Mardini, Feras

2005-12-01

The effect of gas bubbles on platelet behavior is poorly characterized. The authors assessed platelet-bubble and platelet-platelet binding in platelet-rich plasma in the presence and absence of bubbles and three surface-active compounds. Platelet-rich plasma was prepared from blood drawn from 16 volunteers. Experimental groups were surfactant alone, sparging (microbubble embolization) alone, sparging with surfactant, and neither sparging nor surfactant. The surfactants were Pluronic F-127 (Molecular Probes, Eugene, OR), Perftoran (OJSC SPC Perftoran, Moscow, Russia), and Dow Corning Antifoam 1510US (Dow Corning, Midland, MI). Videomicroscopy images of specimens drawn through rectangular glass microcapillaries on an inverted microscope and Coulter counter measurements were used to assess platelet-bubble and platelet-platelet binding, respectively, in calcium-free and recalcified samples. Histamine-induced and adenosine diphosphate-induced platelet-platelet binding were measured in unsparged samples. Differences between groups were considered significant for P < 0.05 using analysis of variance and the Bonferroni correction. Sixty to 100 platelets adhered to bubbles in sparged, surfactant-free samples. With sparging and surfactant, few platelets adhered to bubbles. Numbers of platelet singlets and multimers not adherent to bubbles were different (P < 0.05) compared both with unsparged samples and sparged samples without surfactant. No significant platelet-platelet binding occurred in uncalcified, sparged samples, although 20-30 platelets adhered to bubbles. Without sparging, histamine and adenosine diphosphate provoked platelet-platelet binding with and without surfactants present. Sparging causes platelets to bind to air bubbles and each other. Surfactants added before sparging attenuate platelet-bubble and platelet-platelet binding. Surfactants may have a clinical role in attenuating gas embolism-induced platelet-bubble and platelet-platelet binding.

Correlating folding and signaling in a photoreceptor by single molecule measurements and energy landscape calculations

NASA Astrophysics Data System (ADS)

Hoff, Wouter

2007-03-01

Receptor activation is a fundamental process in biological signaling. We study the structural changes during activation of photoactive yellow protein (PYP). This is triggered by photoisomerization of the p-coumaric acid (pCA) chromophore of PYP, which converts the initial pG state into the activated pB state. Mechanical unfolding of Cys-linked PYP multimers probed by atomic force microscopy (AFM) in the presence and absence of illumination reveals that the core of the protein is extended by 3 nm and destabilized by 30 percent in pB. These results establish a generally applicable single molecule approach for mapping functional conformational changes to selected regions of a protein and indicate that stimulus-induced partial protein unfolding can be employed as a signaling mechanism. Comparative measurements, Jarzynski-Hummer-Szabo analysis of the data, and steered MD simulations of two double-Cys PYP mutants reveal strong anisotropy in the unfolding mechanism along the two axes defined by the Cys residues. Unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously. The other axis displays an unpeaked force profile reflecting a non-cooperative transition, challenging the notion that cooperative unfolding is a universal feature in protein stability. MD simulations with a coarse-grained protein model show that the folding of pG is two-state, consistent with experimental observations. In contrast, the folding free energy surface of a coarse-grained model of pB involves an on-pathway partially unfolded intermediate that closely matches experimental data. The results reveal that interactions between the pCA and its binding pocket can switch the energy landscape for PYP from two- to three-state folding, and show how this can be exploited to trigger large functionally important protein conformational changes.

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L

PubMed Central

Berber, Ergul; Ozbil, Mehmet; Brown, Christine; Baslar, Zafer; Caglayan, S. Hande; Lillicrap, David

2017-01-01

Background Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. Materials and methods The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. Results As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E’ domain, and significant loss of β-sheet secondary structure. Discussion The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion. PMID:27483487

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.

PubMed

Berber, Ergul; Ozbil, Mehmet; Brown, Christine; Baslar, Zafer; Caglayan, S Hande; Lillicrap, David

2017-10-01

Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of β-sheet secondary structure. The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination.

PubMed

Stufano, F; Baronciani, L; Pagliari, M T; Franchi, F; Cozzi, G; Garcia-Oya, I; Bucciarelli, P; Boscarino, M; Peyvandi, F

2015-10-01

Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample. In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines. Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays. The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P < 0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio. Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients. © 2015 International Society on Thrombosis and Haemostasis.

The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation.

PubMed

Sohn, Sook-Young; Hearing, Patrick

2016-06-14

The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses.

The Generation of Human γδT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture.

PubMed

Watanabe, Daisuke; Koyanagi-Aoi, Michiyo; Taniguchi-Ikeda, Mariko; Yoshida, Yukiko; Azuma, Takeshi; Aoi, Takashi

2018-01-01

γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αβT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αβT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer. Stem Cells Translational Medicine 2018;7:34-44. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Detection of Amyloid Beta (Aβ) Oligomeric Composition Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS)

NASA Astrophysics Data System (ADS)

Wang, Jasmine S.-H.; Whitehead, Shawn N.; Yeung, Ken K.-C.

2018-02-01

The use of MALDI MS as a fast and direct method to detect the Aβ oligomers of different masses is examined in this paper. Experimental results suggest that Aβ oligomers are ionized and detected as singly charged ions, and thus, the resulting mass spectrum directly reports the oligomer size distribution. Validation experiments were performed to verify the MS data against artifacts. Mass spectra collected from modified Aβ peptides with different propensities for aggregation were compared. Generally, the relative intensities of multimers were higher from samples where oligomerization was expected to be more favorable, and vice versa. MALDI MS was also able to detect the differences in oligomeric composition before and after the incubation/oligomerization step. Such differences in sample composition were also independently confirmed with an in vitro Aβ toxicity study on primary rat cortical neurons. An additional validation was accomplished through removal of oligomers from the sample using molecular weight cutoff filters; the resulting MS data correctly reflected the removal at the expected cutoff points. The results collectively validated the ability of MALDI MS to assess the monomeric/multimeric composition of Aβ samples. [Figure not available: see fulltext.

Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

PubMed Central

Poon, Betty; Chen, Irvin S. Y.

1998-01-01

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication. PMID:9499049

A background correction algorithm for Van Allen Probes MagEIS electron flux measurements

DOE PAGES

Claudepierre, S. G.; O'Brien, T. P.; Blake, J. B.; ...

2015-07-14

We describe an automated computer algorithm designed to remove background contamination from the Van Allen Probes Magnetic Electron Ion Spectrometer (MagEIS) electron flux measurements. We provide a detailed description of the algorithm with illustrative examples from on-orbit data. We find two primary sources of background contamination in the MagEIS electron data: inner zone protons and bremsstrahlung X-rays generated by energetic electrons interacting with the spacecraft material. Bremsstrahlung X-rays primarily produce contamination in the lower energy MagEIS electron channels (~30–500 keV) and in regions of geospace where multi-M eV electrons are present. Inner zone protons produce contamination in all MagEIS energymore » channels at roughly L < 2.5. The background-corrected MagEIS electron data produce a more accurate measurement of the electron radiation belts, as most earlier measurements suffer from unquantifiable and uncorrectable contamination in this harsh region of the near-Earth space environment. These background-corrected data will also be useful for spacecraft engineering purposes, providing ground truth for the near-Earth electron environment and informing the next generation of spacecraft design models (e.g., AE9).« less

Na+ influx via Orai1 inhibits intracellular ATP-induced mTORC2 signaling to disrupt CD4 T cell gene expression and differentiation

PubMed Central

Miao, Yong; Bhushan, Jaya; Dani, Adish; Vig, Monika

2017-01-01

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napahyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napahyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP]i. Depletion of [ATP]i inhibited mTORC2 dependent NFκB activation in Napahyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napahyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function. DOI: http://dx.doi.org/10.7554/eLife.25155.001 PMID:28492364

Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

NASA Astrophysics Data System (ADS)

Oda, Akifumi; Fukuyoshi, Shuichi

2015-06-01

The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

SdAb heterodimer formation using leucine zippers

NASA Astrophysics Data System (ADS)

Goldman, Ellen R.; Anderson, George P.; Brozozog-Lee, P. Audrey; Zabetakis, Dan

2013-05-01

Single domain antibodies (sdAb) are variable domains cloned from camel, llama, or shark heavy chain only antibodies, and are among the smallest known naturally derived antigen binding fragments. SdAb derived from immunized llamas are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. We hypothesized that the ability to produce heterodimeric sdAb would enable reagents with the robust characteristics of component sdAb, but with dramatically improved overall affinity through increased avidity. Previously we had constructed multimeric sdAb by genetically linking sdAb that bind non-overlapping epitopes on the toxin, ricin. In this work we explored a more flexible approach; the construction of multivalent binding reagents using multimerization domains. We expressed anti-ricin sdAb that recognize different epitopes on the toxin as fusions with differently charged leucine zippers. When the initially produced homodimers are mixed the leucine zipper domains will pair to produce heterodimers. We used fluorescence resonance energy transfer to confirm heterodimer formation. Surface plasmon resonance, circular dichroism, enzyme linked immunosorbent assays, and fluid array assays were used to characterize the multimer constructs, and evaluate their utility in toxin detection.

Modeling impact of small Kansas landfills on underlying aquifers

USGS Publications Warehouse

Sophocleous, M.; Stadnyk, N.G.; Stotts, M.

1996-01-01

Small landfills are exempt from compliance with Resource Conservation and Recovery Act Subtitle D standards for liner and leachate collection. We investigate the ramifications of this exemption under western Kansas semiarid environments and explore the conditions under which naturally occurring geologic settings provide sufficient protection against ground-water contamination. The methodology we employed was to run water budget simulations using the Hydrologic Evaluation of Landfill Performance (HELP) model, and fate and transport simulations using the Multimedia Exposure Assessment Model (MULTIMED) for several western Kansas small landfill scenarios in combination with extensive sensitivity analyses. We demonstrate that requiring landfill cover, leachate collection system (LCS), and compacted soil liner will reduce leachate production by 56%, whereas requiring only a cover without LCS and liner will reduce leachate by half as much. The most vulnerable small landfills are shown to be the ones with no vegetative cover underlain by both a relatively thin vadose zone and aquifer and which overlie an aquifer characterized by cool temperatures and low hydraulic gradients. The aquifer-related physical and chemical parameters proved to be more important than vadose zone and biodegradation parameters in controlling leachate concentrations at the point of compliance. ??ASCE.

MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

PubMed Central

2014-01-01

Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-β superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-β chimeras for activity screen is also discussed. PMID:24559319

Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization

PubMed Central

Vorontsov, Egor; Gallaud, Julien; Malosse, Christian; Michel, Valérie; Cavazza, Christine; Robbe-Saule, Marie; Richaud, Pierre; Chamot-Rooke, Julia; Brochier-Armanet, Céline; De Reuse, Hilde

2015-01-01

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori. PMID:26641249

Coagulation Testing in the Core Laboratory.

PubMed

Winter, William E; Flax, Sherri D; Harris, Neil S

2017-11-08

Primary hemostasis begins with endothelial injury. VWF, produced by endothelial cells, binds to platelets and links them to subendothelial collagen. Platelet-derived ADP and thromboxane activate non-adhered platelets via their GPIIb/IIIa receptors, allowing these platelets to participate in platelet aggregation. Secondary hemostasis is initiated with the binding of factor VII to extravascular tissue factor (TF). Factors II, VII, IX and X are vitamin K-dependent factors. The role of vitamin K is to assist in the addition of gamma carboxylate groups to glutamic acids in the "GLA" domains of these factors.In vitro the intrinsic pathway is initiated when fresh whole blood is placed in a glass tube. The negative charge of the glass initiates the "contact pathway" where FXII is activated and then FXIa cleaves FIX to FIXa. The extrinsic pathway is triggered when tissue factor, phospholipid and calcium are added to plasma anticoagulated with citrate. In vitro, FVII is activated to FVIIa, and TF-FVIIa preferentially converts FX to FXa activating the common pathway.The prothrombin time is commonly used to monitor warfarin anticoagulant therapy. To correct for differences in reagent and instrument, the international normalized ratio was developed to improve standardization of PT reporting globally. The activated partial thromboplastin time (aPTT) is used to evaluate the intrinsic and common pathways of coagulation. The aPTT is useful clinically as a screening test for inherited and acquired factor deficiencies as well as to monitor unfractionated heparin therapy although the anti-Xa assay is now the preferred measure of the effects of unfractionated heparin. The Clauss assay is the most commonly performed fibrinogen assay and uses diluted plasma where clotting is initiated with a high concentration of reagent thrombin.The mixing study assists in the assessment of an abnormally prolonged PT or aPTT. An equal volume of citrated patient plasma is mixed with normal pooled plasma and the PT or aPTT are repeated on the 1:1 mix. Factor activity assays are most commonly performed as a one-stage assay. The patient's citrated plasma is diluted and mixed 1-to-1 with a single factor-deficient substrate plasma. A PT or aPTT is performed on the above mix, depending on the factor being tested.Factor inhibitors are antibodies that are most commonly diagnosed in male patients with severe hemophilia A (FVIII deficiency) where they are induced by factor replacement therapy.Factor inhibitors can also appear in the form of spontaneous autoantibodies in both male and female individuals who were previously well. This is an autoimmune condition called "acquired hemophilia."Most coagulation laboratories can measure the plasma concentration of VWF protein (VWF antigen) by an immunoturbidimetric technique. Testing the functional activity of VWF, utilizes the drug ristocetin.The state of multimerization of VWF is important and is assessed by electrophoresis on agarose gels. Type 2a and 2b VWD are associated with the lack of intermediate- and high molecular weight multimers.The antiphospholipid syndrome (APLS) is an acquired autoimmune phenomenon associated with an increased incidence of both venous and arterial thromboses, as well as fetal loss. Typically, there is a paradoxical prolongation of the aPTT in the absence of any clinical features of bleeding. This is the so-called "lupus anticoagulant (LA) effect." The laboratory definition of the APLS requires the presence of either a "lupus anticoagulant" or a persistent titer of antiphospholipid antibodies.There are now 2 broad classes of direct-acting oral anticoagulants (DOACs): [1] The oral direct thrombin inhibitors (DTIs) such as dabigatran; and [2] The oral direct factor Xa inhibitors such as rivaroxaban and apixaban. The PT and aPTT are variably affected by the DOACs and are generally unhelpful in monitoring their concentrations. Most importantly, a normal PT or aPTT does NOT exclude the presence of any of the DOACs. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evidences for Cooperative Resonance-Assisted Hydrogen Bonds in Protein Secondary Structure Analogs

NASA Astrophysics Data System (ADS)

Zhou, Yu; Deng, Geng; Zheng, Yan-Zhen; Xu, Jing; Ashraf, Hamad; Yu, Zhi-Wu

2016-11-01

Cooperative behaviors of the hydrogen bonding networks in proteins have been discovered for a long time. The structural origin of this cooperativity, however, is still under debate. Here we report a new investigation combining excess infrared spectroscopy and density functional theory calculation on peptide analogs, represented by N-methylformamide (NMF) and N-methylacetamide (NMA). Interestingly, addition of the strong hydrogen bond acceptor, dimethyl sulfoxide, to the pure analogs caused opposite effects, namely red- and blue-shift of the N-H stretching infrared absorption in NMF and NMA, respectively. The contradiction can be reconciled by the marked lowering of the energy levels of the self-associates between NMA molecules due to a cooperative effect of the hydrogen bonds. On the contrary, NMF molecules cannot form long-chain cooperative hydrogen bonds because they tend to form dimers. Even more interestingly, we found excellent linear relationships between changes on bond orders of N-H/N-C/C = O and the hydrogen bond energy gains upon the formation of hydrogen bonding multimers in NMA, suggesting strongly that the cooperativity originates from resonance-assisted hydrogen bonds. Our findings provide insights on the structures of proteins and may also shed lights on the rational design of novel molecular recognition systems.

Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

PubMed Central

2014-01-01

Background Autotransporter proteins represent a treasure trove for molecular engineers who modify Gram-negative bacteria for the export or secretion of foreign proteins across two membrane barriers. A particularly promising direction is the development of autotransporters as antigen display or secretion systems. Immunologists have been using ovalbumin as a reporter antigen for years and have developed sophisticated tools to detect specific T cells that respond to ovalbumin. Although ovalbumin-expressing bacteria are being used to trace T cell responses to colonizing or invading pathogens, current constructs for ovalbumin presentation have not been optimized. Results The activation of T helper cells in response to ovalbumin was improved by displaying the OVA-CD4 reporter epitope as a multimer on the surface of Salmonella and fused to the autotransporter MisL. Expression was optimized by including tandem in vivo promoters and two post-segregational killing systems for plasmid stabilization. Conclusions The use of an autotransporter protein to present relevant epitope repeats on the surface of bacteria, combined with additional techniques favoring stable and efficient in vivo transcription, optimizes antigen presentation to T cells. The technique of multimeric epitope surface display should also benefit the development of new Salmonella or other enterobacterial vaccines. PMID:24898796

Transgenic plants with increased calcium stores

NASA Technical Reports Server (NTRS)

Robertson, Dominique (Inventor); Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Boss, Wendy (Inventor)

2004-01-01

The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

PubMed

Heitner, Tara; Satozawa, Noboru; McLean, Kirk; Vogel, David; Cobb, Ronald R; Liu, Bing; Mahmoudi, Mithra; Finster, Silke; Larsen, Brent; Zhu, Ying; Zhou, Hongxing; Müller-Tiemann, Beate; Monteclaro, Felipe; Zhao, Xiao-Yan; Light, David R

2006-12-01

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Natural Variation at the FRD3 MATE Transporter Locus Reveals Cross-Talk between Fe Homeostasis and Zn Tolerance in Arabidopsis thaliana

PubMed Central

Pineau, Christophe; Loubet, Stéphanie; Lefoulon, Cécile; Chalies, Claude; Fizames, Cécile; Lacombe, Benoit; Ferrand, Marina; Loudet, Olivier; Berthomieu, Pierre; Richard, Odile

2012-01-01

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator. PMID:23236296

Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

PubMed Central

Meng, He; Zhang, Xiaojie; Yu, Genggeng; Lee, Soo Jung; Chen, Y. Eugene; Prudovsky, Igor; Wang, Michael M.

2012-01-01

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells. PMID:23028706

Allostery and the dynamic oligomerization of porphobilinogen synthase

PubMed Central

Jaffe, Eileen K.; Lawrence, Sarah H.

2011-01-01

The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer* ⇔ 6mer*. The “*” represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH; the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer* relative to wild type. The 6mer* has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer*. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects. PMID:22037356

Single-Chain Fv-Based Anti-HIV Proteins: Potential and Limitations

PubMed Central

West, Anthony P.; Galimidi, Rachel P.; Gnanapragasam, Priyanthi N. P.

2012-01-01

The existence of very potent, broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) offers the potential for prophylaxis against HIV-1 infection by passive immunization or gene therapy. Both routes permit the delivery of modified forms of IgGs. Smaller reagents are favored when considering ease of tissue penetration and the limited capacities of gene therapy vectors. Immunoadhesin (single-chain fragment variable [scFv]-Fc) forms of IgGs are one class of relatively small reagent that has been explored for delivery by adeno-associated virus. Here we investigated the neutralization potencies of immunoadhesins compared to those of their parent IgGs. For the antibodies VRC01, PG9, and PG16, the immunoadhesins showed modestly reduced potencies, likely reflecting reduced affinities compared to those of the parent IgG, and the VRC01 immunoadhesin formed dimers and multimers with reduced neutralization potencies. Although scFv forms of neutralizing antibodies may exhibit affinity reductions, they provide a means of building reagents with multiple activities. Attachment of the VRC01 scFv to PG16 IgG yielded a bispecific reagent whose neutralization activity combined activities from both parent antibodies. Although the neutralization activity due to each component was partially reduced, the combined reagent is attractive since fewer strains escaped neutralization. PMID:22013046

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

PubMed Central

Kishko, Iryna; Harish, Balasubramanian; Zayats, Vasilina; Reha, David; Tenner, Brian; Beri, Dhananjay; Gustavsson, Tobias; Ettrich, Rüdiger; Carey, Jannette

2012-01-01

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots. PMID:22952804

Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins

PubMed Central

Prasad, Tuhina; Weiner, Joshua A.

2011-01-01

The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γdel/del null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs. PMID:22275881

Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins.

PubMed

Prasad, Tuhina; Weiner, Joshua A

2011-01-01

The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γ(del/del) null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs.

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

PubMed Central

Schirmer, David; Grünewald, Thomas G. P.; Klar, Richard; Schmidt, Oxana; Wohlleber, Dirk; Rubío, Rebeca Alba; Uckert, Wolfgang; Thiel, Uwe; Bohne, Felix; Busch, Dirk H.; Krackhardt, Angela M.; Burdach, Stefan; Richter, Günther H. S.

2016-01-01

ABSTRACT Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8+ T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells (DC), allo-restricted HLA-A*02:01− CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01− primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2−/−γc−/− mouse model. Initially generated transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01− ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1130-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors. PMID:27471654

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior: A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome

PubMed Central

Alvarez-Cabrera, Ana L.; Delgado, Sandra; Gil-Carton, David; Mortuza, Gulnahar B.; Montoya, Guillermo; Sorzano, Carlos O. S.; Tang, Tang K.; Carazo, Jose M.

2017-01-01

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897–1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis. PMID:28396859

DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II)

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

Bellon, S.F.; Coleman, J.H.; Lippard, S.J.

The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedi-chloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-(Pt(NH{sub 3}){sub 2}(d(GpG))), cis-(Pt(NH){sub 3}{sub 2}(d(ApG))), and cis-(Pt(NH{sub 3}){sub 2}(d(GpTpG))) adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG. Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis. The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymersmore » containing the adducts. The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained. The authors find that the cis-GG and cis-AG adducts both unwind DNA by 13{degrees}, while the cis-GTG adduct unwinds DNA by 23{degrees}. In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts. On the basis of an analysis of the present and other published studies of site-specifically modified DNA. The authors propose that local duplex unwinding is a major determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease. In addition, local duplex unwinding of 13{degrees} and bending by 35{degrees} are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.« less

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system

PubMed Central

Zoltner, Martin; Ng, Wui M.A.V.; Money, Jillian J.; Fyfe, Paul K.; Kneuper, Holger; Palmer, Tracy; Hunter, William N.

2016-01-01

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein. PMID:27130157

Beneficial Effects of High-Density Lipoproteins on Acquired von Willebrand Syndrome in Aortic Valve Stenosis.

PubMed

Gebhard, C; Maafi, F; Stähli, B E; Bonnefoy, A; Gebhard, C E; Nachar, W; de Oliveira Moraes, A Benjamim; Mecteau, M; Mihalache-Avram, T; Lavoie, V; Kernaleguen, A E; Shi, Y; Busseuil, D; Chabot-Blanchet, M; Perrault, L P; Rhainds, D; Rhéaume, E; Tardif, J C

2018-02-01

 Infusions of apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins (HDL), result in aortic valve stenosis (AVS) regression in experimental models. Severe AVS can be complicated by acquired von Willebrand syndrome, a haemorrhagic disorder associated with loss of high-molecular-weight von Willebrand factor (vWF) multimers (HMWM), the latter being a consequence of increased shear stress and enhanced vWF-cleaving protease (ADAMTS-13) activity. Although antithrombotic actions of HDL have been described, its effects on ADAMTS-13 and vWF in AVS are unknown.  We assessed ADAMTS-13 activity in plasma derived from a rabbit model of AVS ( n  = 29) as well as in plasma collected from 64 patients with severe AVS (age 65.0 ± 10.4 years, 44 males) undergoing aortic valve replacement (AVR). In both human and rabbit AVS plasma, ADAMTS-13 activity was higher than that in controls ( p  < 0.05). Accordingly, AVS patients had less HMWM than controls (66.3 ± 27.2% vs. 97.2 ± 24.1%, p  < 0.0001). Both ADAMTS-13 activity and HMWM correlated significantly with aortic transvalvular gradients, thereby showing opposing correlations ( r  = 0.3, p  = 0.018 and r  = -0.4, p  = 0.003, respectively). Administration of an apoA-I mimetic peptide reduced ADAMTS-13 activity in AVS rabbits as compared with the placebo group (2.0 ± 0.5 RFU/sec vs. 3.8 ± 0.4 RFU/sec, p  < 0.05). Similarly, a negative correlation was found between ADAMTS-13 activity and HDL cholesterol levels in patients with AVS ( r  = -0.3, p  = 0.045).  Our data indicate that HDL levels are associated with reduced ADAMTS-13 activity and increased HMWM. HDL-based therapies may reduce the haematologic abnormalities of the acquired von Willebrand syndrome in AVS. Schattauer GmbH Stuttgart.

The end of a monolith: Deconstructing the Cnn-Polo interaction.

PubMed

Eisman, Robert C; Phelps, Melissa A S; Kaufman, Thomas C

2016-04-02

In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.

Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains

PubMed Central

Otikovs, Martins; Landreh, Michael; Nordling, Kerstin; Kronqvist, Nina; Westermark, Per; Jörnvall, Hans; Knight, Stefan; Ridderstråle, Yvonne; Holm, Lena; Meng, Qing; Jaudzems, Kristaps; Chesler, Mitchell; Johansson, Jan; Rising, Anna

2014-01-01

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation. PMID:25093327

Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

PubMed

Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

2015-05-01

Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity. © 2015 John Wiley & Sons Ltd.

Cellular immunotherapy for patients with reactivation of JC and BK polyomaviruses after transplantation.

PubMed

Mani, Jiju; Jin, Nan; Schmitt, Michael

2014-10-01

Immunosuppression of patients after hematopoietic stem cell or kidney transplantation potentially leads to reactivation of JC and BK polyomaviruses. In hematopoietic stem cell transplantation, the reactivation rate of BKV can be up to 60%, resulting in severe complications of the urogenital tract, particularly hemorrhagic cystitis and renal dysfunction. After kidney transplantation, BKV reactivation can cause a loss of the graft. JCV can cause progressive multifocal leukoencephalopathy, a lethal disease. Adoptive transfer of donor-derived polyomavirus-specific T cells is an attractive and promising treatment that restores virus-specific cellular immunity. Pioneering work in the early 1990s on the reconstitution of cellular immunity against cytomegalovirus and recent development in the field of monitoring and isolation of antigen-specific T cells paved the way toward a personalized T-cell therapy. Multimer technology and magnetic beads are available to produce untouched T cells in a single-step, good manufacturing practice-compliant procedure. Another exciting aspect of T-cell therapy against polyomaviruses is the fact that both JCV and BKV can be targeted simultaneously because of their high sequence homology. Finally, "designer T cells" can be redirected to recognize polyomavirus antigens with high-affinity T-cell receptors. This review summarizes the state-of-the art technologies and gives an outlook of future developments in the field. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Traces of pFc' in IVIG interact with human IgG Fc domains and counteract aggregation.

PubMed

Rispens, Theo; Himly, Martin; Ooievaar-De Heer, Pleuni; den Bleker, Tamara H; Aalberse, Rob C

2010-04-16

To prevent multimer formation, intravenous immunoglobulin (IVIG) is often treated with traces of pepsin. So far, the mechanism behind this treatment has been unclear. Recently, we reported that human IgG4 binds other IgG molecules via Fc-Fc interactions. Here we show that IVIG treated with traces of pepsin (Nanogam) inhibits these interactions. We found that--besides IgG4--peptides corresponding to IgG1 and IgG2 pFc' (products of limited pepsin digestion) are responsible for the inhibitory action. Using radiolabeled pFc', it was found that pFc' binds directly to IgG1. Furthermore, recombinant CH3 fragments were found to also possess binding activity, and potencies of inhibition varied over 3 orders of magnitude amongst the subclasses, IgG4 being most potent. We propose that pFc' formation explains how limited pepsin digestion diminishes adverse effects of IVIG. In particular, the presence of this fragment can enhance the stability of IgG products including IVIG and therapeutical monoclonal antibodies. Indeed, using a model system it was found that acid-induced aggregation of IgG is reduced in the presence of pFc', suggesting a 'chaperone-like' activity of this fragment. Thus, pFc' can modulate Fc interactions and may therefore reduce adverse effects of IVIG, in particular by preventing oligomerization. 2010 Elsevier B.V. All rights reserved.

Abalone Hemocyanin Blocks the Entry of Herpes Simplex Virus 1 into Cells: a Potential New Antiviral Strategy

PubMed Central

Talaei Zanjani, Negar; Miranda-Saksena, Monica; Valtchev, Peter; Hueston, Linda; Diefenbach, Eve; Sairi, Fareed; Gomes, Vincent G.

2015-01-01

A marine-derived compound, abalone hemocyanin, from Haliotis rubra was shown to have a unique mechanism of antiviral activity against herpes simplex virus 1 (HSV-1) infections. In vitro assays demonstrated the dose-dependent and inhibitory effect of purified hemocyanin against HSV-1 infection in Vero cells with a 50% effective dose (ED50) of 40 to 50 nM and no significant toxicity. In addition, hemocyanin specifically inhibited viral attachment and entry by binding selectively to the viral surface glycoproteins gD, gB, and gC, probably by mimicking their receptors. However, hemocyanin had no effect on postentry events and did not block infection by binding to cellular receptors for HSV. By the use of different mutants of gD and gB and a competitive heparin binding assay, both protein charge and conformation were shown to be the driving forces of the interaction between hemocyanin and viral glycoproteins. These findings also suggested that hemocyanin may have different motifs for binding to each of the viral glycoproteins B and D. The dimer subunit of hemocyanin with a 10-fold-smaller molecular mass exhibited similar binding to viral surface glycoproteins, showing that the observed inhibition did not require the entire multimer. Therefore, a small hemocyanin analogue could serve as a new antiviral candidate for HSV infections. PMID:26643336

CD80 and CD86 IgC domains are important for quaternary structure, receptor binding and co-signaling function.

PubMed

Girard, Tanya; Gaucher, Denis; El-Far, Mohamed; Breton, Gaëlle; Sékaly, Rafick-Pierre

2014-09-01

CD86 and CD80, the ligands for the co-stimulatory molecules CD28 and CTLA-4, are members of the Ig superfamily. Their structure includes Ig variable-like (IgV) domains, Ig constant-like (IgC) domains and intracellular domains. Although crystallographic studies have clearly identified the IgV domain to be responsible for receptor interactions, earlier studies suggested that both Ig domains are required for full co-signaling function. Herein, we have used deletion and chimeric human CD80 and CD86 molecules in co-stimulation assays to study the impact of the multimeric state of IgV and IgC domains on receptor binding properties and on co-stimulatory function in a peptide-specific T cell activation model. We report for the first time the presence of CD80 dimers and CD86 monomers in living cells. Moreover, we show that the IgC domain of both molecules inhibits multimer formation and greatly affects binding to the co-receptors CD28 and CTLA-4. Finally, both IgC and intracellular domains are required for full co-signaling function. These findings reveal the distinct but complementary roles of CD80 and CD86 IgV and IgC domains in T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs.

PubMed

Zhang, Huifang M; Su, Yue; Guo, Songchuan; Yuan, Ji; Lim, Travis; Liu, Jing; Guo, Peixuan; Yang, Decheng

2009-09-01

Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively.

The end of a monolith: Deconstructing the Cnn-Polo interaction

PubMed Central

2016-01-01

ABSTRACT In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic. PMID:27096551

Mhr1p-dependent concatemeric mitochondrial DNA formation for generating yeast mitochondrial homoplasmic cells.

PubMed

Ling, Feng; Shibata, Takehiko

2004-01-01

Mitochondria carry many copies of mitochondrial DNA (mtDNA), but mt-alleles quickly segregate during mitotic growth through unknown mechanisms. Consequently, all mtDNA copies are often genetically homogeneous within each individual ("homoplasmic"). Our previous study suggested that tandem multimers ("concatemers") formed mainly by the Mhr1p (a yeast nuclear gene-encoded mtDNA-recombination protein)-dependent pathway are required for mtDNA partitioning into buds with concomitant monomerization. The transmission of a few randomly selected clones (as concatemers) of mtDNA into buds is a possible mechanism to establish homoplasmy. The current study provides evidence for this hypothesis as follows: the overexpression of MHR1 accelerates mt-allele-segregation in growing heteroplasmic zygotes, and mhr1-1 (recombination-deficient) causes its delay. The mt-allele-segregation rate correlates with the abundance of concatemers, which depends on Mhr1p. In G1-arrested cells, concatemeric mtDNA was labeled by [14C]thymidine at a much higher density than monomers, indicating concatemers as the immediate products of mtDNA replication, most likely in a rolling circle mode. After releasing the G1 arrest in the absence of [14C]thymidine, the monomers as the major species in growing buds of dividing cells bear a similar density of 14C as the concatemers in the mother cells, indicating that the concatemers in mother cells are the precursors of the monomers in buds.

Functional metagenomic selection of RubisCOs from uncultivated bacteria

USGS Publications Warehouse

Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A.; Witteveen, Briana; Dourado, Manuella N.; Anantharaman, Karthik; Arbing, Mark A.; McCann, Shelley; Oremland, Ronald S.; Banfield, Jillian F.; Wrighton, Kelly C.; Tabita, F. Robert

2016-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2-dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of theGallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possessCO2/O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2-fixing enzymes not previously characterized.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

PubMed

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insights into gelation kinetics and gel front migration in cation-induced polysaccharide hydrogels by viscoelastic and turbidity measurements: Effect of the nature of divalent cations.

PubMed

Huynh, Uyen T D; Chambin, Odile; du Poset, Aline Maire; Assifaoui, Ali

2018-06-15

Polysaccharide-based hydrogels were prepared by the diffusion of various divalent cations (X 2+ ) into the polygalacturonate (polyGal) solution through a dialysis membrane. The diffusion of various divalent cations (Mg 2+ , Ca 2+ , Zn 2+ and Ba 2+ ) was investigated. The polyGal gel growth was studied as a function of the initial cation concentration by both viscoelastic and turbidity measurements. We have demonstrated for the first time that the determination of the spatiotemporal variation of turbidity during the gelation process allowed to study the gel front migration. For Ca-polyGal, Zn-polyGal and Ba-polyGal, the gel front migration was characterized by the presence of a peak at the sol/gel interface. This peak was not observed in the case of Mg-polyGal where the gel was not formed. The apparent diffusion coefficient of the gel front (D app ) which was calculated from the evolution of this peak increased when the initial cation concentration was increased. Moreover, we have suggested a gelation mechanism based on the presence of a threshold molar ratio R* (=[X 2+ ]/[Galacturonic unit]) in which some point-like crosslinks are precursors of the formation of dimers and multimers inducing the contraction of the gel and thus the formation of the gel front. Copyright © 2018 Elsevier Ltd. All rights reserved.

pH-dependent conformational changes of diphtheria toxin adsorbed to lipid monolayers by neutron and X-ray reflection

NASA Astrophysics Data System (ADS)

Kent, Michael; Yim, Hyun; Satija, Sushil; Kuzmenko, Ivan

2006-03-01

Several important bacterial toxins, such as diphtheria, tetanus, and botulinum, invade cells through a process of high affinity binding, internalization via endosome formation, and subsequent membrane penetration of the catalytic domain activated by a pH drop in the endosome. These toxins are composed of three domains: a binding domain, a translocation domain, and an enzyme. The translocation process is not well understood with regard to the detailed conformational changes that occur at each step, To address this, we performed neutron reflectivity measurements for diphtheria toxin bound to lipid monolayers as a function of pH. While the final membrane inserted conformation will not be reproduced with the present monolayer system, important insights can still be gained into several intermediate stages. In particular, we show that no adsorption occurs at pH = 7.6, but strong adsorption occurs over at a pH range from 6.5 to 6.0. Following binding, at least two stages of conformational change occur, as the thickness increases from pH 6.3 to 5.3 and then decreases from pH 5.3 to 4.5. In addition, the dimension of the adsorbed layer substantially exceeds that of the largest dimension in the crystal structure of monomeric diphtheria, suggesting that the toxin may be present as multimers.

Photophysics of Zinc Porphyrin Aggregates in Dilute Water-Ethanol Solutions.

PubMed

Stevens, Amy L; Joshi, Neeraj K; Paige, Matthew F; Steer, Ronald P

2017-12-14

Dimeric and multimeric aggregates of a model metalloporphyrin, zinc tetraphenylporphyrin (ZnTPP), have been produced in a controlled manner by incrementally increasing the water content of dilute aqueous ethanol solutions. Steady state absorption, fluorescence emission, and fluorescence excitation spectra have been measured to identify the aggregates present as a function of solvent composition. The dynamics of the excited states of the aggregates produced initially by excitation in the Soret region have been measured by ultrafast fluorescence upconversion techniques. Only the monomer produces measurable emission from S 2 with a picosecond lifetime; all Soret-excited aggregates, including the dimer, decay radiationlessly on a femtosecond time scale. The S 1 state is the only significant product of the radiationless decay of the S 2 state of the excited monomer, and the aggregates also produce substantial quantum yields of S 1 fluorescence when initially excited in the Soret region. The resulting fluorescent aggregates all decay on a subnanosecond time scale, likely by a mechanism that involves dissociation of the excited monomer from the excitonic multimer. The ZnTPP dimers excited at their ground state geometries in the Soret region exhibit a dynamic behavior that is quite different from those produced following noncoherent triplet-triplet annihilation under the same conditions. The important implications of these observations in determining the aggregation conditions promoting efficient photon upconversion by excitonic annihilation in a variety of media are thoroughly discussed.

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

PubMed

de Groot, A; Koster, M; Gérard-Vincent, M; Gerritse, G; Lazdunski, A; Tommassen, J; Filloux, A

2001-02-01

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Scanning tunneling microscopy and spectroscopy studies of the heavy-electron superconductor TlNi2Se2

NASA Astrophysics Data System (ADS)

Wilfert, Stefan; Schmitt, Martin; Schmidt, Henrik; Mauerer, Tobias; Sessi, Paolo; Wang, Hangdong; Mao, Qianhui; Fang, Minghu; Bode, Matthias

2018-01-01

We report on the structural and superconducting electronic properties of the heavy-electron superconductor TlNi2Se2 . By using a variable-temperature scanning tunneling microscopy (VT-STM) the coexistence of (√{2 }×√{2 }) R 45∘ and (2 ×1 ) surface reconstructions is observed. Similar to earlier observations on the "122" family of Fe-based superconductors, we find that their respective surface fraction strongly depends on the temperature during cleavage, the measurement temperature, and the sample's history. Cleaving at low temperature predominantly results in the (√{2 }×√{2 }) R 45∘ -reconstructed surface. A detailed analysis of the (√{2 }×√{2 }) R 45∘ -reconstructed domains identifies (2 ×1 ) -ordered dimers, tertramers, and higher order even multimers as domain walls. Higher cleaving temperatures and the warming of low-temperature-cleaved samples increases the relative weight of the (2 ×1 ) surface reconstruction. By slowly increasing the sample temperature Ts inside the VT-STM we find that the (√{2 }×√{2 }) R 45∘ surface reconstructions transforms into the (2 ×1 ) structure at Ts=123 K. We identify the polar nature of the TlNi2Se2 (001) surface as the most probable driving mechanism of the two reconstructions, as both lead to a charge density ρ =0.5 e- , thereby avoiding divergent electrostatic potentials and the resulting "polar catastrophe." Low-temperature scanning tunneling spectroscopy (STS) performed with normal metal and superconducting probe tips shows a superconducting gap which is best fit with an isotropic s wave. We could not detect any correlation between the local surface reconstruction, suggesting that the superconductivity is predominantly governed by TlNi2Se2 bulk properties. Correspondingly, temperature- and field-dependent data reveal that both the critical temperature and critical magnetic field are in good agreement with bulk values obtained earlier from transport measurements. In the superconducting state the formation of an Abrikosov lattice is observed without any zero bias anomaly at the vortex core.

Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set.

PubMed

Krueger, Andrew T; Kool, Eric T

2008-03-26

We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.

PBMC are as good a source of tumor-reactive T lymphocytes as TIL after selection by Melan-A/A2 multimer immunomagnetic sorting.

PubMed

Labarrière, Nathalie; Gervois, Nadine; Bonnin, Annabelle; Bouquié, Régis; Jotereau, Francine; Lang, François

2008-02-01

Choosing a reliable source of tumor-specific T lymphocytes and an efficient method to isolate these cells still remains a critical issue in adoptive cellular therapy (ACT). In this study, we assessed the capacity of MHC/peptide based immunomagnetic sorting followed by polyclonal T cell expansion to derive pure polyclonal and tumor-reactive Melan-A specific T cell populations from melanoma patient's PBMC and TIL. We first demonstrated that this approach was extremely efficient and reproducible. We then used this procedure to compare PBMC and TIL-derived cells from three melanoma patients in terms of avidity for Melan-A A27L analog, Melan-A(26-35)and Melan-A(27-35), tumor reactivity (lysis and cytokine production) and repertoire. Regardless of their origin, i.e., fresh PBMC, peptide stimulated PBMC or TIL, all sorted populations (from the three patients) were cytotoxic against HLA-A2+ melanoma cell lines expressing Melan-A. Although some variability in peptide avidity, lytic activity and cytokine production was observed between populations of different origins in a given patient, it differed from one patient to another and thus no correlation could be drawn between T cell source and reactivity. Analysis of Vbeta usage within the sorted populations showed the recurrence of Vbeta3 and Vbeta14 subfamilies in the three patients but differences in the rest of the Melan-A repertoire. In addition, in two patients, we observed major repertoire differences between populations sorted from the three sources. We especially documented that in vitro peptide stimulation of PBMC, used to facilitate the sort by enriching in specific T lymphocytes, could significantly alter their repertoire and reactivity towards tumor cells. We conclude that PBMC which are easily obtained from all melanoma patients, can be as good a source as TIL to derive high amounts of tumor-reactive Melan-A specific T cells, with this selection/amplification procedure. However, the conditions of peptide stimulation should be improved to prevent a possible loss of reactive clonotypes.

Mutants of Yeast Defective in Sucrose Utilization

PubMed Central

Carlson, Marian; Osmond, Barbara C.; Botstein, David

1981-01-01

Utilization of sucrose as a source of carbon and energy in yeast (Saccharomyces) is controlled by the classical SUC genes, which confer the ability to produce the sucrose-degrading enzyme invertase (Mortimer and Hawthorne 1969). Mutants of S. cerevisiae strain S288C (SUC2+) unable to grow anaerobically on sucrose, but still able to use glucose, were isolated. Two major complementation groups were identified: twenty-four recessive mutations at the SUC2 locus (suc2-); and five recessive mutations defining a new locus, SNF1 (for sucrose nonfermenting), essential for sucrose utilization. Two minor complementation groups, each comprising a single member with a leaky sucrose-nonfermenting phenotype, were also identified. The suc2 mutations isolated include four suppressible amber mutations and five mutations apparently exhibiting intragenic complementation; complementation analysis and mitotic mapping studies indicated that all of the suc2 mutations are alleles of a single gene. These results suggest that SUC2 encodes a protein, probably a dimer or multimer. No invertase activity was detected in suc2 mutants.—The SNF1 locus is not tightly linked to SUC2. The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. Although snf1 mutants thus display a petite phenotype, classic petite mutations do not interfere with utilization of sucrose, galactose or maltose. A common feature of all the carbon utilization systems affected by SNF1 is that all are regulated by glucose repression. The snf1 mutants were found to produce the constitutive nonglycosylated form of invertase, but failed to produce the glucose-repressible, glycosylated, secreted invertase. This failure cannot be attributed to a general defect in production of glycosylated and secreted proteins because synthesis of acid phosphatase, a glycosylated secreted protein not subject to glucose repression, was not affected by snf1 mutations. These findings suggest that the SNF1 locus is involved in the regulation of gene expression by glucose repression. PMID:7040163

High-molecular weight adiponectin/HOMA-IR ratio as a biomarker of metabolic syndrome in urban multiethnic Brazilian subjects

PubMed Central

de Abreu, Virgínia Genelhu; Martins, Cyro José de Moraes; de Oliveira, Patricia Aguiar Cardoso; Francischetti, Emilio Antonio

2017-01-01

Metabolic syndrome (MetS) has an important epidemiological relevance due to its increasing prevalence and association with type 2 diabetes and cardiovascular disease. Insulin resistance is a core feature of the MetS. HOMA-IR is a robust clinical and epidemiological marker of MetS. Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions; its levels decrease as number of components of MetS increases. High-molecular weight adiponectin (HMWA) is the multimer responsible for the relationship of adiponectin with insulin sensitivity. HOMA-IR and HMWA are suitable candidates for MetS biomarkers. The ratio of adiponectin to HOMA-IR has been validated as a powerful index of MetS and considered a better marker of its presence, than either HOMA-IR or adiponectin alone, in selected homogeneous populations. We compared the strength of association between HMWA, HOMA-IR and HMWA/HOMA-IR ratio with MetS and its key components. Our data have shown that the median (25th, 75th percentile) of HMWA/HOMA-IR ratio was lower in subjects with MetS [0.51 (0.33, 1.31)] as compared to those without it [2.19 (1.13, 4.71)]. The correlation coefficient (r) was significantly higher for HMWA/HOMA-IR ratio as compared to HMWA for waist circumference (-0.65; -0.40, respectively); mean blood pressure (-0.27; -0.14, respectively); fasting glucose (-0.38; -0.19, respectively); HDL-cholesterol (0.44; 0.40, respectively); and triglycerides (-0.35; -0.18, respectively). In a multivariable logistic regression analysis, the HMWA/HOMA-IR ratio was a sensitive predictor for MetS, being the only marker that was significantly associated with each and all the individual components of the syndrome. These results expand on previous studies in that we used the active circulating form of adiponectin, i.e. HMWA, and represent a typical Brazilian cohort characterized by intense interethnic admixture. Thus, the HMWA/HOMA-IR ratio is a minimally invasive biomarker for MetS that could be clinically useful in prognosing patient outcome. PMID:28746378

High-molecular weight adiponectin/HOMA-IR ratio as a biomarker of metabolic syndrome in urban multiethnic Brazilian subjects.

PubMed

de Abreu, Virgínia Genelhu; Martins, Cyro José de Moraes; de Oliveira, Patricia Aguiar Cardoso; Francischetti, Emilio Antonio

2017-01-01

Metabolic syndrome (MetS) has an important epidemiological relevance due to its increasing prevalence and association with type 2 diabetes and cardiovascular disease. Insulin resistance is a core feature of the MetS. HOMA-IR is a robust clinical and epidemiological marker of MetS. Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions; its levels decrease as number of components of MetS increases. High-molecular weight adiponectin (HMWA) is the multimer responsible for the relationship of adiponectin with insulin sensitivity. HOMA-IR and HMWA are suitable candidates for MetS biomarkers. The ratio of adiponectin to HOMA-IR has been validated as a powerful index of MetS and considered a better marker of its presence, than either HOMA-IR or adiponectin alone, in selected homogeneous populations. We compared the strength of association between HMWA, HOMA-IR and HMWA/HOMA-IR ratio with MetS and its key components. Our data have shown that the median (25th, 75th percentile) of HMWA/HOMA-IR ratio was lower in subjects with MetS [0.51 (0.33, 1.31)] as compared to those without it [2.19 (1.13, 4.71)]. The correlation coefficient (r) was significantly higher for HMWA/HOMA-IR ratio as compared to HMWA for waist circumference (-0.65; -0.40, respectively); mean blood pressure (-0.27; -0.14, respectively); fasting glucose (-0.38; -0.19, respectively); HDL-cholesterol (0.44; 0.40, respectively); and triglycerides (-0.35; -0.18, respectively). In a multivariable logistic regression analysis, the HMWA/HOMA-IR ratio was a sensitive predictor for MetS, being the only marker that was significantly associated with each and all the individual components of the syndrome. These results expand on previous studies in that we used the active circulating form of adiponectin, i.e. HMWA, and represent a typical Brazilian cohort characterized by intense interethnic admixture. Thus, the HMWA/HOMA-IR ratio is a minimally invasive biomarker for MetS that could be clinically useful in prognosing patient outcome.

Humoral immune response of the small-spotted catshark, Scyliorhinus canicula.

PubMed

Crouch, Kathryn; Smith, Lauren E; Williams, Rebecca; Cao, Wei; Lee, Mike; Jensen, Allan; Dooley, Helen

2013-05-01

Cartilaginous fishes are the oldest group in which an adaptive immune system based on immunoglobulin-superfamily members is found. This manuscript compares humoral immune function in small-spotted catshark (Scyliorhinus canicula) with that described for spiny dogfish (Squalus acanthias), another member of the Squalomorphi superorder, and nurse shark, the model for humoral immunity in elasmobranchs and a member of the Galeomorphi superorder. Although small-spotted catshark and nurse shark are separated by over 200 million years we found that immunoglobulin isoforms are well conserved between the two species. However, the plasma protein profile of small-spotted catshark was most similar to that of spiny dogfish, with low levels of pentameric IgM, and IgNAR present as a multimer in plasma rather than a monomer. We show that an antigen-specific monomeric IgM response, with a profile similar to that described previously for nurse sharks, can be raised in small-spotted catshark. Lacking polyclonal or monoclonal antibody reagents for detecting catshark IgNAR we investigated phage-display and recombinant Fc-fusion protein expression as alternative methods to look for an antigen-specific response for this isotype. However, we could find no evidence of an antigen-specific IgNAR in the animals tested using either of these techniques. Thus, unlike nurse sharks where antigen-specific monomeric IgM and IgNAR appear together, it seems there may be a temporal or complete 'uncoupling' of these isotypes during a humoral response in the small-spotted catshark. Copyright © 2013 Elsevier Ltd. All rights reserved.

From Supercomputer Modeling to Highest Mass Resolution in FT-ICR.

PubMed

N Nikolaev, Evgene; N Vladimirov, Gleb; Jertz, Roland; Baykut, Gökhan

2013-01-01

Understanding of behavior of ion ensembles inside FT-ICR cell based on the computer simulation of ion motion gives rise to the new ideas of cell designs. The recently introduced novel FT-ICR cell based on a Penning ion trap with specially shaped excitation and detection electrodes prevents distortion of ion cyclotron motion phases (normally caused by non-ideal electric trapping fields) by averaging the trapping DC electric field during the ion motion in the ICR cell. Detection times of 5 min resulting in resolving power close to 40,000,000 have been reached for reserpine at m/z 609 at a magnetic field of only 7 Tesla. Fine structures of resolved 13Cn isotopic cluster groups could be measured for molecular masses up to 5.7 kDa (insulin) with resolving power of 4,000,000 at 7 Tesla. Based on resolved fine structure patterns atomic compositions can be directly determined using a new developed algorithm for fine structure processing. Mass spectra of proteins and multimers of proteins reaching masses up to 186 kDa (enolase tetramer) could be measured with isotopic resolution. For instance, at 7 Tesla resolving power of 800,000 was achieved for enolase dimer (96 kDa) and 500,000 for molecular masses above 100 kDa. Experimental data indicate that there is practically no limit for the resolving power of this ICR cell except by collisional damping in the ultrahigh vacuum chamber.

A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans

PubMed Central

Lewis, Samantha C.; Joers, Priit; Willcox, Smaranda; Griffith, Jack D.; Jacobs, Howard T.; Hyman, Bradley C.

2015-01-01

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans. PMID:25693201

Robust automated mass spectra interpretation and chemical formula calculation using mixed integer linear programming.

PubMed

Baran, Richard; Northen, Trent R

2013-10-15

Untargeted metabolite profiling using liquid chromatography and mass spectrometry coupled via electrospray ionization is a powerful tool for the discovery of novel natural products, metabolic capabilities, and biomarkers. However, the elucidation of the identities of uncharacterized metabolites from spectral features remains challenging. A critical step in the metabolite identification workflow is the assignment of redundant spectral features (adducts, fragments, multimers) and calculation of the underlying chemical formula. Inspection of the data by experts using computational tools solving partial problems (e.g., chemical formula calculation for individual ions) can be performed to disambiguate alternative solutions and provide reliable results. However, manual curation is tedious and not readily scalable or standardized. Here we describe an automated procedure for the robust automated mass spectra interpretation and chemical formula calculation using mixed integer linear programming optimization (RAMSI). Chemical rules among related ions are expressed as linear constraints and both the spectra interpretation and chemical formula calculation are performed in a single optimization step. This approach is unbiased in that it does not require predefined sets of neutral losses and positive and negative polarity spectra can be combined in a single optimization. The procedure was evaluated with 30 experimental mass spectra and was found to effectively identify the protonated or deprotonated molecule ([M + H](+) or [M - H](-)) while being robust to the presence of background ions. RAMSI provides a much-needed standardized tool for interpreting ions for subsequent identification in untargeted metabolomics workflows.

Tripping Up Trp: Modification of Protein Tryptophan Residues by Reactive Oxygen Species, Modes of Detection, and Biological Consequences

PubMed Central

Ehrenshaft, Marilyn; Deterding, Leesa J.; Mason, Ronald P.

2015-01-01

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone, and/or polymerization of monomers into dimers, multimers and sometimes into insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions which have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion limited rate with hydroxyl radical, and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral while others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and weaknesses of some of the techniques used to detect these oxidative interactions and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants and microbes. PMID:26393422

Immunochemical Localization of GABAA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa).

PubMed

Concas, A; Imperatore, R; Santoru, F; Locci, A; Porcu, P; Cristino, L; Pierobon, P

2016-11-01

γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABA A receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABA A receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ∼50 and ∼52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABA A receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.

Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

PubMed

Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

2016-02-01

A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

PubMed

Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

2008-12-02

A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats☆

PubMed Central

Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

2013-01-01

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. PMID:23648487

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

PubMed

Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

2013-08-01

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Factor Analysis and Counseling Research

ERIC Educational Resources Information Center

Weiss, David J.

1970-01-01

Topics discussed include factor analysis versus cluster analysis, analysis of Q correlation matrices, ipsativity and factor analysis, and tests for the significance of a correlation matrix prior to application of factor analytic techniques. Techniques for factor extraction discussed include principal components, canonical factor analysis, alpha…

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2'-deoxyguanosine.

PubMed

Su, Yan; Patra, Amritraj; Harp, Joel M; Egli, Martin; Guengerich, F Peter

2015-06-26

Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine*

PubMed Central

Su, Yan; Patra, Amritraj; Harp, Joel M.; Egli, Martin; Guengerich, F. Peter

2015-01-01

Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. PMID:25947374

The Gly-54-->Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.

PubMed Central

Matsushita, M; Ezekowitz, R A; Fujita, T

1995-01-01

The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP. Images Figure 1 PMID:7487919

SimGen: A General Simulation Method for Large Systems.

PubMed

Taylor, William R

2017-02-03

SimGen is a stand-alone computer program that reads a script of commands to represent complex macromolecules, including proteins and nucleic acids, in a structural hierarchy that can then be viewed using an integral graphical viewer or animated through a high-level application programming interface in C++. Structural levels in the hierarchy range from α-carbon or phosphate backbones through secondary structure to domains, molecules, and multimers with each level represented in an identical data structure that can be manipulated using the application programming interface. Unlike most coarse-grained simulation approaches, the higher-level objects represented in SimGen can be soft, allowing the lower-level objects that they contain to interact directly. The default motion simulated by SimGen is a Brownian-like diffusion that can be set to occur across all levels of representation in the hierarchy. Links can also be defined between objects, which, when combined with large high-level random movements, result in an effective search strategy for constraint satisfaction, including structure prediction from predicted pairwise distances. The implementation of SimGen makes use of the hierarchic data structure to avoid unnecessary calculation, especially for collision detection, allowing it to be simultaneously run and viewed on a laptop computer while simulating large systems of over 20,000 objects. It has been used previously to model complex molecular interactions including the motion of a myosin-V dimer "walking" on an actin fibre, RNA stem-loop packing, and the simulation of cell motion and aggregation. Several extensions to this original functionality are described. Copyright © 2016 The Francis Crick Institute. Published by Elsevier Ltd.. All rights reserved.

Urea Dependent (15)N NMR-Relaxation Studies on PfP2 Multimers Reveal that the C-Terminal Behaves like an Independent Intrinsically Disordered Peptide.

PubMed

Mishra, Pushpa; Hosur, Ramakrishna V

2015-01-01

Intrinsically disordered proteins or such domains in globular proteins are believed to be playing important roles in protein functions by virtue of their ability to adapt themselves to requirements of different binding partners and thereby accord high specificity to the interaction. Eukaryotic ribosomal stalk is made up of a supramolecular assembly of P0, P1 and P2 proteins. In Plasmodium falciparum, homo-oligomers of P2 are also seen which seem to be involved in many non-ribosomal functions of the protein in the parasite, and in all of these the protein interacts with different interactors. Here we show by extensive (15)N NMR relaxation studies that the C-terminal stretch of about 45 residues of the protein always remains as a flexible disordered domain, regardless of the state of association of the protein. The relaxation behaviors and the derived rotational correlation times for this portion of the protein are essentially the same in the presence of different concentrations of urea which produce different mixtures of PfP2 oligomers in rapid exchange, whereas the rest of the protein shows substantial variations with urea concentration in the relaxation behaviors. In other words, the C-terminal domain behaves as if it were an independent intrinsically disordered peptide. This would augment the notion that the C-terminal domain of PfP2 would be acting as a scavenger for different interactors depending upon the different functions of the protein inside the parasite.

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

PubMed Central

Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

2017-01-01

RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2–5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo. PMID:27794044

Ethanol reduces amyloid aggregation in vitro and prevents toxicity in cell lines.

PubMed

Ormeño, David; Romero, Fernando; López-Fenner, Julio; Avila, Andres; Martínez-Torres, Ataulfo; Parodi, Jorge

2013-01-01

Alzheimer's disease (AD) alters cognitive functions. A mixture of soluble β-amyloid aggregates (Aβ) are known to act as toxic agents. It has been suggested that moderate alcohol intake reduces the development of neurodegenerative diseases, but the molecular mechanisms leading to this type of prevention have been elusive. We show the ethanol effect in the generation of complex Aβ in vitro and the impact on the viability of two cell lines. The effect of ethanol on the kinetics of β-amyloid aggregation in vitro was assessed by turbimetry. Soluble- and ethanol-treated β-amyloid were added to the cell lines HEK and PC-12 to compare their effects on metabolic activity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In addition, we used molecular modeling to assess the impact of exposure to ethanol on the structure of β-amyloid. Exposure to soluble β-amyloid was toxic to both cell lines; however, exposing the cells to β-amyloid aggregated in 10 mmol ethanol prevented the effect. In silico modeling suggested that ethanol alters the dynamics for assembling Aβ by disrupting a critical salt bridge between residues Asp 23 and Lys 28, required for amyloid dimerization. Thus, ethanol prevented the formation of complex short (∼100 nm) Aβ, which are related to higher cell toxicity. Ethanol prevents the formation of stable Aβ dimers in vitro, thus protecting the cells maintained in culture. Accordingly, in silico modelling predicts that soluble β-amyloid molecules do not form stable multimers when exposed to ethanol. Copyright © 2013 IMSS. Published by Elsevier Inc. All rights reserved.

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes

NASA Astrophysics Data System (ADS)

Loo, Rachel R. Ogorzalek; Loo, Joseph A.

2016-06-01

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit "unfolding" to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein's size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size "contractions" seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes.

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes.

PubMed

Loo, Rachel R Ogorzalek; Loo, Joseph A

2016-06-01

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit "unfolding" to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein's size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size "contractions" seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes. Graphical Abstract ᅟ.

Molecular Remodeling of Photosystem II during State Transitions in Chlamydomonas reinhardtii[W

PubMed Central

Iwai, Masakazu; Takahashi, Yuichiro; Minagawa, Jun

2008-01-01

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits. PMID:18757554

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

PubMed

Ling, Feng; Hori, Akiko; Shibata, Takehiko

2007-02-01

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes.

PubMed

Valdes Franco, José A; Wang, Yi; Huo, Naxin; Ponciano, Grisel; Colvin, Howard A; McMahan, Colleen M; Gu, Yong Q; Belknap, William R

2018-04-19

Guayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N 50  = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

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

Kong, Kyoung-Ah; Gadi, Jogeswar; Park, Hyoung Woo

2008-12-05

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminalmore » of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 {mu}M, and appeared to plateau above a concentration of 1 {mu}M. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.« less

Using Horn's Parallel Analysis Method in Exploratory Factor Analysis for Determining the Number of Factors

ERIC Educational Resources Information Center

Çokluk, Ömay; Koçak, Duygu

2016-01-01

In this study, the number of factors obtained from parallel analysis, a method used for determining the number of factors in exploratory factor analysis, was compared to that of the factors obtained from eigenvalue and scree plot--two traditional methods for determining the number of factors--in terms of consistency. Parallel analysis is based on…

Determining the Number of Factors in P-Technique Factor Analysis

ERIC Educational Resources Information Center

Lo, Lawrence L.; Molenaar, Peter C. M.; Rovine, Michael

2017-01-01

Determining the number of factors is a critical first step in exploratory factor analysis. Although various criteria and methods for determining the number of factors have been evaluated in the usual between-subjects R-technique factor analysis, there is still question of how these methods perform in within-subjects P-technique factor analysis. A…

Objective identification of residue ranges for the superposition of protein structures

PubMed Central

2011-01-01

Background The automation of objectively selecting amino acid residue ranges for structure superpositions is important for meaningful and consistent protein structure analyses. So far there is no widely-used standard for choosing these residue ranges for experimentally determined protein structures, where the manual selection of residue ranges or the use of suboptimal criteria remain commonplace. Results We present an automated and objective method for finding amino acid residue ranges for the superposition and analysis of protein structures, in particular for structure bundles resulting from NMR structure calculations. The method is implemented in an algorithm, CYRANGE, that yields, without protein-specific parameter adjustment, appropriate residue ranges in most commonly occurring situations, including low-precision structure bundles, multi-domain proteins, symmetric multimers, and protein complexes. Residue ranges are chosen to comprise as many residues of a protein domain that increasing their number would lead to a steep rise in the RMSD value. Residue ranges are determined by first clustering residues into domains based on the distance variance matrix, and then refining for each domain the initial choice of residues by excluding residues one by one until the relative decrease of the RMSD value becomes insignificant. A penalty for the opening of gaps favours contiguous residue ranges in order to obtain a result that is as simple as possible, but not simpler. Results are given for a set of 37 proteins and compared with those of commonly used protein structure validation packages. We also provide residue ranges for 6351 NMR structures in the Protein Data Bank. Conclusions The CYRANGE method is capable of automatically determining residue ranges for the superposition of protein structure bundles for a large variety of protein structures. The method correctly identifies ordered regions. Global structure superpositions based on the CYRANGE residue ranges allow a clear presentation of the structure, and unnecessary small gaps within the selected ranges are absent. In the majority of cases, the residue ranges from CYRANGE contain fewer gaps and cover considerably larger parts of the sequence than those from other methods without significantly increasing the RMSD values. CYRANGE thus provides an objective and automatic method for standardizing the choice of residue ranges for the superposition of protein structures. PMID:21592348

On the Relations among Regular, Equal Unique Variances, and Image Factor Analysis Models.

ERIC Educational Resources Information Center

Hayashi, Kentaro; Bentler, Peter M.

2000-01-01

Investigated the conditions under which the matrix of factor loadings from the factor analysis model with equal unique variances will give a good approximation to the matrix of factor loadings from the regular factor analysis model. Extends the results to the image factor analysis model. Discusses implications for practice. (SLD)

Application of Factor Analysis on the Financial Ratios of Indian Cement Industry and Validation of the Results by Cluster Analysis

NASA Astrophysics Data System (ADS)

De, Anupam; Bandyopadhyay, Gautam; Chakraborty, B. N.

2010-10-01

Financial ratio analysis is an important and commonly used tool in analyzing financial health of a firm. Quite a large number of financial ratios, which can be categorized in different groups, are used for this analysis. However, to reduce number of ratios to be used for financial analysis and regrouping them into different groups on basis of empirical evidence, Factor Analysis technique is being used successfully by different researches during the last three decades. In this study Factor Analysis has been applied over audited financial data of Indian cement companies for a period of 10 years. The sample companies are listed on the Stock Exchange India (BSE and NSE). Factor Analysis, conducted over 44 variables (financial ratios) grouped in 7 categories, resulted in 11 underlying categories (factors). Each factor is named in an appropriate manner considering the factor loads and constituent variables (ratios). Representative ratios are identified for each such factor. To validate the results of Factor Analysis and to reach final conclusion regarding the representative ratios, Cluster Analysis had been performed.

From nonfinite to finite 1D arrays of origami tiles.

PubMed

Wu, Tsai Chin; Rahman, Masudur; Norton, Michael L

2014-06-17

CONSPECTUS: DNA based nanotechnology provides a basis for high-resolution fabrication of objects almost without physical size limitations. However, the pathway to large-scale production of large objects is currently unclear. Operationally, one method forward is to use high information content, large building blocks, which can be generated with high yield and reproducibility. Although flat DNA origami naturally invites comparison to pixels in zero, one, and two dimensions and voxels in three dimensions and has provided an excellent mechanism for generating blocks of significant size and complexity and a multitude of shapes, the field is young enough that a single "brick" has not become the standard platform used by the majority of researchers in the field. In this Account, we highlight factors we considered that led to our adoption of a cross-shaped, non-space-filling origami species, designed by Dr. Liu of the Seeman laboratory, as the building block ideal for use in the fabrication of finite one-dimensional arrays. Three approaches that can be employed for uniquely coding origami-origami linkages are presented. Such coding not only provides the energetics for tethering the species but also uniquely designates the relative orientation of the origami building blocks. The strength of the coding approach implemented in our laboratory is demonstrated using examples of oligomers ranging from finite multimers composed of four, six, and eight origami structures to semi-infinite polymers (100mers). Two approaches to finite array design and the series of assembly steps that each requires are discussed. The process of AFM observation for array characterization is presented as a critical case study. For these soft species, the array images do not simply present the solution phase geometry projected onto a two-dimensional surface. There are additional perturbations associated with fluidic forces associated with sample preparation. At this time, reconstruction of the "true" or average solution structures for blocks is more readily achieved using computer models than using direct imaging methods. The development of scalable 1D-origami arrays composed of uniquely addressable components is a logical, if not necessary, step in the evolution of higher order fully addressable structures. Our research into the fabrication of arrays has led us to generate a listing of several important areas of future endeavor. Of high importance is the re-enforcement of the mechanical properties of the building blocks and the organization of multiple arrays on a surface of technological importance. While addressing this short list of barriers to progress will prove challenging, coherent development along each of these lines of inquiry will accelerate the appearance of commercial scale molecular manufacturing.

A Review of CEFA Software: Comprehensive Exploratory Factor Analysis Program

ERIC Educational Resources Information Center

Lee, Soon-Mook

2010-01-01

CEFA 3.02(Browne, Cudeck, Tateneni, & Mels, 2008) is a factor analysis computer program designed to perform exploratory factor analysis. It provides the main properties that are needed for exploratory factor analysis, namely a variety of factoring methods employing eight different discrepancy functions to be minimized to yield initial…

Factor analysis of an instrument to measure the impact of disease on daily life.

PubMed

Pedrosa, Rafaela Batista Dos Santos; Rodrigues, Roberta Cunha Matheus; Padilha, Kátia Melissa; Gallani, Maria Cecília Bueno Jayme; Alexandre, Neusa Maria Costa

2016-01-01

to verify the structure of factors of an instrument to measure the Heart Valve Disease Impact on Daily Life (IDCV) when applied to coronary artery disease patients. the study included 153 coronary artery disease patients undergoing outpatient follow-up care. The IDCV structure of factors was initially assessed by means of confirmatory factor analysis and, subsequently, by exploratory factor analysis. The Varimax rotation method was used to estimate the main components of analysis, eigenvalues greater than one for extraction of factors, and factor loading greater than 0.40 for selection of items. Internal consistency was estimated using Cronbach's alpha coefficient. confirmatory factor analysis did not confirm the original structure of factors of the IDCV. Exploratory factor analysis showed three dimensions, which together explained 78% of the measurement variance. future studies with expansion of case selection are necessary to confirm the IDCV new structure of factors.

A comparison study on detection of key geochemical variables and factors through three different types of factor analysis

NASA Astrophysics Data System (ADS)

Hoseinzade, Zohre; Mokhtari, Ahmad Reza

2017-10-01

Large numbers of variables have been measured to explain different phenomena. Factor analysis has widely been used in order to reduce the dimension of datasets. Additionally, the technique has been employed to highlight underlying factors hidden in a complex system. As geochemical studies benefit from multivariate assays, application of this method is widespread in geochemistry. However, the conventional protocols in implementing factor analysis have some drawbacks in spite of their advantages. In the present study, a geochemical dataset including 804 soil samples collected from a mining area in central Iran in order to search for MVT type Pb-Zn deposits was considered to outline geochemical analysis through various fractal methods. Routine factor analysis, sequential factor analysis, and staged factor analysis were applied to the dataset after opening the data with (additive logratio) alr-transformation to extract mineralization factor in the dataset. A comparison between these methods indicated that sequential factor analysis has more clearly revealed MVT paragenesis elements in surface samples with nearly 50% variation in F1. In addition, staged factor analysis has given acceptable results while it is easy to practice. It could detect mineralization related elements while larger factor loadings are given to these elements resulting in better pronunciation of mineralization.

Exploratory Bi-factor Analysis: The Oblique Case.

PubMed

Jennrich, Robert I; Bentler, Peter M

2012-07-01

Bi-factor analysis is a form of confirmatory factor analysis originally introduced by Holzinger and Swineford (Psychometrika 47:41-54, 1937). The bi-factor model has a general factor, a number of group factors, and an explicit bi-factor structure. Jennrich and Bentler (Psychometrika 76:537-549, 2011) introduced an exploratory form of bi-factor analysis that does not require one to provide an explicit bi-factor structure a priori. They use exploratory factor analysis and a bifactor rotation criterion designed to produce a rotated loading matrix that has an approximate bi-factor structure. Among other things this can be used as an aid in finding an explicit bi-factor structure for use in a confirmatory bi-factor analysis. They considered only orthogonal rotation. The purpose of this paper is to consider oblique rotation and to compare it to orthogonal rotation. Because there are many more oblique rotations of an initial loading matrix than orthogonal rotations, one expects the oblique results to approximate a bi-factor structure better than orthogonal rotations and this is indeed the case. A surprising result arises when oblique bi-factor rotation methods are applied to ideal data.

A Brief History of the Philosophical Foundations of Exploratory Factor Analysis.

ERIC Educational Resources Information Center

Mulaik, Stanley A.

1987-01-01

Exploratory factor analysis derives its key ideas from many sources, including Aristotle, Francis Bacon, Descartes, Pearson and Yule, and Kant. The conclusions of exploratory factor analysis are never complete without subsequent confirmatory factor analysis. (Author/GDC)

Exploratory Bi-Factor Analysis: The Oblique Case

ERIC Educational Resources Information Center

Jennrich, Robert I.; Bentler, Peter M.

2012-01-01

Bi-factor analysis is a form of confirmatory factor analysis originally introduced by Holzinger and Swineford ("Psychometrika" 47:41-54, 1937). The bi-factor model has a general factor, a number of group factors, and an explicit bi-factor structure. Jennrich and Bentler ("Psychometrika" 76:537-549, 2011) introduced an exploratory form of bi-factor…

Extension Procedures for Confirmatory Factor Analysis

ERIC Educational Resources Information Center

Nagy, Gabriel; Brunner, Martin; Lüdtke, Oliver; Greiff, Samuel

2017-01-01

We present factor extension procedures for confirmatory factor analysis that provide estimates of the relations of common and unique factors with external variables that do not undergo factor analysis. We present identification strategies that build upon restrictions of the pattern of correlations between unique factors and external variables. The…

Old and New Ideas for Data Screening and Assumption Testing for Exploratory and Confirmatory Factor Analysis

PubMed Central

Flora, David B.; LaBrish, Cathy; Chalmers, R. Philip

2011-01-01

We provide a basic review of the data screening and assumption testing issues relevant to exploratory and confirmatory factor analysis along with practical advice for conducting analyses that are sensitive to these concerns. Historically, factor analysis was developed for explaining the relationships among many continuous test scores, which led to the expression of the common factor model as a multivariate linear regression model with observed, continuous variables serving as dependent variables, and unobserved factors as the independent, explanatory variables. Thus, we begin our paper with a review of the assumptions for the common factor model and data screening issues as they pertain to the factor analysis of continuous observed variables. In particular, we describe how principles from regression diagnostics also apply to factor analysis. Next, because modern applications of factor analysis frequently involve the analysis of the individual items from a single test or questionnaire, an important focus of this paper is the factor analysis of items. Although the traditional linear factor model is well-suited to the analysis of continuously distributed variables, commonly used item types, including Likert-type items, almost always produce dichotomous or ordered categorical variables. We describe how relationships among such items are often not well described by product-moment correlations, which has clear ramifications for the traditional linear factor analysis. An alternative, non-linear factor analysis using polychoric correlations has become more readily available to applied researchers and thus more popular. Consequently, we also review the assumptions and data-screening issues involved in this method. Throughout the paper, we demonstrate these procedures using an historic data set of nine cognitive ability variables. PMID:22403561

Expression of the A56 and K2 proteins is sufficient to inhibit vaccinia virus entry and cell fusion.

PubMed

Wagenaar, Timothy R; Moss, Bernard

2009-02-01

Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease

PubMed Central

Manocha, Gunjan D.; Floden, Angela M.; Rausch, Keiko; Kulas, Joshua A.; McGregor, Brett A.; Rojanathammanee, Lalida; Puig, Kelley R.; Puig, Kendra L.; Karki, Sanjib; Nichols, Michael R.; Darland, Diane C.; Porter, James E.

2016-01-01

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP−/− mice compared with wild-type mice. The mAPP−/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains. PMID:27511018

Total and high molecular weight adiponectin are elevated in patients with Laron syndrome despite marked obesity.

PubMed

Kanety, Hannah; Hemi, Rina; Ginsberg, Shira; Pariente, Clara; Yissachar, Eleanor; Barhod, Ehud; Funahashi, Tohru; Laron, Zvi

2009-12-01

Patients with Laron syndrome (LS; primary GH insensitivity) caused by molecular defects of the GH receptor gene, are characterized by dwarfism, profound obesity, and hyperlipidemia. The aim of the current study was to evaluate adiponectin levels in LS, as obesity is known to be associated with low adiponectin. We studied nine untreated LS adult patients (5 males, 4 females) and six girls with LS receiving once-daily treatment by IGF1. Total and high molecular weight (HMW) adiponectin levels, adiponectin multimers distribution, and metabolic indices were analyzed in serum samples obtained during several years of follow-up. Adiponectin levels in the severely obese adult LS patients (percent body fat; females 61.0+/-2.5%, males 40.6+/-8.1%) were two- to three-fold higher than those reported for subjects of corresponding age, gender and degree of adiposity. Total adiponectin was significantly higher in females compared with males (21.4+/-3.5 vs 10.2+/-4.6 microg/ml, P<0.001). The elevated adiponectin in LS subjects was associated with an increased abundance of the HMW isoform, and positively correlated with body fat percentage (r=0.65, P=0.017) and leptin (r=0.65, P=0.012). There was no correlation between adiponectin levels (total and HMW) and the degree of insulin resistance in LS subjects or their blood lipids levels. Adiponectin was also high in young girls with LS (22.9+/-7.4 microg/ml) and did not change during long-term IGF1 replacement therapy. Adiponectin hypersecretion in LS, despite profound obesity, suggests that GH activity may negatively impact adiponectin secretion from adipocytes.

Insulation of Enhancer-Promoter Communication by a Gypsy Transposon Insert in the Drosophila cut Gene: Cooperation between Suppressor of Hairy-wing and Modifier of mdg4 Proteins

PubMed Central

Gause, Maria; Morcillo, Patrick; Dorsett, Dale

2001-01-01

The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein. PMID:11416154

Bioinformatics Reveal Five Lineages of Oleosins and the Mechanism of Lineage Evolution Related to Structure/Function from Green Algae to Seed Plants1[OPEN

PubMed Central

Huang, Ming-Der; Huang, Anthony H.C.

2015-01-01

Plant cells contain subcellular lipid droplets with a triacylglycerol matrix enclosed by a layer of phospholipids and the small structural protein oleosin. Oleosins possess a conserved central hydrophobic hairpin of approximately 72 residues penetrating into the lipid droplet matrix and amphipathic amino- and carboxyl (C)-terminal peptides lying on the phospholipid surface. Bioinformatics of 1,000 oleosins of green algae and all plants emphasizing biological implications reveal five oleosin lineages: primitive (in green algae, mosses, and ferns), universal (U; all land plants), and three in specific organs or phylogenetic groups, termed seed low-molecular-weight (SL; seed plants), seed high-molecular-weight (SH; angiosperms), and tapetum (T; Brassicaceae) oleosins. Transition from one lineage to the next is depicted from lineage intermediates at junctions of phylogeny and organ distributions. Within a species, each lineage, except the T oleosin lineage, has one to four genes per haploid genome, only approximately two of which are active. Primitive oleosins already possess all the general characteristics of oleosins. U oleosins have C-terminal sequences as highly conserved as the hairpin sequences; thus, U oleosins including their C-terminal peptide exert indispensable, unknown functions. SL and SH oleosin transcripts in seeds are in an approximately 1:1 ratio, which suggests the occurrence of SL-SH oleosin dimers/multimers. T oleosins in Brassicaceae are encoded by rapidly evolved multitandem genes for alkane storage and transfer. Overall, oleosins have evolved to retain conserved hairpin structures but diversified for unique structures and functions in specific cells and plant families. Also, our studies reveal oleosin in avocado (Persea americana) mesocarp and no acyltransferase/lipase motifs in most oleosins. PMID:26232488

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools.

PubMed

Mandacaru, Samuel C; do Vale, Luis H F; Vahidi, Siavash; Xiao, Yiming; Skinner, Owen S; Ricart, Carlos A O; Kelleher, Neil L; de Sousa, Marcelo Valle; Konermann, Lars

2017-03-21

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP1 4 apisimin 4 ) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316 VLFFGLV 322 . Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP1 2 apisimin 1 ) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

2015-01-01

APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

PubMed Central

Tixador, Philippe; Herzog, Laëtitia; Reine, Fabienne; Jaumain, Emilie; Chapuis, Jérôme; Le Dur, Annick; Laude, Hubert; Béringue, Vincent

2010-01-01

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrPSc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics. PMID:20419156

A Simple Negative Interaction in the Positive Transcriptional Feedback of a Single Gene Is Sufficient to Produce Reliable Oscillations

PubMed Central

Miró-Bueno, Jesús M.; Rodríguez-Patón, Alfonso

2011-01-01

Negative and positive transcriptional feedback loops are present in natural and synthetic genetic oscillators. A single gene with negative transcriptional feedback needs a time delay and sufficiently strong nonlinearity in the transmission of the feedback signal in order to produce biochemical rhythms. A single gene with only positive transcriptional feedback does not produce oscillations. Here, we demonstrate that this single-gene network in conjunction with a simple negative interaction can also easily produce rhythms. We examine a model comprised of two well-differentiated parts. The first is a positive feedback created by a protein that binds to the promoter of its own gene and activates the transcription. The second is a negative interaction in which a repressor molecule prevents this protein from binding to its promoter. A stochastic study shows that the system is robust to noise. A deterministic study identifies that the dynamics of the oscillator are mainly driven by two types of biomolecules: the protein, and the complex formed by the repressor and this protein. The main conclusion of this paper is that a simple and usual negative interaction, such as degradation, sequestration or inhibition, acting on the positive transcriptional feedback of a single gene is a sufficient condition to produce reliable oscillations. One gene is enough and the positive transcriptional feedback signal does not need to activate a second repressor gene. This means that at the genetic level an explicit negative feedback loop is not necessary. The model needs neither cooperative binding reactions nor the formation of protein multimers. Therefore, our findings could help to clarify the design principles of cellular clocks and constitute a new efficient tool for engineering synthetic genetic oscillators. PMID:22205920

Adrenocorticotropic Hormone (ACTH) Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane.

PubMed

Malik, Sundeep; Dolan, Terrance M; Maben, Zachary J; Hinkle, Patricia M

2015-11-13

The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (Nout) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Papillomavirus Type 16 E6 Induces Self-Ubiquitination of the E6AP Ubiquitin-Protein Ligase

PubMed Central

Kao, Wynn H.; Beaudenon, Sylvie L.; Talis, Andrea L.; Huibregtse, Jon M.; Howley, Peter M.

2000-01-01

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells. PMID:10864652

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

PubMed

Jahangeer, S; Rodbell, M

1993-10-01

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process.

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

PubMed Central

Jahangeer, S; Rodbell, M

1993-01-01

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process. Images Fig. 1 Fig. 2 PMID:8415607

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

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

Raymond, Donald D.; Piper, Mary E.; Gerrard, Sonja R.

2010-07-13

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-{angstrom} crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA speciesmore » of {approx}100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous {approx}100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.« less

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.

PubMed

Baker, Christopher L; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M; Paigen, Kenneth

2015-09-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

PubMed Central

Baker, Christopher L.; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M.; Paigen, Kenneth

2015-01-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 +/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PMID:26368021

Pigment organization and their interactions in reaction centers of photosystem II: optical spectroscopy at 6 K of reaction centers with modified pheophytin composition.

PubMed

Germano, M; Shkuropatov, A Y; Permentier, H; de Wijn, R; Hoff, A J; Shuvalov, V A; van Gorkom, H J

2001-09-25

Photosystem II reaction centers (RC) with selectively exchanged pheophytin (Pheo) molecules as described in [Germano, M., Shkuropatov, A. Ya., Permentier, H., Khatypov, R. A., Shuvalov, V. A., Hoff, A. J., and van Gorkom, H. J. (2000) Photosynth. Res. 64, 189-198] were studied by low-temperature absorption, linear and circular dichroism, and triplet-minus-singlet absorption-difference spectroscopy. The ratio of extinction coefficients epsilon(Pheo)/epsilon(Chl) for Q(Y) absorption in the RC is approximately 0.40 at 6 K and approximately 0.45 at room temperature. The presence of 2 beta-carotenes, one parallel and one perpendicular to the membrane plane, is confirmed. Absorption at 670 nm is due to the perpendicular Q(Y) transitions of the two peripheral chlorophylls (Chl) and not to either Pheo. The "core" pigments, two Pheo and four Chl absorb in the 676-685 nm range. Delocalized excited states as predicted by the "multimer model" are seen in the active branch. The inactive Pheo and the nearby Chl, however, mainly contribute localized transitions at 676 and 680 nm, respectively, although large CD changes indicate that exciton interactions are present on both branches. Replacement of the active Pheo prevents triplet formation, causes an LD increase at 676 and 681 nm, a blue-shift of 680 nm absorbance, and a bleach of the 685 nm exciton band. The triplet state is mainly localized on the Chl corresponding to B(A) in purple bacteria. Both Pheo Q(Y) transitions are oriented out of the membrane plane. Their Q(X) transitions are parallel to that plane, so that the Pheos in PSII are structurally similar to their homologues in purple bacteria.

Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

PubMed Central

Diaz, Arturo; Gallei, Andreas

2012-01-01

All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell. PMID:22090102

Felix Hoppe-Seyler Lecture 1997. Protective antibody responses against viruses.

PubMed

Zinkernagel, R M

1997-08-01

Neutralizing antibody responses against the acute cytopathic vesicular stomatitis virus (VSV) have been studied in mice to evaluate their general characteristics including specificity, self-/non-self discrimination and memory. IgM responses are generated very early, by day 3 to 4, in a T helper cell-independent fashion and without VSV having polyclonal activating capacities. The order of the glycoprotein tips on the virus envelope (multiple, 8-10 nm distance, paracrystalline) exhibiting the neutralizing determinants are key to this prompt response. These paracrystalline identical multimeric antigens are characteristic of infectious agents and are always reacted against by B cells. Self-antigens that are accessible to B cells in the intact host are either monomeric in serum or mobile multimers on cell surfaces; these configurations need contact dependent or contact independent T help, respectively. Because T help is tolerant against self-antigens, no anti-self B cell responses are usually induced against monomeric self-antigens. If collagen or DNA (rigid multimeric self-antigens) become accessible, however, they may become targets of auto-antibody responses. The antibody repertoire against VSV is partially contained in the germline and partially is generated by somatic mutation; they seem not to undergo affinity-maturation. In any case protection against lethal infection is dependent upon strictly T helper cell dependent IgG generated by day 6 to 7 and reaches a protective level of about 1-10 micrograms/ml. Interesting affinity/avidity and onrate above a minimal threshold are of no apparent advantage for protection in vivo. Maintenance of these antibody levels by antigen depots, and not the presence of memory B cells alone, is key to providing protective immunological memory. Collectively these data suggest that studying biologically important protective antibody responses may modify some of the parameters that have been defined by studying hapten specific antibody responses.

NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

PubMed Central

Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

2013-01-01

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy. PMID:22907642

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

PubMed Central

Spearman, P; Horton, R; Ratner, L; Kuli-Zade, I

1997-01-01

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction. PMID:9261380

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

PubMed Central

Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.

2017-01-01

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state. PMID:28283573

Factor Structure of the Social Appearance Anxiety Scale in Turkish Early Adolescents

ERIC Educational Resources Information Center

Sahin, Ertugrul; Topkaya, Nursel

2015-01-01

Although the Social Appearance Anxiety Scale (SAAS) is most often validated with the use of confirmatory factor analysis (CFA) on undergraduate students, exploratory factor analysis and multiple factor retention decision criteria necessitate the analysis of underlying factor structure to prevent over and under factoring as well as to reveal…

Factor Analysis via Components Analysis

ERIC Educational Resources Information Center

Bentler, Peter M.; de Leeuw, Jan

2011-01-01

When the factor analysis model holds, component loadings are linear combinations of factor loadings, and vice versa. This interrelation permits us to define new optimization criteria and estimation methods for exploratory factor analysis. Although this article is primarily conceptual in nature, an illustrative example and a small simulation show…

Mixture Factor Analysis for Approximating a Nonnormally Distributed Continuous Latent Factor with Continuous and Dichotomous Observed Variables

ERIC Educational Resources Information Center

Wall, Melanie M.; Guo, Jia; Amemiya, Yasuo

2012-01-01

Mixture factor analysis is examined as a means of flexibly estimating nonnormally distributed continuous latent factors in the presence of both continuous and dichotomous observed variables. A simulation study compares mixture factor analysis with normal maximum likelihood (ML) latent factor modeling. Different results emerge for continuous versus…

Factor Analysis of Drawings: Application to College Student Models of the Greenhouse Effect

ERIC Educational Resources Information Center

Libarkin, Julie C.; Thomas, Stephen R.; Ording, Gabriel

2015-01-01

Exploratory factor analysis was used to identify models underlying drawings of the greenhouse effect made by over 200 entering university freshmen. Initial content analysis allowed deconstruction of drawings into salient features, with grouping of these features via factor analysis. A resulting 4-factor solution explains 62% of the data variance,…

Exploratory factor analysis of the 12-item Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being Scale in people newly diagnosed with advanced cancer.

PubMed

Bai, Mei; Dixon, Jane K

2014-01-01

The purpose of this study was to reexamine the factor pattern of the 12-item Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being Scale (FACIT-Sp-12) using exploratory factor analysis in people newly diagnosed with advanced cancer. Principal components analysis (PCA) and 3 common factor analysis methods were used to explore the factor pattern of the FACIT-Sp-12. Factorial validity was assessed in association with quality of life (QOL). Principal factor analysis (PFA), iterative PFA, and maximum likelihood suggested retrieving 3 factors: Peace, Meaning, and Faith. Both Peace and Meaning positively related to QOL, whereas only Peace uniquely contributed to QOL. This study supported the 3-factor model of the FACIT-Sp-12. Suggestions for revision of items and further validation of the identified factor pattern were provided.

Community factors to promote parents' quality of child-nurturing life.

PubMed

Aoyama, Megumi; Wei, Chang Nian; Chang-nian, Wei; Harada, Koichi; Ueda, Kimiyo; Takano, Miyuki; Ueda, Atsushi

2013-01-01

The purpose of this study was to clarify the role of community factors in parents' quality of child-nurturing life (QCNL). We developed a questionnaire to evaluate the degree of QCNL and determine the structural factors related to QCNL as community factors related to parents' QCNL derived from focus group interviews and the Delphi technique. The questionnaire also included the battery of the self-rating depression scale and Tsumori-Inage Infant's Developmental Test. Using the questionnaire, we then conducted a quantitative survey of parents whose children attended nursery schools in Kumamoto Prefecture. Factor analysis, calculation of the mean score and/or ratio to each item, Pearson's correlation coefficient, t test, multiple regression analysis, and covariance structure analysis were performed. The questionnaire we developed consisted of seven items with 75 elements, involving ten elements as community factors. Subjects included 699 parents (mean age 33.6 ± 5.4 years) and 965 children (age range 0-6 years). Factor analysis revealed that community factors consisted of five factors, such as "lifestyle rooted in the ground," "balance of housekeeping and work," "community network," "amenity," and "regeneration of life". These factors may be dominant in a rural area. Finally, we developed a structural model with "community factors," QCNL, QOL, and "child growth" by covariance structural analysis. The analysis revealed that community factors had a positive relation to parents' QCNL (r = 0.81, p < 0.001) and that parental SDS score had a negative relation to parents' QCNL (r = -0.59, p < 0.001). The analysis did show that community factors were positively related to the sound growth of children. The covariance structure analysis revealed that community factors were associated with parents' QCNL, SDS, and "child growth."

Analysis of Performance Factors for Accounting and Finance Related Business Courses in a Distance Education Environment

ERIC Educational Resources Information Center

Benligiray, Serdar; Onay, Ahmet

2017-01-01

The objective of this study is to explore business courses performance factors with a focus on accounting and finance. Course score interrelations are assumed to represent interpretable constructs of these factors. Factor analysis is proposed to identify the constructs that explain the correlations. Factor analysis results identify three…

The Infinitesimal Jackknife with Exploratory Factor Analysis

ERIC Educational Resources Information Center

Zhang, Guangjian; Preacher, Kristopher J.; Jennrich, Robert I.

2012-01-01

The infinitesimal jackknife, a nonparametric method for estimating standard errors, has been used to obtain standard error estimates in covariance structure analysis. In this article, we adapt it for obtaining standard errors for rotated factor loadings and factor correlations in exploratory factor analysis with sample correlation matrices. Both…

A Mapping from the Human Factors Analysis and Classification System (DOD-HFACS) to the Domains of Human Systems Integration (HSI)

DTIC Science & Technology

2009-11-01

Equation Chapter 1 Section 1 A MAPPING FROM THE HUMAN FACTORS ANALYSIS AND CLASSIFICATION SYSTEM (DOD...OMB control number. 1. REPORT DATE NOV 2009 2. REPORT TYPE 3. DATES COVERED 4. TITLE AND SUBTITLE A Mapping from the Human Factors Analysis ...7 The Human Factors Analysis and Classification System .................................................. 7 Mapping of DoD

A new technique for ordering asymmetrical three-dimensional data sets in ecology.

PubMed

Pavoine, Sandrine; Blondel, Jacques; Baguette, Michel; Chessel, Daniel

2007-02-01

The aim of this paper is to tackle the problem that arises from asymmetrical data cubes formed by two crossed factors fixed by the experimenter (factor A and factor B, e.g., sites and dates) and a factor which is not controlled for (the species). The entries of this cube are densities in species. We approach this kind of data by the comparison of patterns, that is to say by analyzing first the effect of factor B on the species-factor A pattern, and second the effect of factor A on the species-factor B pattern. The analysis of patterns instead of individual responses requires a correspondence analysis. We use a method we call Foucart's correspondence analysis to coordinate the correspondence analyses of several independent matrices of species x factor A (respectively B) type, corresponding to each modality of factor B (respectively A). Such coordination makes it possible to evaluate the effect of factor B (respectively A) on the species-factor A (respectively B) pattern. The results obtained by such a procedure are much more insightful than those resulting from a classical single correspondence analysis applied to the global matrix that is obtained by simply unrolling the data cube, juxtaposing for example the individual species x factor A matrices through modalities of factor B. This is because a single global correspondence analysis combines three effects of factors in a way that cannot be determined from factorial maps (factor A, factor B, and factor A x factor B interaction) whereas the applications of Foucart's correspondence analysis clearly discriminate two different issues. Using two data sets, we illustrate that this technique proves to be particularly powerful in the analyses of ecological convergence which include several distinct data sets and in the analyses of spatiotemporal variations of species distributions.

Inventory of File nam.t00z.awiphi00.tm00.grib2

Science.gov Websites

Factor [non-dim] 041 50 mb HGT analysis Geopotential Height [gpm] 042 50 mb TMP analysis Temperature [K /kg] 052 50 mb RIME analysis Rime Factor [non-dim] 053 75 mb HGT analysis Geopotential Height [gpm SNMR analysis Snow Mixing Ratio [kg/kg] 064 75 mb RIME analysis Rime Factor [non-dim] 065 100 mb HGT

Using BMDP and SPSS for a Q factor analysis.

PubMed

Tanner, B A; Koning, S M

1980-12-01

While Euclidean distances and Q factor analysis may sometimes be preferred to correlation coefficients and cluster analysis for developing a typology, commercially available software does not always facilitate their use. Commands are provided for using BMDP and SPSS in a Q factor analysis with Euclidean distances.

Text mining factor analysis (TFA) in green tea patent data

NASA Astrophysics Data System (ADS)

Rahmawati, Sela; Suprijadi, Jadi; Zulhanif

2017-03-01

Factor analysis has become one of the most widely used multivariate statistical procedures in applied research endeavors across a multitude of domains. There are two main types of analyses based on factor analysis: Exploratory Factor Analysis (EFA) and Confirmatory Factor Analysis (CFA). Both EFA and CFA aim to observed relationships among a group of indicators with a latent variable, but they differ fundamentally, a priori and restrictions made to the factor model. This method will be applied to patent data technology sector green tea to determine the development technology of green tea in the world. Patent analysis is useful in identifying the future technological trends in a specific field of technology. Database patent are obtained from agency European Patent Organization (EPO). In this paper, CFA model will be applied to the nominal data, which obtain from the presence absence matrix. While doing processing, analysis CFA for nominal data analysis was based on Tetrachoric matrix. Meanwhile, EFA model will be applied on a title from sector technology dominant. Title will be pre-processing first using text mining analysis.

Psychometric properties of Connor-Davidson Resilience Scale in a Spanish sample of entrepreneurs.

PubMed

Manzano-García, Guadalupe; Ayala Calvo, Juan Carlos

2013-01-01

The literature regarding entrepreneurship suggests that the resilience of entrepreneurs may help to explain entrepreneurial success, but there is no resilience measure widely accepted by researchers. This study analyzes the psychometric properties of the Connor and Davidson Resilience Scale (CD-RISC) in a sample of Spanish entrepreneurs. A telephone survey research method was used. The participants were entrepreneurs operating in the business services sector. Interviewers telephoned a total of 900 entrepreneurs of whom 783 produced usable questionnaires. The CD-RISC was used as data collection instrument. We used principal component analysis factor and confirmatory factor analysis to determine the factor structure of the CD-RISC. Confirmatory factor analysis failed to verify the original five-factor structure of the CD-RISC, whereas principal component analysis factor yielded a 3-factor structure of resilience (hardiness, resourcefulness and optimism). In this research, 47.48% of the total variance was accounted for by three factors, and the obtained factor structure was verified through confirmatory factor analysis. The CD-RISC has been shown to be a reliable and valid tool for measuring entrepreneurs' resilience.

Bootstrap Standard Error Estimates in Dynamic Factor Analysis

ERIC Educational Resources Information Center

Zhang, Guangjian; Browne, Michael W.

2010-01-01

Dynamic factor analysis summarizes changes in scores on a battery of manifest variables over repeated measurements in terms of a time series in a substantially smaller number of latent factors. Algebraic formulae for standard errors of parameter estimates are more difficult to obtain than in the usual intersubject factor analysis because of the…

Factor Analysis of the Aberrant Behavior Checklist in Individuals with Autism Spectrum Disorders

ERIC Educational Resources Information Center

Brinkley, Jason; Nations, Laura; Abramson, Ruth K.; Hall, Alicia; Wright, Harry H.; Gabriels, Robin; Gilbert, John R.; Pericak-Vance, Margaret A. O.; Cuccaro, Michael L.

2007-01-01

Exploratory factor analysis (varimax and promax rotations) of the aberrant behavior checklist-community version (ABC) in 275 individuals with Autism spectrum disorder (ASD) identified four- and five-factor solutions which accounted for greater than 70% of the variance. Confirmatory factor analysis (Lisrel 8.7) revealed indices of moderate fit for…

How Factor Analysis Can Be Used in Classification.

ERIC Educational Resources Information Center

Harman, Harry H.

This is a methodological study that suggests a taxometric technique for objective classification of yeasts. It makes use of the minres method of factor analysis and groups strains of yeast according to their factor profiles. The similarities are judged in the higher-dimensional space determined by the factor analysis, but otherwise rely on the…

A Factor Analysis of Learning Data and Selected Ability Test Scores

ERIC Educational Resources Information Center

Jones, Dorothy L.

1976-01-01

A verbal concept-learning task permitting the externalizing and quantifying of learning behavior and 16 ability tests were administered to female graduate students. Data were analyzed by alpha factor analysis and incomplete image analysis. Six alpha factors and 12 image factors were extracted and orthogonally rotated. Four areas of cognitive…

Emotional Intelligence and Nurse Recruitment: Rasch and confirmatory factor analysis of the trait emotional intelligence questionnaire short form.

PubMed

Snowden, Austyn; Watson, Roger; Stenhouse, Rosie; Hale, Claire

2015-12-01

To examine the construct validity of the Trait Emotional Intelligence Questionnaire Short form. Emotional intelligence involves the identification and regulation of our own emotions and the emotions of others. It is therefore a potentially useful construct in the investigation of recruitment and retention in nursing and many questionnaires have been constructed to measure it. Secondary analysis of existing dataset of responses to Trait Emotional Intelligence Questionnaire Short form using concurrent application of Rasch analysis and confirmatory factor analysis. First year undergraduate nursing and computing students completed Trait Emotional Intelligence Questionnaire-Short Form in September 2013. Responses were analysed by synthesising results of Rasch analysis and confirmatory factor analysis. Participants (N = 938) completed Trait Emotional Intelligence Questionnaire Short form. Rasch analysis showed the majority of the Trait Emotional Intelligence Questionnaire-Short Form items made a unique contribution to the latent trait of emotional intelligence. Five items did not fit the model and differential item functioning (gender) accounted for this misfit. Confirmatory factor analysis revealed a four-factor structure consisting of: self-confidence, empathy, uncertainty and social connection. All five misfitting items from the Rasch analysis belonged to the 'social connection' factor. The concurrent use of Rasch and factor analysis allowed for novel interpretation of Trait Emotional Intelligence Questionnaire Short form. Much of the response variation in Trait Emotional Intelligence Questionnaire Short form can be accounted for by the social connection factor. Implications for practice are discussed. © 2015 John Wiley & Sons Ltd.

The Relation between Factor Score Estimates, Image Scores, and Principal Component Scores

ERIC Educational Resources Information Center

Velicer, Wayne F.

1976-01-01

Investigates the relation between factor score estimates, principal component scores, and image scores. The three methods compared are maximum likelihood factor analysis, principal component analysis, and a variant of rescaled image analysis. (RC)

Exploring the Factor Structure of Neurocognitive Measures in Older Individuals

PubMed Central

Santos, Nadine Correia; Costa, Patrício Soares; Amorim, Liliana; Moreira, Pedro Silva; Cunha, Pedro; Cotter, Jorge; Sousa, Nuno

2015-01-01

Here we focus on factor analysis from a best practices point of view, by investigating the factor structure of neuropsychological tests and using the results obtained to illustrate on choosing a reasonable solution. The sample (n=1051 individuals) was randomly divided into two groups: one for exploratory factor analysis (EFA) and principal component analysis (PCA), to investigate the number of factors underlying the neurocognitive variables; the second to test the “best fit” model via confirmatory factor analysis (CFA). For the exploratory step, three extraction (maximum likelihood, principal axis factoring and principal components) and two rotation (orthogonal and oblique) methods were used. The analysis methodology allowed exploring how different cognitive/psychological tests correlated/discriminated between dimensions, indicating that to capture latent structures in similar sample sizes and measures, with approximately normal data distribution, reflective models with oblimin rotation might prove the most adequate. PMID:25880732

Application of multivariate statistical techniques for differentiation of ripe banana flour based on the composition of elements.

PubMed

Alkarkhi, Abbas F M; Ramli, Saifullah Bin; Easa, Azhar Mat

2009-01-01

Major (sodium, potassium, calcium, magnesium) and minor elements (iron, copper, zinc, manganese) and one heavy metal (lead) of Cavendish banana flour and Dream banana flour were determined, and data were analyzed using multivariate statistical techniques of factor analysis and discriminant analysis. Factor analysis yielded four factors explaining more than 81% of the total variance: the first factor explained 28.73%, comprising magnesium, sodium, and iron; the second factor explained 21.47%, comprising only manganese and copper; the third factor explained 15.66%, comprising zinc and lead; while the fourth factor explained 15.50%, comprising potassium. Discriminant analysis showed that magnesium and sodium exhibited a strong contribution in discriminating the two types of banana flour, affording 100% correct assignation. This study presents the usefulness of multivariate statistical techniques for analysis and interpretation of complex mineral content data from banana flour of different varieties.

Biomotor structures in elite female handball players according to performance.

PubMed

Cavala, Marijana; Rogulj, Nenad; Srhoj, Vatromir; Srhoj, Ljerka; Katić, Ratko

2008-03-01

In order to identify biomotor structures in elite female handball players, factor structures of morphological characteristics and basic motor abilities, and of variables evaluating situation motor abilities of elite female handball players (n = 53) were determined first, followed by determination of differences and relations of the morphological, motor and specific motor space according to handball performance. Factor analysis of 16 morphological measures produced three morphological factors, i.e. factor of absolute voluminosity, i.e. mesoendomorphy, factor of longitudinal skeleton dimensionality, and factor of transverse hand dimensionality. Factor analysis of 15 motor variables yielded five basic motor dimensions, i.e. factor of agility, factor of throwing explosive strength, factor of running explosive strength (sprint), factor of jumping explosive strength and factor of movement frequency rate. Factor analysis of 5 situation motor variables produced two dimensions: factor of specific agility with explosiveness and factor of specific precision with ball manipulation. Analysis of variance yielded greatest differences relative to handball performance in the factor of specific agility and throwing strength, and the factor of basic motoricity that integrates the ability of coordination (agility) with upper extremity throwing explosiveness and lower extremity sprint (30-m sprint) and jumping (standing triple jump). Considering morphological factors, the factor of voluminosity, i.e. mesoendomorphy, which is defined by muscle mass rather than adipose tissue, was found to contribute significantly to the players'performance. Results of regression analysis indicated the handball performance to be predominantly determined by the general specific motor factor based on specific agility and explosiveness, and by the morphological factor based on body mass and volume, i.e. muscle mass. Concerning basic motor abilities, the factor of movement frequency rate, which is associated with the ability of ball manipulation, was observed to predict significantly the handball players' performance.

Bayesian Factor Analysis When Only a Sample Covariance Matrix Is Available

ERIC Educational Resources Information Center

Hayashi, Kentaro; Arav, Marina

2006-01-01

In traditional factor analysis, the variance-covariance matrix or the correlation matrix has often been a form of inputting data. In contrast, in Bayesian factor analysis, the entire data set is typically required to compute the posterior estimates, such as Bayes factor loadings and Bayes unique variances. We propose a simple method for computing…

Factor Retention in Exploratory Factor Analysis: A Comparison of Alternative Methods.

ERIC Educational Resources Information Center

Mumford, Karen R.; Ferron, John M.; Hines, Constance V.; Hogarty, Kristine Y.; Kromrey, Jeffery D.

This study compared the effectiveness of 10 methods of determining the number of factors to retain in exploratory common factor analysis. The 10 methods included the Kaiser rule and a modified Kaiser criterion, 3 variations of parallel analysis, 4 regression-based variations of the scree procedure, and the minimum average partial procedure. The…

What School Psychologists Need to Know about Factor Analysis

ERIC Educational Resources Information Center

McGill, Ryan J.; Dombrowski, Stefan C.

2017-01-01

Factor analysis is a versatile class of psychometric techniques used by researchers to provide insight into the psychological dimensions (factors) that may account for the relationships among variables in a given dataset. The primary goal of a factor analysis is to determine a more parsimonious set of variables (i.e., fewer than the number of…

Evaluation of Parallel Analysis Methods for Determining the Number of Factors

ERIC Educational Resources Information Center

Crawford, Aaron V.; Green, Samuel B.; Levy, Roy; Lo, Wen-Juo; Scott, Lietta; Svetina, Dubravka; Thompson, Marilyn S.

2010-01-01

Population and sample simulation approaches were used to compare the performance of parallel analysis using principal component analysis (PA-PCA) and parallel analysis using principal axis factoring (PA-PAF) to identify the number of underlying factors. Additionally, the accuracies of the mean eigenvalue and the 95th percentile eigenvalue criteria…

A Factor Analysis of Functional Independence and Functional Assessment Measure Scores Among Focal and Diffuse Brain Injury Patients: The Importance of Bifactor Models.

PubMed

Gunn, Sarah; Burgess, Gerald H; Maltby, John

2018-04-30

To explore the factor structure of the UK Functional Independence Measure and Functional Assessment Measure (FIM+FAM) among focal and diffuse acquired brain injury patients. Criterion standard. A National Health Service acute acquired brain injury inpatient rehabilitation hospital. Referred sample of N=447 adults admitted for inpatient treatment following an acquired brain injury significant enough to justify intensive inpatient neurorehabilitation INTERVENTION: Not applicable. Functional Independence Measure and Functional Assessment Measure. Exploratory factor analysis suggested a 2-factor structure to FIM+FAM scores, among both focal-proximate and diffuse-proximate acquired brain injury aetiologies. Confirmatory factor analysis suggested a 3-factor bifactor structure presented the best fit of the FIM+FAM score data across both aetiologies. However, across both analyses, a convergence was found towards a general factor, demonstrated by high correlations between factors in the exploratory factor analysis, and by a general factor explaining the majority of the variance in scores on confirmatory factor analysis. Our findings suggested that although factors describing specific functional domains can be derived from FIM+FAM item scores, there is a convergence towards a single factor describing overall functioning. This single factor informs the specific group factors (eg, motor, psychosocial, and communication function) after brain injury. Further research into the comparative value of the general and group factors as evaluative/prognostic measures is indicated. Copyright © 2018 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Psychometric analysis of the Brisbane Practice Environment Measure (B-PEM).

PubMed

Flint, Anndrea; Farrugia, Charles; Courtney, Mary; Webster, Joan

2010-03-01

To undertake rigorous psychometric testing of the newly developed contemporary work environment measure (the Brisbane Practice Environment Measure [B-PEM]) using exploratory factor analysis and confirmatory factor analysis. Content validity of the 33-item measure was established by a panel of experts. Initial testing involved 195 nursing staff using principal component factor analysis with varimax rotation (orthogonal) and Cronbach's alpha coefficients. Confirmatory factor analysis was conducted using data from a further 983 nursing staff. Principal component factor analysis yielded a four-factor solution with eigenvalues greater than 1 that explained 52.53% of the variance. These factors were then verified using confirmatory factor analysis. Goodness-of-fit indices showed an acceptable fit overall with the full model, explaining 21% to 73% of the variance. Deletion of items took place throughout the evolution of the instrument, resulting in a 26-item, four-factor measure called the Brisbane Practice Environment Measure-Tested. The B-PEM has undergone rigorous psychometric testing, providing evidence of internal consistency and goodness-of-fit indices within acceptable ranges. The measure can be utilised as a subscale or total score reflective of a contemporary nursing work environment. An up-to-date instrument to measure practice environment may be useful for nursing leaders to monitor the workplace and to assist in identifying areas for improvement, facilitating greater job satisfaction and retention.

Likelihood-Based Confidence Intervals in Exploratory Factor Analysis

ERIC Educational Resources Information Center

Oort, Frans J.

2011-01-01

In exploratory or unrestricted factor analysis, all factor loadings are free to be estimated. In oblique solutions, the correlations between common factors are free to be estimated as well. The purpose of this article is to show how likelihood-based confidence intervals can be obtained for rotated factor loadings and factor correlations, by…

Comparisons of Exploratory and Confirmatory Factor Analysis.

ERIC Educational Resources Information Center

Daniel, Larry G.

Historically, most researchers conducting factor analysis have used exploratory methods. However, more recently, confirmatory factor analytic methods have been developed that can directly test theory either during factor rotation using "best fit" rotation methods or during factor extraction, as with the LISREL computer programs developed…

Strategic Analysis and Plan for Implementing Telemedicine at Fort Greely

DTIC Science & Technology

2003-03-01

Analysis The Situational Analysis tool assessed the environmental, market , and organizational factors involved in a Fort Greely telemedicine... Factors ): Medicaid reimbursement is now approved for Alaska regardless of method of healthcare delivery. Market Factors (Customers): The influx of...arrive are Active National Guardsmen and Fort Greely Telemedicine 50 their families. Market Factors (Services): Fairbanks Memorial Hospital (FMH) can

Toward Reflective Judgment in Exploratory Factor Analysis Decisions: Determining the Extraction Method and Number of Factors To Retain.

ERIC Educational Resources Information Center

Knight, Jennifer L.

This paper considers some decisions that must be made by the researcher conducting an exploratory factor analysis. The primary purpose is to aid the researcher in making informed decisions during the factor analysis instead of relying on defaults in statistical programs or traditions of previous researchers. Three decision areas are addressed.…

Factor Analysis of the Brazilian Version of UPPS Impulsive Behavior Scale.

PubMed

Sediyama, Cristina Y N; Moura, Ricardo; Garcia, Marina S; da Silva, Antonio G; Soraggi, Carolina; Neves, Fernando S; Albuquerque, Maicon R; Whiteside, Setephen P; Malloy-Diniz, Leandro F

2017-01-01

Objective: To examine the internal consistency and factor structure of the Brazilian adaptation of the UPPS Impulsive Behavior Scale. Methods: UPPS is a self-report scale composed by 40 items assessing four factors of impulsivity: (a) urgency, (b) lack of premeditation; (c) lack of perseverance; (d) sensation seeking. In the present study 384 participants (278 women and 106 men), who were recruited from schools, universities, leisure centers and workplaces fulfilled the UPPS scale. An exploratory factor analysis was performed by using Varimax factor rotation and Kaiser Normalization, and we also conducted two confirmatory analyses to test the independency of the UPPS components found in previous analysis. Results: Results showed a decrease in mean UPPS total scores with age and this analysis showed that the youngest participants (below 30 years) scored significantly higher than the other groups over 30 years. No difference in gender was found. Cronbach's alpha, results indicated satisfactory values for all subscales, with similar high values for the subscales and confirmatory factor analysis indexes also indicated a poor model fit. The results of two exploratory factor analysis were satisfactory. Conclusion: Our results showed that the Portuguese version has the same four-factor structure of the original and previous translations of the UPPS.

Using factor analysis to identify neuromuscular synergies during treadmill walking

NASA Technical Reports Server (NTRS)

Merkle, L. A.; Layne, C. S.; Bloomberg, J. J.; Zhang, J. J.

1998-01-01

Neuroscientists are often interested in grouping variables to facilitate understanding of a particular phenomenon. Factor analysis is a powerful statistical technique that groups variables into conceptually meaningful clusters, but remains underutilized by neuroscience researchers presumably due to its complicated concepts and procedures. This paper illustrates an application of factor analysis to identify coordinated patterns of whole-body muscle activation during treadmill walking. Ten male subjects walked on a treadmill (6.4 km/h) for 20 s during which surface electromyographic (EMG) activity was obtained from the left side sternocleidomastoid, neck extensors, erector spinae, and right side biceps femoris, rectus femoris, tibialis anterior, and medial gastrocnemius. Factor analysis revealed 65% of the variance of seven muscles sampled aligned with two orthogonal factors, labeled 'transition control' and 'loading'. These two factors describe coordinated patterns of muscular activity across body segments that would not be evident by evaluating individual muscle patterns. The results show that factor analysis can be effectively used to explore relationships among muscle patterns across all body segments to increase understanding of the complex coordination necessary for smooth and efficient locomotion. We encourage neuroscientists to consider using factor analysis to identify coordinated patterns of neuromuscular activation that would be obscured using more traditional EMG analyses.

Product competitiveness analysis for e-commerce platform of special agricultural products

NASA Astrophysics Data System (ADS)

Wan, Fucheng; Ma, Ning; Yang, Dongwei; Xiong, Zhangyuan

2017-09-01

On the basis of analyzing the influence factors of the product competitiveness of the e-commerce platform of the special agricultural products and the characteristics of the analytical methods for the competitiveness of the special agricultural products, the price, the sales volume, the postage included service, the store reputation, the popularity, etc. were selected in this paper as the dimensionality for analyzing the competitiveness of the agricultural products, and the principal component factor analysis was taken as the competitiveness analysis method. Specifically, the web crawler was adopted to capture the information of various special agricultural products in the e-commerce platform ---- chi.taobao.com. Then, the original data captured thereby were preprocessed and MYSQL database was adopted to establish the information library for the special agricultural products. Then, the principal component factor analysis method was adopted to establish the analysis model for the competitiveness of the special agricultural products, and SPSS was adopted in the principal component factor analysis process to obtain the competitiveness evaluation factor system (support degree factor, price factor, service factor and evaluation factor) of the special agricultural products. Then, the linear regression method was adopted to establish the competitiveness index equation of the special agricultural products for estimating the competitiveness of the special agricultural products.

An Evaluation of the Effects of Variable Sampling on Component, Image, and Factor Analysis.

ERIC Educational Resources Information Center

Velicer, Wayne F.; Fava, Joseph L.

1987-01-01

Principal component analysis, image component analysis, and maximum likelihood factor analysis were compared to assess the effects of variable sampling. Results with respect to degree of saturation and average number of variables per factor were clear and dramatic. Differential effects on boundary cases and nonconvergence problems were also found.…

A Comparison of Component and Factor Patterns: A Monte Carlo Approach.

ERIC Educational Resources Information Center

Velicer, Wayne F.; And Others

1982-01-01

Factor analysis, image analysis, and principal component analysis are compared with respect to the factor patterns they would produce under various conditions. The general conclusion that is reached is that the three methods produce results that are equivalent. (Author/JKS)

Inventory of File nam.t00z.awipak00.tm00.grib2

Science.gov Websites

Rime Factor [non-dim] 009 1 hybrid level HGT analysis Geopotential Height [gpm] 010 1 hybrid level TMP [kg/kg] 040 30 mb SNMR analysis Snow Mixing Ratio [kg/kg] 041 30 mb RIME analysis Rime Factor [non-dim Factor [non-dim] 054 75 mb HGT analysis Geopotential Height [gpm] 055 75 mb TMP analysis Temperature [K

On the Likelihood Ratio Test for the Number of Factors in Exploratory Factor Analysis

ERIC Educational Resources Information Center

Hayashi, Kentaro; Bentler, Peter M.; Yuan, Ke-Hai

2007-01-01

In the exploratory factor analysis, when the number of factors exceeds the true number of factors, the likelihood ratio test statistic no longer follows the chi-square distribution due to a problem of rank deficiency and nonidentifiability of model parameters. As a result, decisions regarding the number of factors may be incorrect. Several…

Scale-Free Nonparametric Factor Analysis: A User-Friendly Introduction with Concrete Heuristic Examples.

ERIC Educational Resources Information Center

Mittag, Kathleen Cage

Most researchers using factor analysis extract factors from a matrix of Pearson product-moment correlation coefficients. A method is presented for extracting factors in a non-parametric way, by extracting factors from a matrix of Spearman rho (rank correlation) coefficients. It is possible to factor analyze a matrix of association such that…

Assessing suicide risk among callers to crisis hotlines: a confirmatory factor analysis.

PubMed

Witte, Tracy K; Gould, Madelyn S; Munfakh, Jimmie Lou Harris; Kleinman, Marjorie; Joiner, Thomas E; Kalafat, John

2010-09-01

Our goal was to investigate the factor structure of a risk assessment tool utilized by suicide hotlines and to determine the predictive validity of the obtained factors in predicting subsequent suicidal behavior. We conducted an Exploratory Factor Analysis (EFA), an EFA in a Confirmatory Factor Analysis (EFA/CFA) framework, and a CFA on independent subsamples derived from a total sample of 1,085. Similar to previous studies, we found consistent evidence for a two-factor solution, with one factor representing a more pernicious form of suicide risk (i.e., Resolved Plans and Preparations; RPP) and one factor representing milder suicidal ideation (i.e., Suicidal Desire and Ideation; SDI). The RPP factor trended toward being more predictive of suicidal ideation at follow-up than the SDI factor. (c) 2010 Wiley Periodicals, Inc.

Bootstrap Confidence Intervals for Ordinary Least Squares Factor Loadings and Correlations in Exploratory Factor Analysis

ERIC Educational Resources Information Center

Zhang, Guangjian; Preacher, Kristopher J.; Luo, Shanhong

2010-01-01

This article is concerned with using the bootstrap to assign confidence intervals for rotated factor loadings and factor correlations in ordinary least squares exploratory factor analysis. Coverage performances of "SE"-based intervals, percentile intervals, bias-corrected percentile intervals, bias-corrected accelerated percentile…

Analysis of Factors Influencing Creative Personality of Elementary School Students

ERIC Educational Resources Information Center

Park, Jongman; Kim, Minkee; Jang, Shinho

2017-01-01

This quantitative research examined factors that affect elementary students' creativity and how those factors correlate. Aiming to identify significant factors that affect creativity and to clarify the relationship between these factors by path analysis, this research was designed to be a stepping stone for creativity enhancement studies. Data…

Confirmatory Factor Analysis of the Delirium Rating Scale Revised-98 (DRS-R98).

PubMed

Thurber, Steven; Kishi, Yasuhiro; Trzepacz, Paula T; Franco, Jose G; Meagher, David J; Lee, Yanghyun; Kim, Jeong-Lan; Furlanetto, Leticia M; Negreiros, Daniel; Huang, Ming-Chyi; Chen, Chun-Hsin; Kean, Jacob; Leonard, Maeve

2015-01-01

Principal components analysis applied to the Delirium Rating Scale-Revised-98 contributes to understanding the delirium construct. Using a multisite pooled international delirium database, the authors applied confirmatory factor analysis to Delirium Rating Scale-Revised-98 scores from 859 adult patients evaluated by delirium experts (delirium, N=516; nondelirium, N=343). Confirmatory factor analysis found all diagnostic features and core symptoms (cognitive, language, thought process, sleep-wake cycle, motor retardation), except motor agitation, loaded onto factor 1. Motor agitation loaded onto factor 2 with noncore symptoms (delusions, affective lability, and perceptual disturbances). Factor 1 loading supports delirium as a single construct, but when accompanied by psychosis, motor agitation's role may not be solely as a circadian activity indicator.

A dynamic factor model of the evaluation of the financial crisis in Turkey.

PubMed

Sezgin, F; Kinay, B

2010-01-01

Factor analysis has been widely used in economics and finance in situations where a relatively large number of variables are believed to be driven by few common causes of variation. Dynamic factor analysis (DFA) which is a combination of factor and time series analysis, involves autocorrelation matrices calculated from multivariate time series. Dynamic factor models were traditionally used to construct economic indicators, macroeconomic analysis, business cycles and forecasting. In recent years, dynamic factor models have become more popular in empirical macroeconomics. They have more advantages than other methods in various respects. Factor models can for instance cope with many variables without running into scarce degrees of freedom problems often faced in regression-based analysis. In this study, a model which determines the effect of the global crisis on Turkey is proposed. The main aim of the paper is to analyze how several macroeconomic quantities show an alteration before the evolution of the crisis and to decide if a crisis can be forecasted or not.

49 CFR Appendix D to Part 172 - Rail Risk Analysis Factors

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 2 2011-10-01 2011-10-01 false Rail Risk Analysis Factors D Appendix D to Part... REQUIREMENTS, AND SECURITY PLANS Pt. 172, App. D Appendix D to Part 172—Rail Risk Analysis Factors A. This... safety and security risk analyses required by § 172.820. The risk analysis to be performed may be...

49 CFR Appendix D to Part 172 - Rail Risk Analysis Factors

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Rail Risk Analysis Factors D Appendix D to Part... REQUIREMENTS, AND SECURITY PLANS Pt. 172, App. D Appendix D to Part 172—Rail Risk Analysis Factors A. This... safety and security risk analyses required by § 172.820. The risk analysis to be performed may be...

Critical Factors Analysis for Offshore Software Development Success by Structural Equation Modeling

NASA Astrophysics Data System (ADS)

Wada, Yoshihisa; Tsuji, Hiroshi

In order to analyze the success/failure factors in offshore software development service by the structural equation modeling, this paper proposes to follow two approaches together; domain knowledge based heuristic analysis and factor analysis based rational analysis. The former works for generating and verifying of hypothesis to find factors and causalities. The latter works for verifying factors introduced by theory to build the model without heuristics. Following the proposed combined approaches for the responses from skilled project managers of the questionnaire, this paper found that the vendor property has high causality for the success compared to software property and project property.

[Lake eutrophication modeling in considering climatic factors change: a review].

PubMed

Su, Jie-Qiong; Wang, Xuan; Yang, Zhi-Feng

2012-11-01

Climatic factors are considered as the key factors affecting the trophic status and its process in most lakes. Under the background of global climate change, to incorporate the variations of climatic factors into lake eutrophication models could provide solid technical support for the analysis of the trophic evolution trend of lake and the decision-making of lake environment management. This paper analyzed the effects of climatic factors such as air temperature, precipitation, sunlight, and atmosphere on lake eutrophication, and summarized the research results about the lake eutrophication modeling in considering in considering climatic factors change, including the modeling based on statistical analysis, ecological dynamic analysis, system analysis, and intelligent algorithm. The prospective approaches to improve the accuracy of lake eutrophication modeling with the consideration of climatic factors change were put forward, including 1) to strengthen the analysis of the mechanisms related to the effects of climatic factors change on lake trophic status, 2) to identify the appropriate simulation models to generate several scenarios under proper temporal and spatial scales and resolutions, and 3) to integrate the climatic factors change simulation, hydrodynamic model, ecological simulation, and intelligent algorithm into a general modeling system to achieve an accurate prediction of lake eutrophication under climatic change.

Three Factors Are Critical in Order to Synthesize Intelligible Noise-Vocoded Japanese Speech

PubMed Central

Kishida, Takuya; Nakajima, Yoshitaka; Ueda, Kazuo; Remijn, Gerard B.

2016-01-01

Factor analysis (principal component analysis followed by varimax rotation) had shown that 3 common factors appear across 20 critical-band power fluctuations derived from spoken sentences of eight different languages [Ueda et al. (2010). Fechner Day 2010, Padua]. The present study investigated the contributions of such power-fluctuation factors to speech intelligibility. The method of factor analysis was modified to obtain factors suitable for resynthesizing speech sounds as 20-critical-band noise-vocoded speech. The resynthesized speech sounds were used for an intelligibility test. The modification of factor analysis ensured that the resynthesized speech sounds were not accompanied by a steady background noise caused by the data reduction procedure. Spoken sentences of British English, Japanese, and Mandarin Chinese were subjected to this modified analysis. Confirming the earlier analysis, indeed 3–4 factors were common to these languages. The number of power-fluctuation factors needed to make noise-vocoded speech intelligible was then examined. Critical-band power fluctuations of the Japanese spoken sentences were resynthesized from the obtained factors, resulting in noise-vocoded-speech stimuli, and the intelligibility of these speech stimuli was tested by 12 native Japanese speakers. Japanese mora (syllable-like phonological unit) identification performances were measured when the number of factors was 1–9. Statistically significant improvement in intelligibility was observed when the number of factors was increased stepwise up to 6. The 12 listeners identified 92.1% of the morae correctly on average in the 6-factor condition. The intelligibility improved sharply when the number of factors changed from 2 to 3. In this step, the cumulative contribution ratio of factors improved only by 10.6%, from 37.3 to 47.9%, but the average mora identification leaped from 6.9 to 69.2%. The results indicated that, if the number of factors is 3 or more, elementary linguistic information is preserved in such noise-vocoded speech. PMID:27199790

Logistic regression analysis of factors associated with avascular necrosis of the femoral head following femoral neck fractures in middle-aged and elderly patients.

PubMed

Ai, Zi-Sheng; Gao, You-Shui; Sun, Yuan; Liu, Yue; Zhang, Chang-Qing; Jiang, Cheng-Hua

2013-03-01

Risk factors for femoral neck fracture-induced avascular necrosis of the femoral head have not been elucidated clearly in middle-aged and elderly patients. Moreover, the high incidence of screw removal in China and its effect on the fate of the involved femoral head require statistical methods to reflect their intrinsic relationship. Ninety-nine patients older than 45 years with femoral neck fracture were treated by internal fixation between May 1999 and April 2004. Descriptive analysis, interaction analysis between associated factors, single factor logistic regression, multivariate logistic regression, and detailed interaction analysis were employed to explore potential relationships among associated factors. Avascular necrosis of the femoral head was found in 15 cases (15.2 %). Age × the status of implants (removal vs. maintenance) and gender × the timing of reduction were interactive according to two-factor interactive analysis. Age, the displacement of fractures, the quality of reduction, and the status of implants were found to be significant factors in single factor logistic regression analysis. Age, age × the status of implants, and the quality of reduction were found to be significant factors in multivariate logistic regression analysis. In fine interaction analysis after multivariate logistic regression analysis, implant removal was the most important risk factor for avascular necrosis in 56-to-85-year-old patients, with a risk ratio of 26.00 (95 % CI = 3.076-219.747). The middle-aged and elderly have less incidence of avascular necrosis of the femoral head following femoral neck fractures treated by cannulated screws. The removal of cannulated screws can induce a significantly high incidence of avascular necrosis of the femoral head in elderly patients, while a high-quality reduction is helpful to reduce avascular necrosis.

A Factor Analysis of the BSRI and the PAQ.

ERIC Educational Resources Information Center

Edwards, Teresa A.; And Others

Factor analysis of the Bem Sex Role Inventory (BSRI) and the Personality Attributes Questionnaire (PAQ) was undertaken to study the independence of the masculine and feminine scales within each instrument. Both instruments were administered to undergraduate education majors. Analysis of primary first and second order factors of the BSRI indicated…

Cross-Validation of Levenson's Psychopathy Scale in a Sample of Federal Female Inmates

ERIC Educational Resources Information Center

Brinkley, Chad A.; Diamond, Pamela M.; Magaletta, Philip R.; Heigel, Caron P.

2008-01-01

Levenson, Kiehl, and Fitzpatrick's Self-Report Psychopathy Scale (LSRPS) is evaluated to determine the factor structure and concurrent validity of the instrument among 430 federal female inmates. Confirmatory factor analysis fails to validate the expected 2-factor structure. Subsequent exploratory factor analysis reveals a 3-factor structure…

Confirmatory Factor Analysis of the WISC-III with Child Psychiatric Inpatients.

ERIC Educational Resources Information Center

Tupa, David J.; Wright, Margaret O'Dougherty; Fristad, Mary A.

1997-01-01

Factor models of the Wechsler Intelligence Scale for Children-Third Edition (WISC-III) for one, two, three, and four factors were tested using confirmatory factor analysis with a sample of 177 child psychiatric inpatients. The four-factor model proposed in the WISC-III manual provided the best fit to the data. (SLD)

Spectral compression algorithms for the analysis of very large multivariate images

DOEpatents

Keenan, Michael R.

2007-10-16

A method for spectrally compressing data sets enables the efficient analysis of very large multivariate images. The spectral compression algorithm uses a factored representation of the data that can be obtained from Principal Components Analysis or other factorization technique. Furthermore, a block algorithm can be used for performing common operations more efficiently. An image analysis can be performed on the factored representation of the data, using only the most significant factors. The spectral compression algorithm can be combined with a spatial compression algorithm to provide further computational efficiencies.

A replication of a factor analysis of motivations for trapping

USGS Publications Warehouse

Schroeder, Susan; Fulton, David C.

2015-01-01

Using a 2013 sample of Minnesota trappers, we employed confirmatory factor analysis to replicate an exploratory factor analysis of trapping motivations conducted by Daigle, Muth, Zwick, and Glass (1998).  We employed the same 25 items used by Daigle et al. and tested the same five-factor structure using a recent sample of Minnesota trappers. We also compared motivations in our sample to those reported by Daigle et el.

Factor Analysis of the Brazilian Version of UPPS Impulsive Behavior Scale

PubMed Central

Sediyama, Cristina Y. N.; Moura, Ricardo; Garcia, Marina S.; da Silva, Antonio G.; Soraggi, Carolina; Neves, Fernando S.; Albuquerque, Maicon R.; Whiteside, Setephen P.; Malloy-Diniz, Leandro F.

2017-01-01

Objective: To examine the internal consistency and factor structure of the Brazilian adaptation of the UPPS Impulsive Behavior Scale. Methods: UPPS is a self-report scale composed by 40 items assessing four factors of impulsivity: (a) urgency, (b) lack of premeditation; (c) lack of perseverance; (d) sensation seeking. In the present study 384 participants (278 women and 106 men), who were recruited from schools, universities, leisure centers and workplaces fulfilled the UPPS scale. An exploratory factor analysis was performed by using Varimax factor rotation and Kaiser Normalization, and we also conducted two confirmatory analyses to test the independency of the UPPS components found in previous analysis. Results: Results showed a decrease in mean UPPS total scores with age and this analysis showed that the youngest participants (below 30 years) scored significantly higher than the other groups over 30 years. No difference in gender was found. Cronbach’s alpha, results indicated satisfactory values for all subscales, with similar high values for the subscales and confirmatory factor analysis indexes also indicated a poor model fit. The results of two exploratory factor analysis were satisfactory. Conclusion: Our results showed that the Portuguese version has the same four-factor structure of the original and previous translations of the UPPS. PMID:28484414

Factors affecting job satisfaction in nurse faculty: a meta-analysis.

PubMed

Gormley, Denise K

2003-04-01

Evidence in the literature suggests job satisfaction can make a difference in keeping qualified workers on the job, but little research has been conducted focusing specifically on nursing faculty. Several studies have examined nurse faculty satisfaction in relationship to one or two influencing factors. These factors include professional autonomy, leader role expectations, organizational climate, perceived role conflict and role ambiguity, leadership behaviors, and organizational characteristics. This meta-analysis attempts to synthesize the various studies conducted on job satisfaction in nursing faculty and analyze which influencing factors have the greatest effect. The procedure used for this meta-analysis consisted of reviewing studies to identify factors influencing job satisfaction, research questions, sample size reported, instruments used for measurement of job satisfaction and influencing factors, and results of statistical analysis.

Application of factor analysis to the water quality in reservoirs

NASA Astrophysics Data System (ADS)

Silva, Eliana Costa e.; Lopes, Isabel Cristina; Correia, Aldina; Gonçalves, A. Manuela

2017-06-01

In this work we present a Factor Analysis of chemical and environmental variables of the water column and hydro-morphological features of several Portuguese reservoirs. The objective is to reduce the initial number of variables, keeping their common characteristics. Using the Factor Analysis, the environmental variables measured in the epilimnion and in the hypolimnion, together with the hydromorphological characteristics of the dams were reduced from 63 variables to only 13 factors, which explained a total of 83.348% of the variance in the original data. After performing rotation using the Varimax method, the relations between the factors and the original variables got clearer and more explainable, which provided a Factor Analysis model for these environmental variables using 13 varifactors: Water quality and distance to the source, Hypolimnion chemical composition, Sulfite-reducing bacteria and nutrients, Coliforms and faecal streptococci, Reservoir depth, Temperature, Location, among other factors.

Donor retention in health care in Iran: a factor analysis

PubMed Central

Aghababa, Sara; Nasiripour, Amir Ashkan; Maleki, Mohammadreza; Gohari, Mahmoodreza

2017-01-01

Background: Long-term financial support is essential for the survival of a charitable organization. Health charities need to identify the effective factors influencing donor retention. Methods: In the present study, the items of a questionnaire were derived from both literature review and semi-structured interviews related to donor retention. Using a purposive sampling, 300 academic and executive practitioners were selected. After the follow- up, a total of 243 usable questionnaires were prepared for factor analysis. The questionnaire was validated based on the face and content validity and reliability through Cronbach’s α-coefficient. Results: The results of exploratory factor analysis extracted 2 factors for retention: donor factor (variance = 33.841%; Cronbach’s α-coefficient = 90.2) and charity factor (variance = 29.038%; Cronbach’s α-coefficient = 82.8), respectively. Subsequently, confirmatory factor analysis was applied to support the overall reasonable fit. Conclusions: In this study, it was found that repeated monetary donations are supplied to the charitable organizations when both aspects of donor factor (retention factor and charity factor) for retention are taken into consideration. This model could provide a perspective for making sustainable donations and charitable giving PMID:28955663

Ultrasound-enhanced bioscouring of greige cotton: regression analysis of process factors

USDA-ARS?s Scientific Manuscript database

Process factors of enzyme concentration, time, power and frequency were investigated for ultrasound-enhanced bioscouring of greige cotton. A fractional factorial experimental design and subsequent regression analysis of the process factors were employed to determine the significance of each factor a...

Quantitative analysis of factors that affect oil pipeline network accident based on Bayesian networks: A case study in China

NASA Astrophysics Data System (ADS)

Zhang, Chao; Qin, Ting Xin; Huang, Shuai; Wu, Jian Song; Meng, Xin Yan

2018-06-01

Some factors can affect the consequences of oil pipeline accident and their effects should be analyzed to improve emergency preparation and emergency response. Although there are some qualitative analysis models of risk factors' effects, the quantitative analysis model still should be researched. In this study, we introduce a Bayesian network (BN) model of risk factors' effects analysis in an oil pipeline accident case that happened in China. The incident evolution diagram is built to identify the risk factors. And the BN model is built based on the deployment rule for factor nodes in BN and the expert knowledge by Dempster-Shafer evidence theory. Then the probabilities of incident consequences and risk factors' effects can be calculated. The most likely consequences given by this model are consilient with the case. Meanwhile, the quantitative estimations of risk factors' effects may provide a theoretical basis to take optimal risk treatment measures for oil pipeline management, which can be used in emergency preparation and emergency response.

The School Counseling Program Implementation Survey: Initial Instrument Development and Exploratory Factor Analysis

ERIC Educational Resources Information Center

Clemens, Elysia V.; Carey, John C.; Harrington, Karen M.

2010-01-01

This article details the initial development of the School Counseling Program Implementation Survey and psychometric results including reliability and factor structure. An exploratory factor analysis revealed a three-factor model that accounted for 54% of the variance of the intercorrelation matrix and a two-factor model that accounted for 47% of…

Affective Outcomes of Schooling: Full-Information Item Factor Analysis of a Student Questionnaire.

ERIC Educational Resources Information Center

Muraki, Eiji; Engelhard, George, Jr.

Recent developments in dichotomous factor analysis based on multidimensional item response models (Bock and Aitkin, 1981; Muthen, 1978) provide an effective method for exploring the dimensionality of questionnaire items. Implemented in the TESTFACT program, this "full information" item factor analysis accounts not only for the pairwise joint…

Item Factor Analysis: Current Approaches and Future Directions

ERIC Educational Resources Information Center

Wirth, R. J.; Edwards, Michael C.

2007-01-01

The rationale underlying factor analysis applies to continuous and categorical variables alike; however, the models and estimation methods for continuous (i.e., interval or ratio scale) data are not appropriate for item-level data that are categorical in nature. The authors provide a targeted review and synthesis of the item factor analysis (IFA)…

Spatial Dependence and Heterogeneity in Bayesian Factor Analysis: A Cross-National Investigation of Schwartz Values

ERIC Educational Resources Information Center

Stakhovych, Stanislav; Bijmolt, Tammo H. A.; Wedel, Michel

2012-01-01

In this article, we present a Bayesian spatial factor analysis model. We extend previous work on confirmatory factor analysis by including geographically distributed latent variables and accounting for heterogeneity and spatial autocorrelation. The simulation study shows excellent recovery of the model parameters and demonstrates the consequences…

Q-Type Factor Analysis of Healthy Aged Men.

ERIC Educational Resources Information Center

Kleban, Morton H.

Q-type factor analysis was used to re-analyze baseline data collected in 1957, on 47 men aged 65-91. Q-type analysis is the use of factor methods to study persons rather than tests. Although 550 variables were originally studied involving psychiatry, medicine, cerebral metabolism and chemistry, personality, audiometry, dichotic and diotic memory,…

Simulating the Effects of Common and Specific Abilities on Test Performance: An Evaluation of Factor Analysis

ERIC Educational Resources Information Center

McFarland, Dennis J.

2014-01-01

Purpose: Factor analysis is a useful technique to aid in organizing multivariate data characterizing speech, language, and auditory abilities. However, knowledge of the limitations of factor analysis is essential for proper interpretation of results. The present study used simulated test scores to illustrate some characteristics of factor…

On the stability analysis of approximate factorization methods for 3D Euler and Navier-Stokes equations

NASA Technical Reports Server (NTRS)

Demuren, A. O.; Ibraheem, S. O.

1993-01-01

The convergence characteristics of various approximate factorizations for the 3D Euler and Navier-Stokes equations are examined using the von-Neumann stability analysis method. Three upwind-difference based factorizations and several central-difference based factorizations are considered for the Euler equations. In the upwind factorizations both the flux-vector splitting methods of Steger and Warming and van Leer are considered. Analysis of the Navier-Stokes equations is performed only on the Beam and Warming central-difference scheme. The range of CFL numbers over which each factorization is stable is presented for one-, two-, and three-dimensional flow. Also presented for each factorization is the CFL number at which the maximum eigenvalue is minimized, for all Fourier components, as well as for the high frequency range only. The latter is useful for predicting the effectiveness of multigrid procedures with these schemes as smoothers. Further, local mode analysis is performed to test the suitability of using a uniform flow field in the stability analysis. Some inconsistencies in the results from previous analyses are resolved.

Measuring Prosocial Tendencies in Germany: Sources of Validity and Reliablity of the Revised Prosocial Tendency Measure

PubMed Central

Rodrigues, Johannes; Ulrich, Natalie; Mussel, Patrick; Carlo, Gustavo; Hewig, Johannes

2017-01-01

The prosocial tendencies measure (PTM; Carlo and Randall, 2002) is a widely used measurement for prosocial tendencies in English speaking participants. This instrument distinguishes between six different types of prosocial tendencies that partly share some common basis, but also can be opposed to each other. To examine these constructs in Germany, a study with 1067 participants was conducted. The study investigated the structure of this German version of the PTM-R via exploratory factor analysis, confirmatory factor analysis, correlations with similar constructs in subsamples as well as via measurement invariance test concerning the original English version. The German translation showed a similar factor structure to the English version in exploratory factor analysis and in confirmatory factor analysis. Measurement invariance was found between the English and German language versions of the PTM and support for the proposed six-factor structure (altruistic, anonymous, compliant, dire, emotional and public prosocial behavior) was also found in confirmatory factor analysis. Furthermore, the expected interrelations of these factors of prosocial behavior tendencies were obtained. Finally, correlations of the prosocial behavior tendencies with validating constructs and behaviors were found. Thus, the findings stress the importance of seeing prosocial behavior not as a single dimension construct, but as a factored construct which now can also be assessed in German speaking participants. PMID:29270144

Instruments measuring perceived racism/racial discrimination: review and critique of factor analytic techniques.

PubMed

Atkins, Rahshida

2014-01-01

Several compendiums of instruments that measure perceived racism and/or discrimination are present in the literature. Other works have reviewed the psychometric properties of these instruments in terms of validity and reliability and have indicated if the instrument was factor analyzed. However, little attention has been given to the quality of the factor analysis performed. The aim of this study was to evaluate the exploratory factor analyses done on instruments measuring perceived racism/racial discrimination using guidelines from experts in psychometric theory. The techniques used for factor analysis were reviewed and critiqued and the adequacy of reporting was evaluated. Internet search engines and four electronic abstract databases were used to identify 16 relevant instruments that met the inclusion/exclusion criteria. Principal component analysis was the most frequent method of extraction (81%). Sample sizes were adequate for factor analysis in 81 percent of studies. The majority of studies reported appropriate criteria for the acceptance of un-rotated factors (81%) and justified the rotation method (75%). Exactly 94 percent of studies reported partially acceptable criteria for the acceptance of rotated factors. The majority of articles (69%) reported adequate coefficient alphas for the resultant subscales. In 81 percent of the studies, the conceptualized dimensions were supported by factor analysis.

INSTRUMENTS MEASURING PERCEIVED RACISM/RACIAL DISCRIMINATION: REVIEW AND CRITIQUE OF FACTOR ANALYTIC TECHNIQUES

PubMed Central

Atkins, Rahshida

2015-01-01

Several compendiums of instruments that measure perceived racism and/or discrimination are present in the literature. Other works have reviewed the psychometric properties of these instruments in terms of validity and reliability and have indicated if the instrument was factor analyzed. However, little attention has been given to the quality of the factor analysis performed. The aim of this study was to evaluate the exploratory factor analyses done on instruments measuring perceived racism/racial discrimination using guidelines from experts in psychometric theory. The techniques used for factor analysis were reviewed and critiqued and the adequacy of reporting was evaluated. Internet search engines and four electronic abstract databases were used to identify 16 relevant instruments that met the inclusion/exclusion criteria. Principal component analysis was the most frequent method of extraction (81%). Sample sizes were adequate for factor analysis in 81 percent of studies. The majority of studies reported appropriate criteria for the acceptance of un-rotated factors (81%) and justified the rotation method (75%). Exactly 94 percent of studies reported partially acceptable criteria for the acceptance of rotated factors. The majority of articles (69%) reported adequate coefficient alphas for the resultant subscales. In 81 percent of the studies, the conceptualized dimensions were supported by factor analysis. PMID:25626225

Factor Analysis of Intern Effectiveness

ERIC Educational Resources Information Center

Womack, Sid T.; Hannah, Shellie Louise; Bell, Columbus David

2012-01-01

Four factors in teaching intern effectiveness, as measured by a Praxis III-similar instrument, were found among observational data of teaching interns during the 2010 spring semester. Those factors were lesson planning, teacher/student reflection, fairness & safe environment, and professionalism/efficacy. This factor analysis was as much of a…

Applying Cognitive Work Analysis to Time Critical Targeting Functionality

DTIC Science & Technology

2004-10-01

Cognitive Task Analysis , CTA, Cognitive Task Analysis , Human Factors, GUI, Graphical User Interface, Heuristic Evaluation... Cognitive Task Analysis MITRE Briefing January 2000 Dynamic Battle Management Functional Architecture 3-1 Section 3 Human Factors...clear distinction between Cognitive Work Analysis (CWA) and Cognitive Task Analysis (CTA), therefore this document will refer to these

Evaluating voice characteristics of first-year acting students in Israel: factor analysis.

PubMed

Amir, Ofer; Primov-Fever, Adi; Kushnir, Tami; Kandelshine-Waldman, Osnat; Wolf, Michael

2013-01-01

Acting students require diverse, high-quality, and high-intensity vocal performance from early stages of their training. Demanding vocal activities, before developing the appropriate vocal skills, put them in high risk for developing vocal problems. A retrospective analysis of voice characteristics of first-year acting students using several voice evaluation tools. A total of 79 first-year acting students (55 women and 24 men) were assigned into two study groups: laryngeal findings (LFs) and no laryngeal findings, based on stroboscopic findings. Their voice characteristics were evaluated using acoustic analysis, aerodynamic examination, perceptual scales, and self-report questionnaires. Results obtained from each set of measures were examined using a factor analysis approach. Significant differences between the two groups were found for a single fundamental frequency (F(0))-Regularity factor; a single Grade, Roughness, Breathiness, Asthenia, Strain perceptual factor; and the three self-evaluation factors. Gender differences were found for two acoustic analysis factors, which were based on F(0) and its derivatives, namely an aerodynamic factor that represents expiratory volume measurements and a single self-evaluation factor that represents the tendency to seek therapy. Approximately 50% of the first-year acting students had LFs. These students differed from their peers in the control group in a single acoustic analysis factor, as well as perceptual and self-report factors. No group differences, however, were found for the aerodynamic factors. Early laryngeal examination and voice evaluation of future professional voice users could provide a valuable individual baseline, to which later examinations could be compared, and assist in providing personally tailored treatment. Copyright © 2013 The Voice Foundation. Published by Mosby, Inc. All rights reserved.

Risky Business: Factor Analysis of Survey Data – Assessing the Probability of Incorrect Dimensionalisation

PubMed Central

van der Eijk, Cees; Rose, Jonathan

2015-01-01

This paper undertakes a systematic assessment of the extent to which factor analysis the correct number of latent dimensions (factors) when applied to ordered-categorical survey items (so-called Likert items). We simulate 2400 data sets of uni-dimensional Likert items that vary systematically over a range of conditions such as the underlying population distribution, the number of items, the level of random error, and characteristics of items and item-sets. Each of these datasets is factor analysed in a variety of ways that are frequently used in the extant literature, or that are recommended in current methodological texts. These include exploratory factor retention heuristics such as Kaiser’s criterion, Parallel Analysis and a non-graphical scree test, and (for exploratory and confirmatory analyses) evaluations of model fit. These analyses are conducted on the basis of Pearson and polychoric correlations. We find that, irrespective of the particular mode of analysis, factor analysis applied to ordered-categorical survey data very often leads to over-dimensionalisation. The magnitude of this risk depends on the specific way in which factor analysis is conducted, the number of items, the properties of the set of items, and the underlying population distribution. The paper concludes with a discussion of the consequences of over-dimensionalisation, and a brief mention of alternative modes of analysis that are much less prone to such problems. PMID:25789992

An Analysis of Factors that Influence Enlistment Decisions in the U.S. Army

DTIC Science & Technology

1998-03-01

NAVAL POSTGRADUATE SCHOOL Monterey, California CM THESIS AN ANALYSIS OF FACTORS THAT INFLUENCE ENLISTMENT DECISIONS IN THE U.S. ARMY by Young...TITLE AND SUBTITLE : AN ANALYSIS OF FACTORS THAT INFLUENCE ENLISTMENT DECISIONS IN THE U.S. ARMY 6. AUTHOR(S) Oh, Young Yeol 7...200 words) The purpose of this thesis is to analyze factors that influence decisions to enlist in the U.S. Army. This thesis uses 1997 New Recruit

Secondary School Burnout Scale (SSBS)

ERIC Educational Resources Information Center

Aypay, Ayse

2012-01-01

The purpose of this study is to develop "Secondary School Burnout Scale." Study group included 728 students out of 14 schools in four cities in Turkey. Both Exploratory Factor Analysis and Confirmatory Factor Analysis were conducted on the data. A seven-factor solution emerged. The seven factors explained 61% of the total variance. The…

Analyse Factorielle d'une Batterie de Tests de Comprehension Orale et Ecrite (Factor Analysis of a Battery of Tests of Listening and Reading Comprehension). Melanges Pedagogiques, 1971.

ERIC Educational Resources Information Center

Lonchamp, F.

This is a presentation of the results of a factor analysis of a battery of tests intended to measure listening and reading comprehension in English as a second language. The analysis sought to answer the following questions: (1) whether the factor analysis method yields results when applied to tests which are not specifically designed for this…

Confirmatory Factor Analysis of the Trinity Inventory of Precursors to Suicide (TIPS) and Its Relationship to Hopelessness and Depression

ERIC Educational Resources Information Center

Smyth, Caroline L.; MacLachlan, Malcolm

2005-01-01

Numerous existing measures assess attitudes toward suicide yet fail to account for contextual factors. The Trinity Inventory of Precursors to Suicide (TIPS) is presented as an alternative, with implications for the development of prevention programs. Having previously reported exploratory analysis of the TIPS; confirmatory factor analysis and…

Examining Evolving Performance on the Force Concept Inventory Using Factor Analysis

ERIC Educational Resources Information Center

Semak, M. R.; Dietz, R. D.; Pearson, R. H.; Willis, C. W

2017-01-01

The application of factor analysis to the "Force Concept Inventory" (FCI) has proven to be problematic. Some studies have suggested that factor analysis of test results serves as a helpful tool in assessing the recognition of Newtonian concepts by students. Other work has produced at best ambiguous results. For the FCI administered as a…

Prediction of quality attributes of chicken breast fillets by using Vis/NIR spectroscopy combined with factor analysis method

USDA-ARS?s Scientific Manuscript database

Visible/near-infrared (Vis/NIR) spectroscopy with wavelength range between 400 and 2500 nm combined with factor analysis method was tested to predict quality attributes of chicken breast fillets. Quality attributes, including color (L*, a*, b*), pH, and drip loss were analyzed using factor analysis ...

Factor Analysis Methods and Validity Evidence: A Systematic Review of Instrument Development across the Continuum of Medical Education

ERIC Educational Resources Information Center

Wetzel, Angela Payne

2011-01-01

Previous systematic reviews indicate a lack of reporting of reliability and validity evidence in subsets of the medical education literature. Psychology and general education reviews of factor analysis also indicate gaps between current and best practices; yet, a comprehensive review of exploratory factor analysis in instrument development across…

Dimensions of temperament: an analysis.

PubMed

Lorr, M; Stefic, E C

1976-01-01

The TDOT recast into a single stimulus format was administered to 150 college Ss. A factor analysis of the items followed by an analysis of item clusters that define each factor indicated the presence of 14 dimensions. Of the 10 bipolar scales of the TDOT, 3 were confirmed as independent dimensions, and 5 were confirmed in part or split into unipolar factors.

Assessing School Work Culture: A Higher-Order Analysis and Strategy.

ERIC Educational Resources Information Center

Johnson, William L.; Johnson, Annabel M.; Zimmerman, Kurt J.

This paper reviews a work culture productivity model and reports the development of a work culture instrument based on the culture productivity model. Higher order principal components analysis was used to assess work culture, and a third-order factor analysis shows how the first-order factors group into higher-order factors. The school work…

A Primer on Bootstrap Factor Analysis as Applied to Health Studies Research

ERIC Educational Resources Information Center

Lu, Wenhua; Miao, Jingang; McKyer, E. Lisako J.

2014-01-01

Objectives: To demonstrate how the bootstrap method could be conducted in exploratory factor analysis (EFA) with a syntax written in SPSS. Methods: The data obtained from the Texas Childhood Obesity Prevention Policy Evaluation project (T-COPPE project) were used for illustration. A 5-step procedure to conduct bootstrap factor analysis (BFA) was…

Exploratory Factor Analysis of a Force Concept Inventory Data Set

ERIC Educational Resources Information Center

Scott, Terry F.; Schumayer, Daniel; Gray, Andrew R.

2012-01-01

We perform a factor analysis on a "Force Concept Inventory" (FCI) data set collected from 2109 respondents. We address two questions: the appearance of conceptual coherence in student responses to the FCI and some consequences of this factor analysis on the teaching of Newtonian mechanics. We will highlight the apparent conflation of Newton's…

On the Extraction of Components and the Applicability of the Factor Model.

ERIC Educational Resources Information Center

Dziuban, Charles D.; Harris, Chester W.

A reanalysis of Shaycroft's matrix of intercorrelations of 10 test variables plus 4 random variables is discussed. Three different procedures were used in the reanalysis: (1) Image Component Analysis, (2) Uniqueness Rescaling Factor Analysis, and (3) Alpha Factor Analysis. The results of these analyses are presented in tables. It is concluded from…

A Markov Chain Monte Carlo Approach to Confirmatory Item Factor Analysis

ERIC Educational Resources Information Center

Edwards, Michael C.

2010-01-01

Item factor analysis has a rich tradition in both the structural equation modeling and item response theory frameworks. The goal of this paper is to demonstrate a novel combination of various Markov chain Monte Carlo (MCMC) estimation routines to estimate parameters of a wide variety of confirmatory item factor analysis models. Further, I show…

Developing Multidimensional Likert Scales Using Item Factor Analysis: The Case of Four-Point Items

ERIC Educational Resources Information Center

Asún, Rodrigo A.; Rdz-Navarro, Karina; Alvarado, Jesús M.

2016-01-01

This study compares the performance of two approaches in analysing four-point Likert rating scales with a factorial model: the classical factor analysis (FA) and the item factor analysis (IFA). For FA, maximum likelihood and weighted least squares estimations using Pearson correlation matrices among items are compared. For IFA, diagonally weighted…

Links between CD147 function, glycosylation, and caveolin-1.

PubMed

Tang, Wei; Chang, Sharon B; Hemler, Martin E

2004-09-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. It was shown previously that caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction; now we show that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, we have 1) determined the biochemical basis for the unusual size variation in CD147, 2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and 3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, we now have a mechanism to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1.

T-Cell Therapy Using Interleukin-21–Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression

PubMed Central

Chapuis, Aude G.; Roberts, Ilana M.; Thompson, John A.; Margolin, Kim A.; Bhatia, Shailender; Lee, Sylvia M.; Sloan, Heather L.; Lai, Ivy P.; Farrar, Erik A.; Wagener, Felecia; Shibuya, Kendall C.; Cao, Jianhong; Wolchok, Jedd D.; Greenberg, Philip D.

2016-01-01

Purpose Peripheral blood–derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti–CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy. PMID:27269940

T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

PubMed

Chapuis, Aude G; Roberts, Ilana M; Thompson, John A; Margolin, Kim A; Bhatia, Shailender; Lee, Sylvia M; Sloan, Heather L; Lai, Ivy P; Farrar, Erik A; Wagener, Felecia; Shibuya, Kendall C; Cao, Jianhong; Wolchok, Jedd D; Greenberg, Philip D; Yee, Cassian

2016-11-01

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8 + T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

PubMed Central

Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

1992-01-01

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

PubMed Central

Ferron, François; Li, Zongli; Danek, Eric I.; Luo, Dahai; Wong, Yeehwa; Coutard, Bruno; Lantez, Violaine; Charrel, Rémi; Canard, Bruno; Walz, Thomas; Lescar, Julien

2011-01-01

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs. PMID:21589902

MVA vaccine encoding CMV antigens safely induces durable expansion of CMV-specific T cells in healthy adults

PubMed Central

La Rosa, Corinna; Longmate, Jeff; Martinez, Joy; Zhou, Qiao; Kaltcheva, Teodora I.; Tsai, Weimin; Drake, Jennifer; Carroll, Mary; Wussow, Felix; Chiuppesi, Flavia; Hardwick, Nicola; Dadwal, Sanjeet; Aldoss, Ibrahim; Nakamura, Ryotaro; Zaia, John A.

2017-01-01

Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933. PMID:27760761

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

DOE PAGES

He, Wei; Felderman, Martina; Evans, Angela C.; ...

2017-07-24

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, andmore » protein misfolding. For this study, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.« less

Influence of Coulombic repulsion on the dissociation pathways and energetics of multiprotein complexes in the gas phase.

PubMed

Sinelnikov, Igor; Kitova, Elena N; Klassen, John S

2007-04-01

Thermal dissociation experiments, implemented with blackbody infrared radiative dissociation and Fourier-transform ion cyclotron resonance mass spectrometry, are performed on gaseous protonated and deprotonated ions of the homopentameric B subunits of Shiga toxin 1 (Stx1 B5) and Shiga toxin 2 (Stx2 B5) and the homotetramer streptavidin (S4). Dissociation of the gaseous, multisubunit complexes proceeds predominantly by the loss of a single subunit. Notably, the fractional partitioning of charge between the product ions, i.e., the leaving subunit and the resulting multimer, for a given complex is, within error, constant over the range of charge states investigated. The Arrhenius activation parameters (E(a), A) measured for the loss of subunit decrease with increasing charge state of the complex. However, the parameters for the protonated and deprotonated ions, with the same number of charges, are indistinguishable. The influence of the complex charge state on the dissociation pathways and the magnitude of the dissociation E(a) are modeled theoretically with the discrete charge droplet model (DCDM) and the protein structure model (PSM), wherein the structure of the subunits is considered. Importantly, the major subunit charge states observed experimentally for the Stx1 B5(n+/-) ions correspond to the minimum energy charge distribution predicted by DCDM and PSM assuming a late dissociative transition-state (TS); while for structurally-related Stx2 B5(n+) ions, the experimental charge distribution corresponds to an early TS. It is proposed that the lateness of the TS is related, in part, to the degree of unfolding of the leaving subunit, with Stx1 B being more unfolded than Stx2 B. PSM, incorporating significant subunit unfolding is necessary to account for the product ions observed for the S4(n+) ions. The contribution of Coulombic repulsion to the dissociation E(a) is quantified and the intrinsic activation energy is estimated for the first time.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

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

He, Wei; Felderman, Martina; Evans, Angela C.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, andmore » protein misfolding. For this study, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.« less

The Role of Sigma-1 Receptor, an Intracellular Chaperone in Neurodegenerative Diseases.

PubMed

Penke, Botond; Fulop, Livia; Szucs, Maria; Frecska, Ede

2018-01-01

Widespread protein aggregation occurs in the living system under stress or during aging, owing to disturbance of endoplasmic reticulum (ER) proteostasis. Many neurodegenerative diseases may have a common mechanism: the failure of protein homeostasis. Perturbation of ER results in unfolded protein response (UPR). Prolonged chronical UPR may activate apoptotic pathways and cause cell death. Research articles on Sigma-1 receptor were reviewed. ER is associated to mitochondria by the mitochondria-associated ER-membrane, MAM. The sigma-1 receptor (Sig-1R), a well-known ER-chaperone localizes in the MAM. It serves for Ca2+-signaling between the ER and mitochondria, involved in ion channel activities and especially important during neuronal differentiation. Sig-1R acts as central modulator in inter-organelle signaling. Sig-1R helps cell survival by attenuating ER-stress. According to sequence based predictions Sig-1R is a 223 amino acid protein with two transmembrane (2TM) domains. The X-ray structure of the Sig-1R [1] showed a membrane-bound trimeric assembly with one transmembrane (1TM) region. Despite the in vitro determined assembly, the results of in vivo studies are rather consistent with the 2TM structure. The receptor has unique and versatile pharmacological profile. Dimethyl tryptamine (DMT) and neuroactive steroids are endogenous ligands that activate Sig-1R. The receptor has a plethora of interacting client proteins. Sig-1R exists in oligomeric structures (dimer-trimer-octamer-multimer) and this fact may explain interaction with diverse proteins. Sig-1R agonists have been used in the treatment of different neurodegenerative diseases, e.g. Alzheimer's and Parkinson's diseases (AD and PD) and amyotrophic lateral sclerosis. Utilization of Sig-1R agents early in AD and similar other diseases has remained an overlooked therapeutic opportunity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Role of tissue transglutaminase type 2 in calbindin-D28k interaction with ataxin-1

PubMed Central

Vig, P.J.S.; Wei, J.; Shao, Q.; Hebert, M.D.; Subramony, S.H.; Sutton, L.T.

2007-01-01

Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeats within the disease protein, ataxin-1. The mutant ataxin-1 precipitates as large intranuclear aggregates in the affected neurons. These aggregates may protect neurons from mutant protein and/or trigger neuronal degeneration by encouraging recruitment of other essential proteins. Our previous studies have shown that calcium binding protein calbindin-D28k (CaB) associated with SCA1 pathogenesis is recruited to ataxin-1 aggregates in Purkinje cells of SCA1 mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in cross-linking and aggregation of ataxin-1, the present study was initiated to determine if TG2 has any role in CaB-ataxin-1 interaction. The guinea pig TG2 covalently cross-linked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. CaB interaction with ataxin-1 was studied using HeLa cell lysates expressing GFP and GFP tagged ataxin-1 with normal and expanded polyglutamine repeats (Q2, Q30 and Q82). The reaction products were analyzed by Western blots using anti- polyglutamine, CaB or GFP antibodies. CaB interacted with ataxin-1 independent of TG2 as the protein-protein cross-linker DSS stabilized CaB-ataxin-1 complex. TG2 cross-linked CaB preferentially with Q82 ataxin-1. The cross-linking was inhibited with EGTA or TG2 inhibitor cystamine. The present data indicate that CaB may be a TG2 substrate. In addition, aggregates of mutant ataxin-1 may recruit CaB via TG2 mediated covalent cross-linking, further supporting the argument that ataxin-1 aggregates may be toxic to neurons. PMID:17442486

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

PubMed

Hong, Weimin Conrad; Yano, Hideaki; Hiranita, Takato; Chin, Frederick T; McCurdy, Christopher R; Su, Tsung-Ping; Amara, Susan G; Katz, Jonathan L

2017-07-07

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ 1 R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ 1 R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di- o -tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the B max values of [ 3 H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ 1 R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ 1 R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ 1 R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ 1 R antagonist CM304. Moreover, σ 1 R ligands had distinct effects on σ 1 R multimerization. CM304 increased the proportion of multimeric σ 1 Rs, whereas (+)-pentazocine increased monomeric σ 1 Rs. Together these results support the hypothesis that σ 1 R agonists promote dissociation of σ 1 R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ 1 R agonists in animal models. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

PubMed

Haldar, Suranjana; Bevers, Loes E; Tosha, Takehiko; Theil, Elizabeth C

2011-07-22

Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³⁺O-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O](n) transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

Normal and frictional interactions of purified human statherin adsorbed on molecularly-smooth solid substrata.

PubMed

Harvey, Neale M; Carpenter, Guy H; Proctor, Gordon B; Klein, Jacob

2011-09-01

Human salivary statherin was purified from parotid saliva and adsorbed to bare hydrophilic (HP) mica and STAI-coated hydrophobic (HB) mica in a series of Surface Force Balance experiments that measured the normal (F(n)) and friction forces (F(s)*) between statherin-coated mica substrata. Readings were taken both in the presence of statherin solution (HP and HB mica) and after rinsing (HP mica). F(n) measurements showed, for both substrata, monotonic steric repulsion that set on at a surface separation D ~20 nm, indicating an adsorbed layer whose unperturbed thickness was ca 10 nm. An additional longer-ranged repulsion, probably of electrostatic double-layer origin, was observed for rinsed surfaces under pure water. Under applied pressures of ~1 MPa, each surface layer was compressed to a thickness of ca 2 nm on both types of substratum, comparable with earlier estimates of the size of the statherin molecule. Friction measurements, in contrast with F(n) observations, were markedly different on the two different substrata: friction coefficients, μ ≡ ∂F(s)*/∂F(n), on the HB substratum (μ ≈ 0.88) were almost an order of magnitude higher than on the HP substratum (μ ≈ 0.09 and 0.12 for unrinsed and rinsed, respectively), and on the HB mica there was a lower dependence of friction on sliding speed than on the HP mica. The observations were attributed to statherin adsorbing to the mica in multimer aggregates, with internal re-arrangement of the protein molecules within the aggregate dependent on the substratum to which the aggregate adsorbed. This internal re-arrangement permitted aggregates to be of similar size on HP and HB mica but to have different internal molecular orientations, thus exposing different moieties to the solution in each case and accounting for the very different friction behaviour.

Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

PubMed

Clock, Sarah A; Planet, Paul J; Perez, Brenda A; Figurski, David H

2008-02-01

Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.

β-Hairpin-Mediated Formation of Structurally Distinct Multimers of Neurotoxic Prion Peptides

PubMed Central

Gill, Andrew C.

2014-01-01

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A117V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies. PMID:24498083

Ensino de Astronomia no Ensino Médio

NASA Astrophysics Data System (ADS)

Albrecht, E.; Voelzke, M. R.

2008-09-01

O presente trabalho de intervenção foi realizado junto a Escola Estadual Colônia dos Pescadores na cidade de Caraguatatuba, com três turmas do terceiro ano do Ensino Médio, envolvendo 119 alunos, 40 na turma A, 40 na turma B e 39 na turma C. A fase inicial foi composta de um questionário de vinte questões dissertativas e objetivas para diagnosticar nos educandos os conceitos prévios sobre Astronomia e, partindo destes realizar uma interferência nas classes envolvidas utilizando metodologias diferentes sendo elas: a tradicional, onde o professor é um repassador de informações, fazendo uso exclusivo de lousa e giz; a segunda também de forma tradicional, porém com auxílio de multimídia para desenvolvimento das aulas e a terceira sob forma de seminários, elaborados e apresentados pelos educandos, no qual o educador faz apenas as intervenções necessárias. Ao final do trabalho as mesmas turmas da fase inicial orientadas pelo mesmo professor responderam novamente ao questionário proposto para diagnosticar dentre as três metodologias utilizadas qual apresentou melhores resultados, sendo os iniciais comparados com os finais. Quando questionados a respeito do significado de Astronomia observou-se inicialmente que os acertos na turma A foram de 100%,turma B: 64%, turma C: 84%, após a intervenção os acertos foram: 100%, 97% e 85% respectivamente, demonstrando claramente uma absorção de conhecimentos. Quando interrogados sobre quantos planetas você acha que existem em nosso Sistema Solar? os acertos foram: turma A: 39%, turma B: 48% e turma C: 46%, após o desenvolvimento do trabalho os acertos foram 94%, 97% e 90% respectivamente.Dentro das respostas obtidas observa-se que a metodologia tradicional com o auxílio de multimeios, aplicada na turma B, demonstrou melhores resultados, foi a mais significativa.

Multiplication factor versus regression analysis in stature estimation from hand and foot dimensions.

PubMed

Krishan, Kewal; Kanchan, Tanuj; Sharma, Abhilasha

2012-05-01

Estimation of stature is an important parameter in identification of human remains in forensic examinations. The present study is aimed to compare the reliability and accuracy of stature estimation and to demonstrate the variability in estimated stature and actual stature using multiplication factor and regression analysis methods. The study is based on a sample of 246 subjects (123 males and 123 females) from North India aged between 17 and 20 years. Four anthropometric measurements; hand length, hand breadth, foot length and foot breadth taken on the left side in each subject were included in the study. Stature was measured using standard anthropometric techniques. Multiplication factors were calculated and linear regression models were derived for estimation of stature from hand and foot dimensions. Derived multiplication factors and regression formula were applied to the hand and foot measurements in the study sample. The estimated stature from the multiplication factors and regression analysis was compared with the actual stature to find the error in estimated stature. The results indicate that the range of error in estimation of stature from regression analysis method is less than that of multiplication factor method thus, confirming that the regression analysis method is better than multiplication factor analysis in stature estimation. Copyright © 2012 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Success Probability Analysis for Shuttle Based Microgravity Experiments

NASA Technical Reports Server (NTRS)

Liou, Ying-Hsin Andrew

1996-01-01

Presented in this report are the results of data analysis of shuttle-based microgravity flight experiments. Potential factors were identified in the previous grant period, and in this period 26 factors were selected for data analysis. In this project, the degree of success was developed and used as the performance measure. 293 of the 391 experiments in Lewis Research Center Microgravity Database were assigned degrees of success. The frequency analysis and the analysis of variance were conducted to determine the significance of the factors that effect the experiment success.

Assessing Suicide Risk Among Callers to Crisis Hotlines: A Confirmatory Factor Analysis

PubMed Central

Witte, Tracy K.; Gould, Madelyn S.; Munfakh, Jimmie Lou Harris; Kleinman, Marjorie; Joiner, Thomas E.; Kalafat, John

2012-01-01

Our goal was to investigate the factor structure of a risk assessment tool utilized by suicide hotlines and to determine the predictive validity of the obtained factors in predicting subsequent suicidal behavior. 1,085 suicidal callers to crisis hotlines were divided into three sub-samples, which allowed us to conduct an independent Exploratory Factor Analysis (EFA), EFA in a Confirmatory Factor Analysis (EFA/CFA) framework, and CFA. Similar to previous factor analytic studies (Beck et al., 1997; Holden & DeLisle, 2005; Joiner, Rudd, & Rajab, 1997; Witte et al., 2006), we found consistent evidence for a two-factor solution, with one factor representing a more pernicious form of suicide risk (i.e., Resolved Plans and Preparations) and one factor representing more mild suicidal ideation (i.e., Suicidal Desire and Ideation). Using structural equation modeling techniques, we found preliminary evidence that the Resolved Plans and Preparations factor trended toward being more predictive of suicidal ideation than the Suicidal Desire and Ideation factor. This factor analytic study is the first longitudinal study of the obtained factors. PMID:20578186

A decision model for selecting sustainable drinking water supply and greywater reuse systems for developing communities with a case study in Cimahi, Indonesia.

PubMed

Henriques, Justin J; Louis, Garrick E

2011-01-01

Capacity Factor Analysis is a decision support system for selection of appropriate technologies for municipal sanitation services in developing communities. Developing communities are those that lack the capability to provide adequate access to one or more essential services, such as water and sanitation, to their residents. This research developed two elements of Capacity Factor Analysis: a capacity factor based classification for technologies using requirements analysis, and a matching policy for choosing technology options. First, requirements analysis is used to develop a ranking for drinking water supply and greywater reuse technologies. Second, using the Capacity Factor Analysis approach, a matching policy is developed to guide decision makers in selecting the appropriate drinking water supply or greywater reuse technology option for their community. Finally, a scenario-based informal hypothesis test is developed to assist in qualitative model validation through case study. Capacity Factor Analysis is then applied in Cimahi Indonesia as a form of validation. The completed Capacity Factor Analysis model will allow developing communities to select drinking water supply and greywater reuse systems that are safe, affordable, able to be built and managed by the community using local resources, and are amenable to expansion as the community's management capacity increases. Copyright © 2010 Elsevier Ltd. All rights reserved.

The School Counselor Leadership Survey: Instrument Development and Exploratory Factor Analysis

ERIC Educational Resources Information Center

Young, Anita; Bryan, Julia

2015-01-01

This study examined the factor structure of the School Counselor Leadership Survey (SCLS). Survey development was a threefold process that resulted in a 39-item survey of 801 school counselors and school counselor supervisors. The exploratory factor analysis indicated a five-factor structure that revealed five key dimensions of school counselor…

A Confirmatory Factor Analysis of the Academic Motivation Scale with Black College Students

ERIC Educational Resources Information Center

Cokley, Kevin

2015-01-01

The factor structure of the Academic Motivation Scale (AMS) was examined with a sample of 578 Black college students. A confirmatory factor analysis of the AMS was conducted. Results indicated that the hypothesized seven-factor model did not fit the data. Implications for future research with the AMS are discussed.

Hierarchical Factoring Based On Image Analysis And Orthoblique Rotations.

PubMed

Stankov, L

1979-07-01

The procedure for hierarchical factoring suggested by Schmid and Leiman (1957) is applied within the framework of image analysis and orthoblique rotational procedures. It is shown that this approach necessarily leads to correlated higher order factors. Also, one can obtain a smaller number of factors than produced by typical hierarchical procedures.

The Factor Structure of the English Language Development Assessment: A Confirmatory Factor Analysis

ERIC Educational Resources Information Center

Kuriakose, Anju

2011-01-01

This study investigated the internal factor structure of the English language development Assessment (ELDA) using confirmatory factor analysis. ELDA is an English language proficiency test developed by a consortium of multiple states and is used to identify and reclassify English language learners in kindergarten to grade 12. Scores on item…

Factors Predictive of Mathematics Achievement in Kindergarten, First and Third Grades: An Opportunity-Propensity Analysis

ERIC Educational Resources Information Center

Byrnes, James P.; Wasik, Barbara A.

2009-01-01

A secondary analysis of the Early Childhood Longitudinal Study-Kindergarten Sample (N = 17,401) was conducted to determine the factors that are most strongly associated with math achievement during kindergarten, first grade, and third grade. Factors from the following three categories were considered: antecedent factors (e.g., family…

A Comparison of Distribution Free and Non-Distribution Free Factor Analysis Methods

ERIC Educational Resources Information Center

Ritter, Nicola L.

2012-01-01

Many researchers recognize that factor analysis can be conducted on both correlation matrices and variance-covariance matrices. Although most researchers extract factors from non-distribution free or parametric methods, researchers can also extract factors from distribution free or non-parametric methods. The nature of the data dictates the method…

Psychometric analysis of the new ADHD DSM-V derived symptoms.

PubMed

Ghanizadeh, Ahmad

2012-03-20

Following the agreements on the reformulating and revising of ADHD diagnostic criteria, recently, the proposed revision for ADHD added 4 new symptoms to the hyperactivity and Impulsivity aspect in DSM-V. This study investigates the psychometric properties of the proposed ADHD diagnostic criteria. ADHD diagnosis was made according to DSM-IV. The parents completed the screening test of ADHD checklist of Child Symptom Inventory-4 and the 4 items describing the new proposed symptoms in DSM-V. The confirmatory factor analysis of the ADHD DSM-V derived items supports the loading of two factors including inattentiveness and hyperactivity/impulsivity. There is a sufficient reliability for the items. However, confirmatory factor analysis showed that the three-factor model is better fitted than the two-factor one. Moreover, the results of the exploratory analysis raised some concerns about the factor loading of the four new items. The current results support the two-factor model of the DSM-V ADHD diagnostic criteria including inattentiveness and hyperactivity/impulsivity. However, the four new items can be considered as a third factor.

Sexual Harassment Retaliation Climate DEOCS 4.1 Construct Validity Summary

DTIC Science & Technology

2017-08-01

exploratory factor analysis, and bivariate correlations (sample 1) 2) To determine the factor structure of the remaining (final) questions via...statistics, reliability analysis, exploratory factor analysis, and bivariate correlations of the prospective Sexual Harassment Retaliation Climate...reported by the survey requester). For information regarding the composition of sample, refer to Table 1. Table 1. Sample 1 Demographics n

A comprehensive probabilistic analysis model of oil pipelines network based on Bayesian network

NASA Astrophysics Data System (ADS)

Zhang, C.; Qin, T. X.; Jiang, B.; Huang, C.

2018-02-01

Oil pipelines network is one of the most important facilities of energy transportation. But oil pipelines network accident may result in serious disasters. Some analysis models for these accidents have been established mainly based on three methods, including event-tree, accident simulation and Bayesian network. Among these methods, Bayesian network is suitable for probabilistic analysis. But not all the important influencing factors are considered and the deployment rule of the factors has not been established. This paper proposed a probabilistic analysis model of oil pipelines network based on Bayesian network. Most of the important influencing factors, including the key environment condition and emergency response are considered in this model. Moreover, the paper also introduces a deployment rule for these factors. The model can be used in probabilistic analysis and sensitive analysis of oil pipelines network accident.

Global sensitivity analysis for urban water quality modelling: Terminology, convergence and comparison of different methods

NASA Astrophysics Data System (ADS)

Vanrolleghem, Peter A.; Mannina, Giorgio; Cosenza, Alida; Neumann, Marc B.

2015-03-01

Sensitivity analysis represents an important step in improving the understanding and use of environmental models. Indeed, by means of global sensitivity analysis (GSA), modellers may identify both important (factor prioritisation) and non-influential (factor fixing) model factors. No general rule has yet been defined for verifying the convergence of the GSA methods. In order to fill this gap this paper presents a convergence analysis of three widely used GSA methods (SRC, Extended FAST and Morris screening) for an urban drainage stormwater quality-quantity model. After the convergence was achieved the results of each method were compared. In particular, a discussion on peculiarities, applicability, and reliability of the three methods is presented. Moreover, a graphical Venn diagram based classification scheme and a precise terminology for better identifying important, interacting and non-influential factors for each method is proposed. In terms of convergence, it was shown that sensitivity indices related to factors of the quantity model achieve convergence faster. Results for the Morris screening method deviated considerably from the other methods. Factors related to the quality model require a much higher number of simulations than the number suggested in literature for achieving convergence with this method. In fact, the results have shown that the term "screening" is improperly used as the method may exclude important factors from further analysis. Moreover, for the presented application the convergence analysis shows more stable sensitivity coefficients for the Extended-FAST method compared to SRC and Morris screening. Substantial agreement in terms of factor fixing was found between the Morris screening and Extended FAST methods. In general, the water quality related factors exhibited more important interactions than factors related to water quantity. Furthermore, in contrast to water quantity model outputs, water quality model outputs were found to be characterised by high non-linearity.

Proposal for a recovery prediction method for patients affected by acute mediastinitis

PubMed Central

2012-01-01

Background An attempt to find a prediction method of death risk in patients affected by acute mediastinitis. There is not such a tool described in available literature for that serious disease. Methods The study comprised 44 consecutive cases of acute mediastinitis. General anamnesis and biochemical data were included. Factor analysis was used to extract the risk characteristic for the patients. The most valuable results were obtained for 8 parameters which were selected for further statistical analysis (all collected during few hours after admission). Three factors reached Eigenvalue >1. Clinical explanations of these combined statistical factors are: Factor1 - proteinic status (serum total protein, albumin, and hemoglobin level), Factor2 - inflammatory status (white blood cells, CRP, procalcitonin), and Factor3 - general risk (age, number of coexisting diseases). Threshold values of prediction factors were estimated by means of statistical analysis (factor analysis, Statgraphics Centurion XVI). Results The final prediction result for the patients is constructed as simultaneous evaluation of all factor scores. High probability of death should be predicted if factor 1 value decreases with simultaneous increase of factors 2 and 3. The diagnostic power of the proposed method was revealed to be high [sensitivity =90%, specificity =64%], for Factor1 [SNC = 87%, SPC = 79%]; for Factor2 [SNC = 87%, SPC = 50%] and for Factor3 [SNC = 73%, SPC = 71%]. Conclusion The proposed prediction method seems a useful emergency signal during acute mediastinitis control in affected patients. PMID:22574625

Independent Prognostic Factors for Acute Organophosphorus Pesticide Poisoning.

PubMed

Tang, Weidong; Ruan, Feng; Chen, Qi; Chen, Suping; Shao, Xuebo; Gao, Jianbo; Zhang, Mao

2016-07-01

Acute organophosphorus pesticide poisoning (AOPP) is becoming a significant problem and a potential cause of human mortality because of the abuse of organophosphate compounds. This study aims to determine the independent prognostic factors of AOPP by using multivariate logistic regression analysis. The clinical data for 71 subjects with AOPP admitted to our hospital were retrospectively analyzed. This information included the Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, 6-h post-admission blood lactate levels, post-admission 6-h lactate clearance rates, admission blood cholinesterase levels, 6-h post-admission blood cholinesterase levels, cholinesterase activity, blood pH, and other factors. Univariate analysis and multivariate logistic regression analyses were conducted to identify all prognostic factors and independent prognostic factors, respectively. A receiver operating characteristic curve was plotted to analyze the testing power of independent prognostic factors. Twelve of 71 subjects died. Admission blood lactate levels, 6-h post-admission blood lactate levels, post-admission 6-h lactate clearance rates, blood pH, and APACHE II scores were identified as prognostic factors for AOPP according to the univariate analysis, whereas only 6-h post-admission blood lactate levels, post-admission 6-h lactate clearance rates, and blood pH were independent prognostic factors identified by multivariate logistic regression analysis. The receiver operating characteristic analysis suggested that post-admission 6-h lactate clearance rates were of moderate diagnostic value. High 6-h post-admission blood lactate levels, low blood pH, and low post-admission 6-h lactate clearance rates were independent prognostic factors identified by multivariate logistic regression analysis. Copyright © 2016 by Daedalus Enterprises.

Estimation of the behavior factor of existing RC-MRF buildings

NASA Astrophysics Data System (ADS)

Vona, Marco; Mastroberti, Monica

2018-01-01

In recent years, several research groups have studied a new generation of analysis methods for seismic response assessment of existing buildings. Nevertheless, many important developments are still needed in order to define more reliable and effective assessment procedures. Moreover, regarding existing buildings, it should be highlighted that due to the low knowledge level, the linear elastic analysis is the only analysis method allowed. The same codes (such as NTC2008, EC8) consider the linear dynamic analysis with behavior factor as the reference method for the evaluation of seismic demand. This type of analysis is based on a linear-elastic structural model subject to a design spectrum, obtained by reducing the elastic spectrum through a behavior factor. The behavior factor (reduction factor or q factor in some codes) is used to reduce the elastic spectrum ordinate or the forces obtained from a linear analysis in order to take into account the non-linear structural capacities. The behavior factors should be defined based on several parameters that influence the seismic nonlinear capacity, such as mechanical materials characteristics, structural system, irregularity and design procedures. In practical applications, there is still an evident lack of detailed rules and accurate behavior factor values adequate for existing buildings. In this work, some investigations of the seismic capacity of the main existing RC-MRF building types have been carried out. In order to make a correct evaluation of the seismic force demand, actual behavior factor values coherent with force based seismic safety assessment procedure have been proposed and compared with the values reported in the Italian seismic code, NTC08.

Exploratory factor analysis of borderline personality disorder criteria in hospitalized adolescents.

PubMed

Becker, Daniel F; McGlashan, Thomas H; Grilo, Carlos M

2006-01-01

The authors examined the factor structure of borderline personality disorder (BPD) in hospitalized adolescents and also sought to add to the theoretical and clinical understanding of any homogeneous components by determining whether they may be related to specific forms of Axis I pathology. Subjects were 123 adolescent inpatients, who were reliably assessed with structured diagnostic interviews for Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition Axes I and II disorders. Exploratory factor analysis identified BPD components, and logistic regression analyses tested whether these components were predictive of specific Axis I disorders. Factor analysis revealed a 4-factor solution that accounted for 67.0% of the variance. Factor 1 ("suicidal threats or gestures" and "emptiness or boredom") predicted depressive disorders and alcohol use disorders. Factor 2 ("affective instability," "uncontrolled anger," and "identity disturbance") predicted anxiety disorders and oppositional defiant disorder. Factor 3 ("unstable relationships" and "abandonment fears") predicted only anxiety disorders. Factor 4 ("impulsiveness" and "identity disturbance") predicted conduct disorder and substance use disorders. Exploratory factor analysis of BPD criteria in adolescent inpatients revealed 4 BPD factors that appear to differ from those reported for similar studies of adults. The factors represent components of self-negation, irritability, poorly modulated relationships, and impulsivity--each of which is associated with characteristic Axis I pathology. These findings shed light on the nature of BPD in adolescents and may also have implications for treatment.

Factor analysis of the Zung self-rating depression scale in a large sample of patients with major depressive disorder in primary care.

PubMed

Romera, Irene; Delgado-Cohen, Helena; Perez, Teresa; Caballero, Luis; Gilaberte, Immaculada

2008-01-14

The aim of this study was to examine the symptomatic dimensions of depression in a large sample of patients with major depressive disorder (MDD) in the primary care (PC) setting by means of a factor analysis of the Zung self-rating depression scale (ZSDS). A factor analysis was performed, based on the polychoric correlations matrix, between ZSDS items using promax oblique rotation in 1049 PC patients with a diagnosis of MDD (DSM-IV). A clinical interpretable four-factor solution consisting of a core depressive factor (I); a cognitive factor (II); an anxiety factor (III) and a somatic factor (IV) was extracted. These factors accounted for 36.9% of the variance on the ZSDS. The 4-factor structure was validated and high coefficients of congruence were obtained (0.98, 0.95, 0.92 and 0.87 for factors I, II, III and IV, respectively). The model seemed to fit the data well with fit indexes within recommended ranges (GFI = 0.9330, AGFI = 0.9112 and RMR = 0.0843). Our findings suggest that depressive symptoms in patients with MDD in the PC setting cluster into four dimensions: core depressive, cognitive, anxiety and somatic, by means of a factor analysis of the ZSDS. Further research is needed to identify possible diagnostic, therapeutic or prognostic implications of the different depressive symptomatic profiles.

Factor analysis of the Zung self-rating depression scale in a large sample of patients with major depressive disorder in primary care

PubMed Central

Romera, Irene; Delgado-Cohen, Helena; Perez, Teresa; Caballero, Luis; Gilaberte, Immaculada

2008-01-01

Background The aim of this study was to examine the symptomatic dimensions of depression in a large sample of patients with major depressive disorder (MDD) in the primary care (PC) setting by means of a factor analysis of the Zung self-rating depression scale (ZSDS). Methods A factor analysis was performed, based on the polychoric correlations matrix, between ZSDS items using promax oblique rotation in 1049 PC patients with a diagnosis of MDD (DSM-IV). Results A clinical interpretable four-factor solution consisting of a core depressive factor (I); a cognitive factor (II); an anxiety factor (III) and a somatic factor (IV) was extracted. These factors accounted for 36.9% of the variance on the ZSDS. The 4-factor structure was validated and high coefficients of congruence were obtained (0.98, 0.95, 0.92 and 0.87 for factors I, II, III and IV, respectively). The model seemed to fit the data well with fit indexes within recommended ranges (GFI = 0.9330, AGFI = 0.9112 and RMR = 0.0843). Conclusion Our findings suggest that depressive symptoms in patients with MDD in the PC setting cluster into four dimensions: core depressive, cognitive, anxiety and somatic, by means of a factor analysis of the ZSDS. Further research is needed to identify possible diagnostic, therapeutic or prognostic implications of the different depressive symptomatic profiles. PMID:18194524

Factor structure of the Japanese version of the Edinburgh Postnatal Depression Scale in the postpartum period.

PubMed

Kubota, Chika; Okada, Takashi; Aleksic, Branko; Nakamura, Yukako; Kunimoto, Shohko; Morikawa, Mako; Shiino, Tomoko; Tamaji, Ai; Ohoka, Harue; Banno, Naomi; Morita, Tokiko; Murase, Satomi; Goto, Setsuko; Kanai, Atsuko; Masuda, Tomoko; Ando, Masahiko; Ozaki, Norio

2014-01-01

The Edinburgh Postnatal Depression Scale (EPDS) is a widely used screening tool for postpartum depression (PPD). Although the reliability and validity of EPDS in Japanese has been confirmed and the prevalence of PPD is found to be about the same as Western countries, the factor structure of the Japanese version of EPDS has not been elucidated yet. 690 Japanese mothers completed all items of the EPDS at 1 month postpartum. We divided them randomly into two sample sets. The first sample set (n = 345) was used for exploratory factor analysis, and the second sample set was used (n = 345) for confirmatory factor analysis. The result of exploratory factor analysis indicated a three-factor model consisting of anxiety, depression and anhedonia. The results of confirmatory factor analysis suggested that the anxiety and anhedonia factors existed for EPDS in a sample of Japanese women at 1 month postpartum. The depression factor varies by the models of acceptable fit. We examined EPDS scores. As a result, "anxiety" and "anhedonia" exist for EPDS among postpartum women in Japan as already reported in Western countries. Cross-cultural research is needed for future research.

Factor analysis of serogroups botanica and aurisina of Leptospira biflexa.

PubMed

Cinco, M

1977-11-01

Factor analysis is performed on serovars of Botanica and Aurisina serogroup of Leptospira biflexa. The results show the arrangement of main factors serovar and serogroup specific, as well as the antigens common with serovars of heterologous serogroups.

Identifying Items to Assess Methodological Quality in Physical Therapy Trials: A Factor Analysis

PubMed Central

Cummings, Greta G.; Fuentes, Jorge; Saltaji, Humam; Ha, Christine; Chisholm, Annabritt; Pasichnyk, Dion; Rogers, Todd

2014-01-01

Background Numerous tools and individual items have been proposed to assess the methodological quality of randomized controlled trials (RCTs). The frequency of use of these items varies according to health area, which suggests a lack of agreement regarding their relevance to trial quality or risk of bias. Objective The objectives of this study were: (1) to identify the underlying component structure of items and (2) to determine relevant items to evaluate the quality and risk of bias of trials in physical therapy by using an exploratory factor analysis (EFA). Design A methodological research design was used, and an EFA was performed. Methods Randomized controlled trials used for this study were randomly selected from searches of the Cochrane Database of Systematic Reviews. Two reviewers used 45 items gathered from 7 different quality tools to assess the methodological quality of the RCTs. An exploratory factor analysis was conducted using the principal axis factoring (PAF) method followed by varimax rotation. Results Principal axis factoring identified 34 items loaded on 9 common factors: (1) selection bias; (2) performance and detection bias; (3) eligibility, intervention details, and description of outcome measures; (4) psychometric properties of the main outcome; (5) contamination and adherence to treatment; (6) attrition bias; (7) data analysis; (8) sample size; and (9) control and placebo adequacy. Limitation Because of the exploratory nature of the results, a confirmatory factor analysis is needed to validate this model. Conclusions To the authors' knowledge, this is the first factor analysis to explore the underlying component items used to evaluate the methodological quality or risk of bias of RCTs in physical therapy. The items and factors represent a starting point for evaluating the methodological quality and risk of bias in physical therapy trials. Empirical evidence of the association among these items with treatment effects and a confirmatory factor analysis of these results are needed to validate these items. PMID:24786942

Identifying items to assess methodological quality in physical therapy trials: a factor analysis.

PubMed

Armijo-Olivo, Susan; Cummings, Greta G; Fuentes, Jorge; Saltaji, Humam; Ha, Christine; Chisholm, Annabritt; Pasichnyk, Dion; Rogers, Todd

2014-09-01

Numerous tools and individual items have been proposed to assess the methodological quality of randomized controlled trials (RCTs). The frequency of use of these items varies according to health area, which suggests a lack of agreement regarding their relevance to trial quality or risk of bias. The objectives of this study were: (1) to identify the underlying component structure of items and (2) to determine relevant items to evaluate the quality and risk of bias of trials in physical therapy by using an exploratory factor analysis (EFA). A methodological research design was used, and an EFA was performed. Randomized controlled trials used for this study were randomly selected from searches of the Cochrane Database of Systematic Reviews. Two reviewers used 45 items gathered from 7 different quality tools to assess the methodological quality of the RCTs. An exploratory factor analysis was conducted using the principal axis factoring (PAF) method followed by varimax rotation. Principal axis factoring identified 34 items loaded on 9 common factors: (1) selection bias; (2) performance and detection bias; (3) eligibility, intervention details, and description of outcome measures; (4) psychometric properties of the main outcome; (5) contamination and adherence to treatment; (6) attrition bias; (7) data analysis; (8) sample size; and (9) control and placebo adequacy. Because of the exploratory nature of the results, a confirmatory factor analysis is needed to validate this model. To the authors' knowledge, this is the first factor analysis to explore the underlying component items used to evaluate the methodological quality or risk of bias of RCTs in physical therapy. The items and factors represent a starting point for evaluating the methodological quality and risk of bias in physical therapy trials. Empirical evidence of the association among these items with treatment effects and a confirmatory factor analysis of these results are needed to validate these items. © 2014 American Physical Therapy Association.

Factor analysis and multiple regression between topography and precipitation on Jeju Island, Korea

NASA Astrophysics Data System (ADS)

Um, Myoung-Jin; Yun, Hyeseon; Jeong, Chang-Sam; Heo, Jun-Haeng

2011-11-01

SummaryIn this study, new factors that influence precipitation were extracted from geographic variables using factor analysis, which allow for an accurate estimation of orographic precipitation. Correlation analysis was also used to examine the relationship between nine topographic variables from digital elevation models (DEMs) and the precipitation in Jeju Island. In addition, a spatial analysis was performed in order to verify the validity of the regression model. From the results of the correlation analysis, it was found that all of the topographic variables had a positive correlation with the precipitation. The relations between the variables also changed in accordance with a change in the precipitation duration. However, upon examining the correlation matrix, no significant relationship between the latitude and the aspect was found. According to the factor analysis, eight topographic variables (latitude being the exception) were found to have a direct influence on the precipitation. Three factors were then extracted from the eight topographic variables. By directly comparing the multiple regression model with the factors (model 1) to the multiple regression model with the topographic variables (model 3), it was found that model 1 did not violate the limits of statistical significance and multicollinearity. As such, model 1 was considered to be appropriate for estimating the precipitation when taking into account the topography. In the study of model 1, the multiple regression model using factor analysis was found to be the best method for estimating the orographic precipitation on Jeju Island.

Examining evolving performance on the Force Concept Inventory using factor analysis

NASA Astrophysics Data System (ADS)

Semak, M. R.; Dietz, R. D.; Pearson, R. H.; Willis, C. W.

2017-06-01

The application of factor analysis to the Force Concept Inventory (FCI) has proven to be problematic. Some studies have suggested that factor analysis of test results serves as a helpful tool in assessing the recognition of Newtonian concepts by students. Other work has produced at best ambiguous results. For the FCI administered as a pre- and post-test, we see factor analysis as a tool by which the changes in conceptual associations made by our students may be gauged given the evolution of their response patterns. This analysis allows us to identify and track conceptual linkages, affording us insight as to how our students have matured due to instruction. We report on our analysis of 427 pre- and post-tests. The factor models for the pre- and post-tests are explored and compared along with the methodology by which these models were fit to the data. The post-test factor pattern is more aligned with an expert's interpretation of the questions' content, as it allows for a more readily identifiable relationship between factors and physical concepts. We discuss this evolution in the context of approaching the characteristics of an expert with force concepts. Also, we find that certain test items do not significantly contribute to the pre- or post-test factor models and attempt explanations as to why this is so. This may suggest that such questions may not be effective in probing the conceptual understanding of our students.

Exploratory factor analysis in Rehabilitation Psychology: a content analysis.

PubMed

Roberson, Richard B; Elliott, Timothy R; Chang, Jessica E; Hill, Jessica N

2014-11-01

Our objective was to examine the use and quality of exploratory factor analysis (EFA) in articles published in Rehabilitation Psychology. Trained raters examined 66 separate exploratory factor analyses in 47 articles published between 1999 and April 2014. The raters recorded the aim of the EFAs, the distributional statistics, sample size, factor retention method(s), extraction and rotation method(s), and whether the pattern coefficients, structure coefficients, and the matrix of association were reported. The primary use of the EFAs was scale development, but the most widely used extraction and rotation method was principle component analysis, with varimax rotation. When determining how many factors to retain, multiple methods (e.g., scree plot, parallel analysis) were used most often. Many articles did not report enough information to allow for the duplication of their results. EFA relies on authors' choices (e.g., factor retention rules extraction, rotation methods), and few articles adhered to all of the best practices. The current findings are compared to other empirical investigations into the use of EFA in published research. Recommendations for improving EFA reporting practices in rehabilitation psychology research are provided.

Factor Structure of the Student-Teacher Relationship Scale for Norwegian School-Age Children Explored with Confirmatory Factor Analysis

ERIC Educational Resources Information Center

Drugli, May Britt; Hjemdal, Odin

2013-01-01

The validity of the Student-Teacher Relationship Scale (STRS) was examined in a national sample of 863 Norwegian schoolchildren in grades 1-7 (aged 6-13). The original factor structure of the STRS was tested by confirmatory factor analysis (CFA). The CFA results did not support the original three-factor structure of the STRS. Subsequent CFA of the…

Intrinsic disorder in transcription factors†

PubMed Central

Liu, Jiangang; Perumal, Narayanan B.; Oldfield, Christopher J.; Su, Eric W.; Uversky, Vladimir N.; Dunker, A. Keith

2008-01-01

Intrinsic disorder (ID) is highly abundant in eukaryotes, which reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported, no systematic analysis has been reported so far. To test for a general prevalence of intrinsic disorder in transcriptional regulation, we used the Predictor Of Natural Disorder Regions (PONDR) to analyze the abundance of intrinsic disorder in three transcription factor datasets and two control sets. This analysis revealed that from 94.13% to 82.63% of transcription factors posses extended regions of intrinsic disorder, relative to 54.51% and 18.64% of the proteins in two control datasets, which indicates the significant prevalence of intrinsic disorder in transcription factors. This propensity of transcription factors for intrinsic disorder was confirmed by cumulative distribution function analysis and charge-hydropathy plots. The amino acid composition analysis showed that all three transcription factor datasets were substantially depleted in order-promoting residues, and significantly enriched in disorder-promoting residues. Our analysis of the distribution of disorder within the transcription factor datasets revealed that: (a) The AT-hooks and basic regions of transcription factor DNA-binding domains are highly disordered; (b) The degree of disorder in transcription factor activation regions is much higher than that in DNA-binding domains; (c) The degree of disorder is significantly higher in eukaryotic transcription factors than in prokaryotic transcription factors; (d) The level of α-MoRFs (molecular recognition feature) prediction is much higher in transcription factors. Overall, our data reflected the fact that the eukaryotes with well-developed gene transcription machinery require transcription factor flexibility to be more efficient. PMID:16734424

Logistic Regression and Path Analysis Method to Analyze Factors influencing Students’ Achievement

NASA Astrophysics Data System (ADS)

Noeryanti, N.; Suryowati, K.; Setyawan, Y.; Aulia, R. R.

2018-04-01

Students' academic achievement cannot be separated from the influence of two factors namely internal and external factors. The first factors of the student (internal factors) consist of intelligence (X1), health (X2), interest (X3), and motivation of students (X4). The external factors consist of family environment (X5), school environment (X6), and society environment (X7). The objects of this research are eighth grade students of the school year 2016/2017 at SMPN 1 Jiwan Madiun sampled by using simple random sampling. Primary data are obtained by distributing questionnaires. The method used in this study is binary logistic regression analysis that aims to identify internal and external factors that affect student’s achievement and how the trends of them. Path Analysis was used to determine the factors that influence directly, indirectly or totally on student’s achievement. Based on the results of binary logistic regression, variables that affect student’s achievement are interest and motivation. And based on the results obtained by path analysis, factors that have a direct impact on student’s achievement are students’ interest (59%) and students’ motivation (27%). While the factors that have indirect influences on students’ achievement, are family environment (97%) and school environment (37).

Statistical analysis of microgravity experiment performance using the degrees of success scale

NASA Technical Reports Server (NTRS)

Upshaw, Bernadette; Liou, Ying-Hsin Andrew; Morilak, Daniel P.

1994-01-01

This paper describes an approach to identify factors that significantly influence microgravity experiment performance. Investigators developed the 'degrees of success' scale to provide a numerical representation of success. A degree of success was assigned to 293 microgravity experiments. Experiment information including the degree of success rankings and factors for analysis was compiled into a database. Through an analysis of variance, nine significant factors in microgravity experiment performance were identified. The frequencies of these factors are presented along with the average degree of success at each level. A preliminary discussion of the relationship between the significant factors and the degree of success is presented.

On the Factor Structure of a Reading Comprehension Test

ERIC Educational Resources Information Center

Salehi, Mohammad

2011-01-01

To investigate the construct validly of a section of a high stakes test, an exploratory factor analysis using principal components analysis was employed. The rotation used was varimax with the suppression level of 0.30. Eleven factors were extracted out of 35 reading comprehension items. The fact that these factors emerged speak to the construct…

Multilevel Confirmatory Factor Analysis of a Scale Measuring Interagency Collaboration of Children's Mental Health Agencies

ERIC Educational Resources Information Center

Dedrick, Robert F.; Greenbaum, Paul E.

2011-01-01

Multilevel confirmatory factor analysis was used to evaluate the factor structure underlying the 12-item, three-factor "Interagency Collaboration Activities Scale" (ICAS) at the informant level and at the agency level. Results from 378 professionals (104 administrators, 201 service providers, and 73 case managers) from 32 children's mental health…

Understanding Older Adults' Perceptions of Internet Use: An Exploratory Factor Analysis

ERIC Educational Resources Information Center

Zheng, Robert; Spears, Jeffrey; Luptak, Marilyn; Wilby, Frances

2015-01-01

The current study examined factors related to older adults' perceptions of Internet use. Three hundred ninety five older adults participated in the study. The factor analysis revealed four factors perceived by older adults as critical to their Internet use: social connection, self-efficacy, the need to seek financial information, and the need to…