Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish.

NARCIS (Netherlands)

Meijer, A.H.; Krens, SF Gabby; Rodriguez, IA Medina; He, S; Bitter, W.; Snaar-Jagalska, B Ewa; Spaink, H.P.

2004-01-01

The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the

Surgical approach to TIR3 cytology class A prospective evaluation.

Science.gov (United States)

D'Alessandro, Nicola; Fasano, Giovanni Michele; Gilio, Francesco; Iside, Giovanni; Izzo, Maria Lucia; Loffredo, Andrea; Pinto, Margherita; Tramontano, Salvatore; Tramutola, Giuseppe; Citro, Giuseppe

2014-01-01

Fine-needle aspiration (FNA) has proven to be a safe and reliable method of investigation of thyroid lesions. Referencing to European classification, the associated risk of malignancy for TIR3, category reserved for aspirates that contain architectural and/or nuclear atypia, is variable in such studies. Aims of study were evaluating safety of surgical approach, assessing perioperative parameters surgically related, and estimating neoplastic rate for TIR3 group. A prospective evaluation of all TIR3 submitted to thyroidectomy was conducted by assessing histopatohologic results between January 2005 and December 2012, considering two categories, positive (neoplastic) and negative (not neoplastic) group. Intraoperative and complication rate was analyzed on TIR3 population. A total of 1514 total thyroidectomy was performed from 2005 to 2012: a total of 148 cases was considered on TIR3 group. Positive cases amounted to 64 (43.2%), 29 of which were carcinoma (19.6% of total population) and 35 of which were adenoma, while negative cases amounted to 84 (56.8%). Sensitivity and specificity of TIR3 as neoplastic screening was 43.2% and 82.1%. A total of 32 linfectomies was performed (21.6% of group). Positive group presented a significant lower mean age than negative group (42.1 vs 56.2 years) TIR3 group represents a various category, with probably different malignancy risk. Our results and neoplasms rate confirmed that surgical option should be gold standard, in order to define atypical pattern and reduce delayed diagnoses. Choice of a second FNA or a imaging monitoring should be adopted for specific condition. Fine-needle aspiration, Thyroidectomy, TIR3, Thyroid cancer.

Forgetting in C. elegans Is Accelerated by Neuronal Communication via the TIR-1/JNK-1 Pathway

Directory of Open Access Journals (Sweden)

Akitoshi Inoue

2013-03-01

Full Text Available The control of memory retention is important for proper responses to constantly changing environments, but the regulatory mechanisms underlying forgetting have not been fully elucidated. Our genetic analyses in C. elegans revealed that mutants of the TIR-1/JNK-1 pathway exhibited prolonged retention of olfactory adaptation and salt chemotaxis learning. In olfactory adaptation, conditioning induces attenuation of odor-evoked Ca2+ responses in olfactory neurons, and this attenuation is prolonged in the TIR-1/JNK-1-pathway mutant animals. We also found that a pair of neurons in which the pathway functions is required for the acceleration of forgetting, but not for sensation or adaptation, in wild-type animals. In addition, the neurosecretion from these cells is important for the acceleration of forgetting. Therefore, we propose that these neurons accelerate forgetting through the TIR-1/JNK-1 pathway by sending signals that directly or indirectly stimulate forgetting.

On the composition of ISO 25964 hierarchical relations (BTG, BTP, BTI)

NARCIS (Netherlands)

Alexiev, A; Isaac, A.H.J.C.A.; Lindenthal, J

2015-01-01

Knowledge organization systems (KOS) can use different types of hierarchical relations: broader generic (BTG), broader partitive (BTP), and broader instantial (BTI). The latest ISO standard on thesauri (ISO 25964) has formalized these relations in a corresponding OWL ontology (De Smedt et al., ISO

Analysis of TIR- and non-TIR-NBS-LRR disease resistance gene analogous in pepper: characterization, genetic variation, functional divergence and expression patterns

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Wan Hongjian

2012-09-01

Full Text Available Abstract Background Pepper (Capsicum annuum L. is one of the most important vegetable crops worldwide. However, its yield and fruit quality can be severely threatened by several pathogens. The plant nucleotide-binding site (NBS-leucine-rich repeat (LRR gene family is the largest class of known disease resistance genes (R genes effective against such pathogens. Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs. Results A total of 78 R gene analogues (CaRGAs were identified in pepper by degenerate PCR amplification and database mining. Phylogenetic tree analysis of the deduced amino acid sequences for 51 of these CaRGAs with typically conserved motifs ( P-loop, kinase-2 and GLPL along with some known R genes from Arabidopsis and tomato grouped these CaRGAs into the non-Toll interleukin-1 receptor (TIR-NBS-LRR (CaRGAs I to IV and TIR-NBS-LRR (CaRGAs V to VII subfamilies. The presence of consensus motifs (i.e. P-loop, kinase-2 and hydrophobic domain is typical of the non-TIR- and TIR-NBS-LRR gene subfamilies. This finding further supports the view that both subfamilies are widely distributed in dicot species. Functional divergence analysis provided strong statistical evidence of altered selective constraints during protein evolution between the two subfamilies. Thirteen critical amino acid sites involved in this divergence were also identified using DIVERGE version 2 software. Analyses of non-synonymous and synonymous substitutions per site showed that purifying selection can play a critical role in the evolutionary processes of non-TIR- and TIR-NBS-LRR RGAs in pepper. In addition, four specificity-determining positions were predicted to be responsible for functional specificity. qRT-PCR analysis showed that both salicylic and abscisic acids induce the expression of CaRGA genes, suggesting that they may primarily be involved in

A TIR domain variant of MyD88 adapter-like (Mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling.

NARCIS (Netherlands)

Nagpal, K.; Plantinga, T.S.; Wong, J.; Monks, B.G.; Gay, N.J.; Netea, M.G.; Fitzgerald, K.A.; Golenbock, D.

2009-01-01

The adapter protein MyD88 adapter-like (Mal), encoded by TIR-domain containing adapter protein (Tirap) (MIM 606252), is the most polymorphic of the five adapter proteins involved in Toll-like receptor signaling, harboring eight non-synonymous single nucleotide polymorphisms in its coding region. We

Early Transcriptional Responses of Bovine Chorioallantoic Membrane Explants to Wild Type, ΔvirB2 or ΔbtpB Brucella abortus Infection

Science.gov (United States)

Mol, Juliana P. S.; Costa, Erica A.; Carvalho, Alex F.; Sun, Yao-Hui; Tsolis, Reneé M.; Paixão, Tatiane A.; Santos, Renato L.

2014-01-01

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; Pabortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion. PMID:25259715

Effect of pipe rupture loads inside containment in the break exclusionary piping outside containment

International Nuclear Information System (INIS)

Weiss, G.

1987-01-01

The plant design for protection against piping failures outside containment should make sure that fluid system piping in containment penetration areas are designed to meet the break exclusionary provisions contained in the BTP MEB 3-1. According to these provisions, following a piping failure (main steam line) inside containment, the part of the flued head connected to the piping outside containment, should not exceed the ASME Code stress limits for the appropriate load combinations. A finite element analysis has been performed to evaluate the stress level in this area. (orig./HP)

[Family of ribosomal proteins S1 contains unique conservative domain].

Science.gov (United States)

Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

2010-01-01

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

Thermal Imaging Performance of TIR Onboard the Hayabusa2 Spacecraft

Science.gov (United States)

Arai, Takehiko; Nakamura, Tomoki; Tanaka, Satoshi; Demura, Hirohide; Ogawa, Yoshiko; Sakatani, Naoya; Horikawa, Yamato; Senshu, Hiroki; Fukuhara, Tetsuya; Okada, Tatsuaki

2017-07-01

The thermal infrared imager (TIR) is a thermal infrared camera onboard the Hayabusa2 spacecraft. TIR will perform thermography of a C-type asteroid, 162173 Ryugu (1999 JU3), and estimate its surface physical properties, such as surface thermal emissivity ɛ , surface roughness, and thermal inertia Γ, through remote in-situ observations in 2018 and 2019. In prelaunch tests of TIR, detector calibrations and evaluations, along with imaging demonstrations, were performed. The present paper introduces the experimental results of a prelaunch test conducted using a large-aperture collimator in conjunction with TIR under atmospheric conditions. A blackbody source, controlled at constant temperature, was measured using TIR in order to construct a calibration curve for obtaining temperatures from observed digital data. As a known thermal emissivity target, a sandblasted black almite plate warmed from the back using a flexible heater was measured by TIR in order to evaluate the accuracy of the calibration curve. As an analog target of a C-type asteroid, carbonaceous chondrites (50 mm × 2 mm in thickness) were also warmed from the back and measured using TIR in order to clarify the imaging performance of TIR. The calibration curve, which was fitted by a specific model of the Planck function, allowed for conversion to the target temperature within an error of 1°C (3σ standard deviation) for the temperature range of 30 to 100°C. The observed temperature of the black almite plate was consistent with the temperature measured using K-type thermocouples, within the accuracy of temperature conversion using the calibration curve when the temperature variation exhibited a random error of 0.3 °C (1σ ) for each pixel at a target temperature of 50°C. TIR can resolve the fine surface structure of meteorites, including cracks and pits with the specified field of view of 0.051°C (328 × 248 pixels). There were spatial distributions with a temperature variation of 3°C at the setting

A novel role for the TIR domain in association with pathogen-derived elicitors.

Directory of Open Access Journals (Sweden)

Tessa M Burch-Smith

2007-03-01

Full Text Available Plant innate immunity is mediated by Resistance (R proteins, which bear a striking resemblance to animal molecules of similar function. Tobacco N is a TIR-NB-LRR R gene that confers resistance to Tobacco mosaic virus, specifically the p50 helicase domain. An intriguing question is how plant R proteins recognize the presence of pathogen-derived Avirulence (Avr elicitor proteins. We have used biochemical cell fraction and immunoprecipitation in addition to confocal fluorescence microscopy of living tissue to examine the association between N and p50. Surprisingly, both N and p50 are cytoplasmic and nuclear proteins, and N's nuclear localization is required for its function. We also demonstrate an in planta association between N and p50. Further, we show that N's TIR domain is critical for this association, and indeed, it alone can associate with p50. Our results differ from current models for plant innate immunity that propose detection is mediated solely through the LRR domains of these molecules. The data we present support an intricate process of pathogen elicitor recognition by R proteins involving multiple subcellular compartments and the formation of multiple protein complexes.

Spatial Resolution Assessment of the Telops Airborne TIR Imagery

Science.gov (United States)

Mousakhani, S.; Eslami, M.; Saadatseresht, M.

2017-09-01

Having a high spatial resolution of Thermal InfraRed (TIR) Sensors is a challenge in remote sensing applications. Airborne high spatial resolution TIR is a novel source of data that became available lately. Recent developments in spatial resolution of the TIR sensors have been an interesting topic for scientists. TIR sensors are very sensitive to the energies emitted from objects. Past researches have been shown that increasing the spatial resolution of an airborne image will decrease the spectral content of the data and will reduce the Signal to Noise Ratio (SNR). Therefore, in this paper a comprehensive assessment is adapted to estimate an appropriate spatial resolution of the TIR data (TELOPS TIR data), in consideration of the SNR. So, firstly, a low-pass filter is applied on TIR data and the achieved products fed to a classification method for analysing of the accuracy improvement. The obtained results show that, there is no significant change in classification accuracy by applying low-pass filter. Furthermore, estimation of the appropriate spatial resolution of the TIR data is evaluated for obtaining higher spectral content and SNR. For this purpose, different resolutions of the TIR data are created and fed to the maximum likelihood classification method separately. The results illustrated in the case of using images with ground pixel size four times greater than the original image, the classification accuracy is not reduced. Also, SNR and spectral contents are improved. But the corners sharpening is declined.

Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

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Hyunjeong Yang

Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

Landsat 8 Operational Land Imager (OLI)_Thermal Infared Sensor (TIRS) V1

Data.gov (United States)

National Aeronautics and Space Administration — Abstract:The Operational Land Imager (OLI) and Thermal Infrared Sensor (TIRS) are instruments onboard the Landsat 8 satellite, which was launched in February of...

The Prototype HyspIRI Thermal Infrared Radiometer (PHyTIR): A High Speed, Multispectral, Thermal Instrument Development in Support of HyspIRI-TIR

Science.gov (United States)

Hook, Simon

2011-01-01

The Prototype HyspIRI Thermal Infrared Radiometer (PHyTIR) is being developed as part of the risk reduction activities associated with the Hyperspectral Infrared Imager (HyspIRI). The HyspIRI mission was recommended by the National Research Council Decadal Survey and includes a visible shortwave infrared (SWIR) pushboom spectrometer and a multispectral whiskbroom thermal infrared (TIR) imager. Data from the HyspIRI mission will be used to address key science questions related to the Solid Earth and Carbon Cycle and Ecosystems focus areas of the NASA Science Mission Directorate. The HyspIRI TIR system will have 60m ground resolution, better than 200mK noise equivalent delta temperature (NEDT), 0.5C absolute temperature resolution with a 5-day repeat from LEO orbit. PHyTIR addresses the technology readiness level (TRL) of certain key subsystems of the TIR imager, primarily the detector assembly and scanning mechanism. PHyTIR will use Mercury Cadmium Telluride (MCT) technology at the focal plane and operate in time delay integration mode. A custom read out integrated circuit (ROIC) will provide the high speed readout hence allowing the high data rates needed for the 5 day repeat. PHyTIR will also demonstrate a newly developed interferometeric metrology system. This system will provide an absolute measurement of the scanning mirror to an order of magnitude better than conventional optical encoders. This will minimize the reliance on ground control points hence minimizing post-processing (e.g. geo-rectification computations).

PHyTIR - A Prototype Thermal Infrared Radiometer

Science.gov (United States)

Jau, Bruno M.; Hook, Simon J.; Johnson, William R.; Foote, Marc C.; Paine, Christopher G.; Pannell, Zack W.; Smythe, Robert F.; Kuan, Gary M.; Jakoboski, Julie K.; Eng, Bjorn T.

2013-01-01

This paper describes the PHyTIR (Prototype HyspIRI Thermal Infrared Radiometer) instrument, which is the engineering model for the proposed HyspIRI (Hyperspectral Infrared Imager) earth observing instrument. The HyspIRI mission would be comprised of the HyspIRI TIR (Thermal Infrared Imager), and a VSWIR (Visible Short-Wave Infra-Red Imaging Spectrometer). Both instruments would be used to address key science questions related to the earth's carbon cycle, ecosystems, climate, and solid earth properties. Data gathering of volcanic activities, earthquakes, wildfires, water use and availability, urbanization, and land surface compositions and changes, would aid the predictions and evaluations of such events and the impact they create. Even though the proposed technology for the HyspIRI imager is mature, the PHyTIR prototype is needed to advance the technology levels for several of the instrument's key components, and to reduce risks, in particular to validate 1) the higher sensitivity, spatial resolution, and higher throughput required for this focal plane array, 2) the pointing accuracy, 2) the characteristics of several spectral channels, and 4) the use of ambient temperature optics. The PHyTIR telescope consists of the focal plane assembly that is housed within a cold housing located inside a vacuum enclosure; all mounted to a bulkhead, and an optical train that consists of 3 powered mirrors; extending to both sides of the bulkhead. A yoke connects the telescope to a scan mirror. The rotating mirror enables to scan- a large track on the ground. This structure is supported by kinematic mounts, linking the telescope assembly to a base plate that would also become the spacecraft interface for HyspIRI. The focal plane's cooling units are also mounted to the base plate, as is an overall enclosure that has two viewing ports with large exterior baffles, shielding the focal plane from incoming stray light. PHyTIR's electronics is distributed inside and near the vacuum

Insight into Phosphatidylinositol-Dependent Membrane Localization of the Innate Immune Adaptor Protein Toll/Interleukin 1 Receptor Domain-Containing Adaptor Protein

Directory of Open Access Journals (Sweden)

Mahesh Chandra Patra

2018-01-01

Full Text Available The toll/interleukin 1 receptor (TIR domain-containing adaptor protein (TIRAP plays an important role in the toll-like receptor (TLR 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI 4,5-bisphosphate (PIP2 on the plasma membrane and PI (3,4,5-trisphosphate (PIP3 on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP’s membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein–protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32, additional positively charged residues (K34, K35, and R36 showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP’s affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP’s membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases.

ASTER L2 Surface Radiance TIR V003

Data.gov (United States)

National Aeronautics and Space Administration — The ASTER L2 Surface Radiance TIR is an on-demand product generated using the five thermal infra-red (TIR) Bands (acquired either during the day or night time)...

Genome-wide comparative analysis reveals similar types of NBS genes in hybrid Citrus sinensis genome and original Citrus clementine genome and provides new insights into non-TIR NBS genes.

Directory of Open Access Journals (Sweden)

Yunsheng Wang

Full Text Available In this study, we identified and compared nucleotide-binding site (NBS domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China. Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.

GOSAT TIR spectral validation with High/Low temperature target using Aircraft base-FTS S-HIS

Science.gov (United States)

Kataoka, F.; Knuteson, R.; Taylor, J. K.; Kuze, A.; Shiomi, K.; Suto, H.; Yoshida, J.

2017-12-01

The Greenhouse gases Observing SATellite (GOSAT) was launched on January 2009. The GOSAT is equipped with TANSO-FTS (Fourier-Transform Spectrometer), which observe reflected solar radiation from the Earth's surface with shortwave infrared (SWIR) band and thermal emission from the Earth's surface and atmosphere with thermal infrared (TIR) band. The TIR band cover wide spectral range (650 - 1800 [cm-1]) with a high spectral resolution (0.2 [cm-1]). The TIR spectral information provide vertical distribution of CO2 and CH4. GOSAT has been operation more than eight years. In this long operation, GOSAT had experienced two big accidents; Rotation of one of the solar paddles stopped and sudden TANSO-FTS operation stop in May 2014 and cryocooler shutdown and restart in August - September 2015. These events affected the operation condition of the TIR photo-conductive (PC)-MCT detector. FTS technology using multiplex wide spectra needs wide dynamic range. PC detector has nonlinearity. Its correction needs accurate estimation of time-dependent offset. In current TIR Level 1B product version (V201), the non-photon level offset (Vdc_offset) estimated from on-orbit deep space calibration data and pre-launch background radiation model. But the background radiation and detector temperature have changed after cryocooler shutdown events. These changes are too small to detect from onboard temperature sensors. The next TIR Level 1B product uses cross calibration data together with deep space calibration data and instrument radiation model has been updated. This work describes the evaluation of new TIR Level 1B spectral quality with aircraft-based FTS; Scanning High-resolution Interferometer Sounder (S-HIS). The S-HIS mounted on the high-altitude ER-2 aircraft and flew at about 20km altitude. Because the observation geometry of GOSAT and S-HIS are quite different, we used the double difference method using atmospheric transfer model. GOSAT TIR band cover wide dynamic range, so we check

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Science.gov (United States)

Mistry, Pragnesh; Laird, Michelle H. W.; Schwarz, Ryan S.; Greene, Shannon; Dyson, Tristan; Snyder, Greg A.; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y.; MacKerell, Alexander D.; Vogel, Stefanie N.

2015-01-01

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

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

Science.gov (United States)

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

2018-06-19

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

Plan de mejoramiento a la empresa BTP Medidores y Accesorios S.A.

OpenAIRE

Prieto García, Carolina

2013-01-01

Este proyecto se realizó con el fin de poner en marcha los conocimientos aprendidos en la carrera, como es el realizar y desarrollar herramientas que permitan crear soluciones a los problemas que se puedan presentar en una empresa. Para ello se seleccionó la empresa de producción “BTP Medidores y Accesorios”, en donde a partir de la aplicación del MMGO y el análisis interno de la metodología DOFA, se pudo conocer e identificar las falencias y/o oportunidades de mejora que existían. En el m...

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein*

Science.gov (United States)

Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V.; Sampey, Gavin C.; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

2014-01-01

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. PMID:24939845

Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

Science.gov (United States)

Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V; Sampey, Gavin C; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

2014-08-08

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Subarachnoidal-pleural fistula (SAPF) as an unusual cause of persistent pleural effusion. Beta-trace protein as a marker for SAPF. Case report and review of the literature.

Science.gov (United States)

Deseyne, S; Vanhouteghem, K; Hallaert, G; Delanghe, J; Malfait, T

2015-02-01

We describe a case of a 56-year-old woman who developed a recurrent pleural effusion after a thoracoscopic resection of an anterior bulging thoracic disc hernia (level D9-D10). Despite several evacuating pleural punctions, dyspnea reoccurred due to recurrent pleural effusion, the same side as the disc resection. Because of increasing headache after each punction, a subarachnoidal pleural fistula (SAPF) was suspected. Although magnetic resonance imaging (MRI) showed features suggestive of SAPF, there was not enough evidence to justify a new thorascopy. Cerebrospinal fluid (CSF) leakage into the thoracic and abdominal cavity has been described as a result of trauma or surgery. Detection of beta-trace protein (BTP, a brain-specific protein) has been described to detect CSF fistulae causing rhino- and otoliquorrhea. Similarly, BTP determination could be used to identify the presence of CSF at other anatomical sites such as the thoracic cavity. Therefore, we decided to determine the concentration of BTP in the pleural effusion of this patient. BTP was assayed using immunonephelometry. The patient's BTP pleural fluid concentration was 14·0 mg/l, which was a 25-fold increase compared with the BTP serum concentration. After insertion of a subarachnoidal lumbal catheter, a video-assisted thorascopy was performed. Leakage of liquor through the parietal pleura into the thoracic cavity was observed. The SAPF was closed using a durasis patch and DuraSeal®. Postoperatively, there was no reoccurrence of pleural fluid. SAPF has to be included to the differential diagnosis of patients with persistent pleural effusion after spinal surgery. This case illustrates the importance of BTP in diagnosing SAPF, especially in cases where major therapeutic consequences may need to be drawn.

Computing UV/vis spectra using a combined molecular dynamics and quantum chemistry approach: bis-triazin-pyridine (BTP) ligands studied in solution.

Science.gov (United States)

Höfener, Sebastian; Trumm, Michael; Koke, Carsten; Heuser, Johannes; Ekström, Ulf; Skerencak-Frech, Andrej; Schimmelpfennig, Bernd; Panak, Petra J

2016-03-21

We report a combined computational and experimental study to investigate the UV/vis spectra of 2,6-bis(5,6-dialkyl-1,2,4-triazin-3-yl)pyridine (BTP) ligands in solution. In order to study molecules in solution using theoretical methods, force-field parameters for the ligand-water interaction are adjusted to ab initio quantum chemical calculations. Based on these parameters, molecular dynamics (MD) simulations are carried out from which snapshots are extracted as input to quantum chemical excitation-energy calculations to obtain UV/vis spectra of BTP ligands in solution using time-dependent density functional theory (TDDFT) employing the Tamm-Dancoff approximation (TDA). The range-separated CAM-B3LYP functional is used to avoid large errors for charge-transfer states occurring in the electronic spectra. In order to study environment effects with theoretical methods, the frozen-density embedding scheme is applied. This computational procedure allows to obtain electronic spectra calculated at the (range-separated) DFT level of theory in solution, revealing solvatochromic shifts upon solvation of up to about 0.6 eV. Comparison to experimental data shows a significantly improved agreement compared to vacuum calculations and enables the analysis of relevant excitations for the line shape in solution.

Plasma Beta-Trace Protein as a Marker of Residual Renal Function: The Effect of Different Hemodialysis Modalities and Intra-Individual Variability over Time

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Amaryllis H. van Craenenbroeck

2017-11-01

Full Text Available Background/Aims: Beta-trace protein (BTP is a low-molecular-weight molecule, which may be used to assess residual renal function (RRF in dialysis patients. Here we evaluated the influence of hemodialysis (HD and hemodiafiltration (HDF on plasma BTP, and analyzed the inter- and intra-individual variability of plasma BTP over time in HD and peritoneal dialysis (PD patients. Methods: In 12 prevalent HD patients, the effect of a single session of low-flux HD, high-flux HD and HDF on plasma BTP was studied. Blood samples were taken at baseline, after 120 and 240 minutes, and at the start of the next dialysis session. In 13 HD patients and 10 PD patients, inter- and intra-individual variability over three months was studied (monthly and weekly, respectively. Plasma BTP was measured using a nephelometric method. Results: No significant decrease in plasma BTP was seen following a session of low-flux HD. Both high-flux HD and HDF resulted in a significant decrease immediately after dialysis (22% and 61% median decrease, respectively. A significant reduction of the molecule persisted only in HDF and a significant decrease (-15% was still found immediately before the start of the next dialysis session. In both HD and PD patients, the reproducibility over time was excellent with intra-class correlation coefficient of 0.96 (0.93-0.99 and 0.92 (0.86-0.99 respectively. In a small cohort of PD patients, fair agreement existed between mGFR (average of renal urea and creatinine clearance from a 24 hours urine collection and the BTP-based GFR estimation. Conclusion: BTP is a stable marker and a promising tool for RRF estimations in PD and HD patients. In patients receiving HDF, plasma levels of BTP should be interpreted with caution.

Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity.

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Meenu S Padmanabhan

2013-03-01

Full Text Available Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the SQUAMOSA PROMOTER BINDING PROTEIN (SBP-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the Nicotiana TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to Tobacco mosaic virus. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against Pseudomonas syringae carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5.

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

Science.gov (United States)

An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

2008-09-05

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

Science.gov (United States)

Nakamura, Masayoshi; Yagi, Noriyoshi; Kato, Takehide; Fujita, Satoshi; Kawashima, Noriyuki; Ehrhardt, David W; Hashimoto, Takashi

2012-07-01

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Use of the Vis-SWIR to Aid Atmospheric Correction of Multispectral and Hyperspectral Thermal Infrared (TIR) Imagery: The TIR Model

National Research Council Canada - National Science Library

Gruninger, John; Fox, Marsha; Lee, Jamine; Ratkowski, Anthony J; Hoke, Michael L

2006-01-01

The atmospheric correction of thermal infrared (TIR) imagery involves the combined tasks of separation of atmospheric transmittance, downwelling flux and upwelling radiance from the surface material spectral emissivity and temperature...

Nucleic acid-binding properties of the RRM-containing protein RDM1

International Nuclear Information System (INIS)

Hamimes, Samia; Bourgeon, Dominique; Stasiak, Alicja Z.; Stasiak, Andrzej; Van Dyck, Eric

2006-01-01

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L 119 GF → AAA mutation affects the mode of RDM1 binding to single-stranded DNA

Venemaa keelab osa kaupade sisseveo TIR-märkmiku katte all / Lauri Lusti

Index Scriptorium Estoniae

Lusti, Lauri

1997-01-01

1998. aastast rakendab Venemaa olulisi piiranguid seoses TIR-süsteemi kasutamisega kauba sisseveol. Lisatud Vene Föderatsiooni Riikliku Tollikomitee käskkiri 22. augustist 1997 nr. 513 "TIR-karneti kasutamine mõne kategooria kaupade veoks"

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

Science.gov (United States)

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

Selective adsorption of thiophene and 1-benzothiophene on metal-ion-exchanged zeolites in organic medium.

Science.gov (United States)

Xue, Mei; Chitrakar, Ramesh; Sakane, Kohji; Hirotsu, Takahiro; Ooi, Kenta; Yoshimura, Yuji; Feng, Qi; Sumida, Naoto

2005-05-15

Adsorption of the organic sulfur compounds thiophene (TP) and 1-benzothiophene (1-BTP) in an organic model solution of hydrodesulfurizated gasoline (heptane with 1 wt% toluene and 0.156 mM (5 ppmw as sulfur) TP or 1-BTP) was studied by a batch method at 80 degrees C using metal-ion-exchanged Y-zeolites. Although NaY-zeolite or its acid-treated material rarely adsorbed the organic sulfur compounds, NaY-zeolites exchanged with Ag+, Cu2+, and Ce3+ ions and NH(4)Y-zeolites exchanged with Ce3+ ions showed markedly high adsorptive capacities for TP and 1-BTP. The sulfur uptake increased in the order CuY-zeolite(Na)1-BTP followed the Langmuir's relationship and the saturation capacities by CeY-zeolite(Na) were calculated as 0.022 and 0.033 mmol/g, respectively. The mole ratios of TP/Ce and 1-BTP/Ce were 0.031 and 0.047, respectively. CeY-zeolite(NH4) which was prepared from NH4Y-zeolite showed less uptake of TP and 1-BTP than CeY-zeolite(Na), probably due to its lower cerium content.

Multi-layer Retrievals of Greenhouse Gases from a Combined Use of GOSAT TANSO-FTS SWIR and TIR

Science.gov (United States)

Kikuchi, N.; Kuze, A.; Kataoka, F.; Shiomi, K.; Hashimoto, M.; Suto, H.; Knuteson, R. O.; Iraci, L. T.; Yates, E. L.; Gore, W.; Tanaka, T.; Yokota, T.

2016-12-01

The TANSO-FTS sensor onboard GOSAT has three frequency bands in the shortwave infrared (SWIR) and the fourth band in the thermal infrared (TIR). Observations of high-resolution spectra of reflected sunlight in the SWIR are extensively utilized to retrieve column-averaged concentrations of the major greenhouse gases such as carbon dioxide (XCO2) and methane (XCH4). Although global XCO2 and XCH4 distribution retrieved from SWIR data can reduce the uncertainty in the current knowledge about sources and sinks of these gases, information on the vertical profiles would be more useful to constrain the surface flux and also to identify the local emission sources. Based on the degrees of freedom for signal, Kulawik et al. (2016, IWGGMS-12 presentation) shows that 2-layer information on the concentration of CO2 can be extracted from TANSO-FTS SWIR measurements, and the retrieval error is predicted to be about 5 ppm in the lower troposphere. In this study, we present multi-layer retrievals of CO2 and CH4 from a combined use of measurements of TANSO-FTS SWIR and TIR. We selected GOSAT observations at Railroad Valley Playa in Nevada, USA, which is a vicarious calibration site for TANSO-FTS, as we have various ancillary data including atmospheric temperature and humidity taken by a radiosonde, surface temperature, and surface emissivity with a ground based FTS. All of these data are useful especially for retrievals using TIR spectra. Currently, we use the 700-800 cm-1 and 1200-1300 cm-1 TIR windows for CO2 and CH4 retrievals, respectively, in addition to the SWIR bands. We found that by adding TIR windows, 3-layer information can be extracted, and the predicted retrieval error in the CO2 concentration was reduced about 1 ppm in the lower troposphere. We expect that the retrieval error could be further reduced by optimizing TIR windows and by reducing systematic forward model errors.

Evaluation des risques-projet par une approche AHP/WPM: Application à un projet réel BTP

OpenAIRE

LMOUSSAOUI , Hafida; JAMOULI , Hicham

2015-01-01

International audience; La complexité et l'incertitude de l'environnement sont à l'origine de nouveaux défis auxquels les entreprises du domaine BTP font face quotidiennement. La maîtrise des paramètres des projets devient donc une nécessité pour leur pérennité. L'objectif du management des risques est de maîtriser ces paramètres pendant toutes les phases du projet. Il s'appuie sur un processus continu et itératif permettant successivement d'identifier et analyser les risques encourus, les év...

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

Science.gov (United States)

Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

Design and testing of a uniformly solar energy TIR-R concentration lenses for HCPV systems.

Science.gov (United States)

Shen, S C; Chang, S J; Yeh, C Y; Teng, P C

2013-11-04

In this paper, total internal reflection-refraction (TIR-R) concentration (U-TIR-R-C) lens module were designed for uniformity using the energy configuration method to eliminate hot spots on the surface of solar cell and increase conversion efficiency. The design of most current solar concentrators emphasizes the high-power concentration of solar energy, however neglects the conversion inefficiency resulting from hot spots generated by uneven distributions of solar energy concentrated on solar cells. The energy configuration method proposed in this study employs the concept of ray tracing to uniformly distribute solar energy to solar cells through a U-TIR-R-C lens module. The U-TIR-R-C lens module adopted in this study possessed a 76-mm diameter, a 41-mm thickness, concentration ratio of 1134 Suns, 82.6% optical efficiency, and 94.7% uniformity. The experiments demonstrated that the U-TIR-R-C lens module reduced the core temperature of the solar cell from 108 °C to 69 °C and the overall temperature difference from 45 °C to 10 °C, and effectively relative increased the conversion efficiency by approximately 3.8%. Therefore, the U-TIR-R-C lens module designed can effectively concentrate a large area of sunlight onto a small solar cell, and the concentrated solar energy can be evenly distributed in the solar cell to achieve uniform irradiance and effectively eliminate hot spots.

SpecTIR and SEBASS analysis of the National Mining District, Humboldt County, Nevada

Science.gov (United States)

Morken, Todd O.

The purpose of this study was to evaluate the minerals and materials that could be uniquely identified and mapped from measurements made with airborne hyperspectral SpecTIR VNIR/SWIR and SEBASS TIR sensors over areas in the National Mining District. SpecTIR Corporation and Aerospace Corporation acquired Hyperspectral measurements on June 26, 2008 using their ProSpecTIR and SEBASS sensors respectively. In addition the effects of vegetation, elevation, the atmosphere on spectral measurements were evaluated to determine their impact upon the data analysis and target identification. The National Mining District is located approximately 75 miles northeast of Winnemucca, Nevada at the northern end of the Santa Rosa Mountains. Precious metal mining has been dormant in this area since the 1940's, however with increased metal prices over the last decade economic interest in the region has increased substantially. Buckskin Mountain has a preserved alteration assemblage that is exposed in topographically steep terrain, ideal for exploring what hydrothermal alteration products can be identified and mapped in these datasets. These Visible Near Infrared (VNIR), Short Wave Infrared (SWIR), and Long Wave Infrared (LWIR) hyperspectral datasets were used to identify and map kaolinite, alunite, quartz, opal, and illite/muscovite, all of which are useful exploration target identifiers and can indicate regions of alteration. These mapping results were then combined with and compared to other geospatial data in a geographic information systems (GIS) database. The TIR hyperspectral data provided significant additional information that can benefit geologic exploration and demonstrated its usefulness as an additional tool for geological exploration.

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1 protein with a unique regulatory C-terminus.

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Fabio Schneider Ribeiro

Full Text Available The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1. The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension.

Directory of Open Access Journals (Sweden)

Alan Y Deng

Full Text Available Multiple quantitative trait loci (QTLs for blood pressure (BP have been detected in rat models of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1/activator of G protein signaling 8 (Ags8 to be the strongest candidate for C1QTL1, since numerous non-synonymous mutations are found. Moreover, the 5' Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evidence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or targeted-gene modifications, it might represent a part of the BP genetic architecture that operates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could validate the concept that a QTL in itself can influence BP 'indirectly' by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 variations will reveal novel insights into the BP modulation via a regulatory hierarchy.

Thermodynamic and structural description of europium complexation in 1-octanol - H2O solutions

International Nuclear Information System (INIS)

Vu, T.H.; Charbonnel, M.C.; Boubals, N.; Couston, L.; Arnaud, F.

2008-01-01

Polydentate N-bearing ligands such as bis-triazinyl-pyridines (BTPs) are interesting extractants for actinide(III)/lanthanide(III) separation. A description of europium complexation in 1-octanol solutions was undertaken to enhance the knowledge of the extraction mechanisms. The first solvation shell for europium(III) nitrate, chloride, and perchlorate with different amounts of water was determined by Time-Resolved Laser-Induced Fluorescence (TRLIF) spectroscopy. Europium nitrate complexation by iPr-BTP was then studied by TRLIF and micro-calorimetry; similar stability constants related to the formation of Eu(BTP) 2 3+ and Eu(BTP) 3 3+ were obtained by both techniques (log(β 2 ) = 9.0 ± 0.3 and log(β 3 ) = 13.8 ± 0.2). The presence of water in the octanol diluent has an influence on solvation of europium and also on the [Eu(BTP) 2 3+ ] / [Eu(BTP) 3 3+ ] ratio. (authors)

Impact of line parameter database and continuum absorption on GOSAT TIR methane retrieval

Science.gov (United States)

Yamada, A.; Saitoh, N.; Nonogaki, R.; Imasu, R.; Shiomi, K.; Kuze, A.

2017-12-01

The current methane retrieval algorithm (V1) at wavenumber range from 1210 cm-1 to 1360 cm-1 including CH4 ν 4 band from the thermal infrared (TIR) band of Thermal and Near-infrared Sensor for Carbon Observation Fourier Transform Spectrometer (TANSO-FTS) onboard Greenhouse Gases Observing Satellite (GOSAT) uses LBLRTM V12.1 with AER V3.1 line database and MT CKD 2.5.2 continuum absorption model to calculate optical depth. Since line parameter databases have been updated and the continuum absorption may have large uncertainty, the purpose of this study is to assess the impact on {CH}4 retrieval from the choice of line parameter databases and the uncertainty of continuum absorption. We retrieved {CH}4 profiles with replacement of line parameter database from AER V3.1 to AER v1.0, HITRAN 2004, HITRAN 2008, AER V3.2, or HITRAN 2012 (Rothman et al. 2005, 2009, and 2013. Clough et al., 2005), we assumed 10% larger continuum absorption coefficients and 50% larger temperature dependent coefficient of continuum absorption based on the report by Paynter and Ramaswamy (2014). We compared the retrieved CH4 with the HIPPO CH4 observation (Wofsy et al., 2012). The difference from HIPPO observation of AER V3.2 was the smallest and 24.1 ± 45.9 ppbv. The differences of AER V1.0, HITRAN 2004, HITRAN 2008, and HITRAN 2012 were 35.6 ± 46.5 ppbv, 37.6 ± 46.3 ppbv, 32.1 ± 46.1 ppbv, and 35.2 ± 46.0 ppbv, respectively. Maximum {CH}4 retrieval differences were -0.4 ppbv at the layer of 314 hPa when we used 10% larger absorption coefficients of {H}2O foreign continuum. Comparing AER V3.2 case to HITRAN 2008 case, the line coupling effect reduced difference by 8.0 ppbv. Line coupling effects were important for GOSAT TIR {CH}4 retrieval. Effects from the uncertainty of continuum absorption were negligible small for GOSAT TIR CH4 retrieval.

Effects of Weathering on TIR Spectra and Rock Classification

Science.gov (United States)

McDowell, M. L.; Hamilton, V. E.; Riley, D.

2006-03-01

Changes in mineralogy due to weathering are detectable in the TIR and cause misclassification of rock types. We survey samples over a range of lithologies and attempt to provide a method of correction for rock identification from weathered spectra.

Identifying Pre-Seismic TIR Anomalies: A Long Term (2004-2015) Of RST Analysis Over Turkish Area

Science.gov (United States)

Perrone, A.; Tramutoli, V.; Corrado, A.; Filizzola, C.; Genzano, N.; Lisi, M.; Paciello, R.; Pergola, N.

2017-12-01

Since eighties, fluctuations of Earth's thermally emitted radiation, measured by satellite sensors operating in the thermal infrared (TIR) spectral range (i.e. 10-12 µm), have been associated with the complex process of preparation of earthquakes. Several theories have been proposed to explain their origin and their space-time evolution. In this paper, the Earth's emitted radiation in the Thermal Infra-Red spectral region is considered for its possible correlation with M≥4 earthquakes occurred in Turkey in between 2004 and 2015. Robust Satellite Technique (RST) and RETIRA (Robust Estimator of TIR Anomalies) index were used to preliminarily define, and then to identify, Significant Sequences of TIR Anomalies (SSTAs) in the period 1 April 2004- 31 October 2015 (12 years) of daily TIR images acquired by the Spinning Enhanced Visible and Infrared Imager (SEVIRI) on board the Meteosat Second Generation (MSG) satellite. The performed analysis shows that more than 67% of all identified SSTAs occur in the pre-fixed space-time window around the occurrence time and location of earthquakes (M≥4), with a false positive rate smaller than 33%. Moreover, Molchan error diagram analysis gave a clear indication of non-casualty of such a correlation, in comparison with the random guess function. Notwithstanding the huge amount of missed events due to frequent space/time data gaps produced by the presence of clouds over the scene the achieved results, and particularly the low rate of false positives registered on a so long testing period, seems sufficient (at least) to qualify TIR anomalies (identified by RST approach and RETIRA index) among the parameters to be considered in the framework of a multi-parametric approach to time-Dependent Assessment of Seismic Hazard (t-DASH).

Structural requirements for cub domain containing protein 1 (CDCP1 and Src dependent cell transformation.

Directory of Open Access Journals (Sweden)

Gwendlyn Kollmorgen

Full Text Available Cub domain containing protein 1 (CDCP1 is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762 abolished transformation, and mutation of a palmitoylation motif (C689,690G strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.

Derivation of Land Surface Temperature for Landsat-8 TIRS Using a Split Window Algorithm

Directory of Open Access Journals (Sweden)

Offer Rozenstein

2014-03-01

Full Text Available Land surface temperature (LST is one of the most important variables measured by satellite remote sensing. Public domain data are available from the newly operational Landsat-8 Thermal Infrared Sensor (TIRS. This paper presents an adjustment of the split window algorithm (SWA for TIRS that uses atmospheric transmittance and land surface emissivity (LSE as inputs. Various alternatives for estimating these SWA inputs are reviewed, and a sensitivity analysis of the SWA to misestimating the input parameters is performed. The accuracy of the current development was assessed using simulated Modtran data. The root mean square error (RMSE of the simulated LST was calculated as 0.93 °C. This SWA development is leading to progress in the determination of LST by Landsat-8 TIRS.

Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1.

Science.gov (United States)

Csorba, Tibor; Lózsa, Rita; Hutvágner, György; Burgyán, József

2010-05-01

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein-protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA-RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0.

Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

Science.gov (United States)

Hsieh, Ming-Chun; Ho, Yu-Cheng; Lai, Cheng-Yuan; Wang, Hsueh-Hsiao; Lee, An-Sheng; Cheng, Jen-Kun; Chau, Yat-Pang; Peng, Hsien-Yu

2017-11-01

Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

Directory of Open Access Journals (Sweden)

Hsiao Chiu-Bin

2006-11-01

Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

Thermodynamic and structural description of europium complexation in 1-octanol - H{sub 2}O solutions

Energy Technology Data Exchange (ETDEWEB)

Vu, T.H.; Charbonnel, M.C.; Boubals, N.; Couston, L. [CEA Marcoule, DEN/DRCP/SCPS/LCAM, BP 17171, 30207 Bagnols-sur-Ceze (France); Arnaud, F. [Laboratoire de Chimie Physique, IPHC, 25 rue Becquerel, 67087 Strasbourg (France)

2008-07-01

Polydentate N-bearing ligands such as bis-triazinyl-pyridines (BTPs) are interesting extractants for actinide(III)/lanthanide(III) separation. A description of europium complexation in 1-octanol solutions was undertaken to enhance the knowledge of the extraction mechanisms. The first solvation shell for europium(III) nitrate, chloride, and perchlorate with different amounts of water was determined by Time-Resolved Laser-Induced Fluorescence (TRLIF) spectroscopy. Europium nitrate complexation by iPr-BTP was then studied by TRLIF and micro-calorimetry; similar stability constants related to the formation of Eu(BTP){sub 2}{sup 3+} and Eu(BTP){sub 3}{sup 3+} were obtained by both techniques (log({beta}{sub 2}) = 9.0 {+-} 0.3 and log({beta}{sub 3}) = 13.8 {+-} 0.2). The presence of water in the octanol diluent has an influence on solvation of europium and also on the [Eu(BTP){sub 2}{sup 3+}] / [Eu(BTP){sub 3}{sup 3+}] ratio. (authors)

Photochemistry of modified proteins benzophenone-containing bovine serum albumin

International Nuclear Information System (INIS)

Mariano, P.S.; Glover, G.I.; Wilkinson, T.J.

1976-01-01

The results of exploratory and mechanistic studies of the photochemistry of poly-p-benzoyl-acetimido-bovine serum albumin, a modified protein containing photoreactive and photosensitizing groups, are reported. Specifically described are recent findings concerning (1) the synthesis and characterization of a modified bovine serum albumin that contains benzophenone-like moieties, (2) the photochemistry of this modified protein which appeared to involve photoreductive coupling of the benzophenone chromophores to the protein backbone, and (3) triplet energy transfer from modified bovine serum albumin to small molecule acceptors resulting in quenching of the photoreaction. (author)

High-resolution gulf water skin temperature estimation using TIR/ASTER

Digital Repository Service at National Institute of Oceanography (India)

Kunte, P.D.; ManiMurali, R.; Mahender, K.

to separate geomorphic features. It is demonstrated that high resolution water skin temperature of small water bodies can be determined correctly, economically and less laboriously using space-based TIR/ASTER and that estimated temperature can be effectively...

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Science.gov (United States)

Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

2016-06-10

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

Synthesis and characterisation of nitrogen-containing poly heterocyclic molecules for the complexation of polluting metal cations

International Nuclear Information System (INIS)

Leconte, N.

2007-12-01

The recovery of actinides(III) from the mixture actinides(III)/lanthanides(III) is a great deal of the nuclear waste management. Experiences have shown that derivatives of 2,6- bis(1,2,4-triazine-3-yl)pyridine (BTP) are able to extract selectively actinides(III) from lanthanides(III). But the properties of these molecules need to be reinforced to exhibit best extraction performances and to resist to the harsh conditions of the extracting processes. Originally functionalized ligands were synthesized and tested in a radioactive medium. In a second part, this work focuses on the control and the detection of actinides(III). This could be done thanks to the use of a chemical sensor such as quartz crystal microbalance. The study of this apparatus required the preliminary synthesis of original complexing BTP-derived structures. The last part of this work deals with the study of new synthetic ways to afford the BTP moiety. The palladium-catalyzed cross-coupling reaction of 3-methylthio-1,2,4-triazine with various 2-stannylated pyridines have been explored. Our investigations have also allowed to develop a method for synthesizing unsymmetrical 1,2,4,5-tetrazine from the cross-coupling reaction between 3-methylthio-6-(morpholine-N-yl)-1,2,4,5-tetrazine and organoboron / organo-stannane derivatives. (author)

Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

Directory of Open Access Journals (Sweden)

Begoña Barriuso

2016-06-01

Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

Antitumor Active Protein-containing Glycans from the Body of Ganoderma tsugae

Institute of Scientific and Technical Information of China (English)

LIU Ying; LI Yue-fei; ZHENG Ke-yan; FEI Xiao-fang

2012-01-01

To explore the effects of traditional herbal medicine Ganoderma tsugae(G.tsugae) on immunomodulatory and antitumor activities,the crude polysaccharides ofG.tsugae were purified by filtration,diethylaminoethyl(DEAE)sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography.Two main fractions,protein-containing glycans CSSLP-I and CSSLP-2,were obtained via the gradient elution.The protein content,molecular weight,and monosaccharide composition of the two fractions were analyzed.Furthermore,the influence of the protein-containing glycans from G.tsugae on the activation of human acute monocytic leukemia cell line(THP-1 ) and their antitumor activities to the human hepatocellular liver carcinoma cell(HepG-2) in vitro were evaluated.The results indicate that CSSLP-I and CSSLP-2 could increase the pinocytic activity of THP-1 cells and induce THP-1 cells to produce the cytokines of TNFa and IL-2,significantly.CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell(NepG-2).Moreover,the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the participation of TNFa and 1L-2 or other antitumor factors induced from THP-1 cclls by G.tsugae protein-containing glycan fractions.

Robust Satellite Techniques analysis of ten years (2004-2013) of MSG/SEVIRI TIR radiances over Greece region

Science.gov (United States)

Genzano, N.; Eleftheriou, A.; Filizzola, C.; Paciello, R.; Pergola, N.; Vallianatos, F.; Tramutoli, V.

2014-12-01

Space-time fluctuations of Earth's emitted Thermal InfraRed (TIR) radiation have been observed from satellite months to weeks before earthquakes occurrence. Among the different approach proposed to discern transient anomalous signals possibly associated to seismic activity from normal TIR signal fluctuations (i.e. related to the change of natural factor and/or observation conditions), since 2001 the Robust Satellite Techniques (RST) were used to investigate tens of earthquakes with a wide range of magnitudes (from 4.0 to 7.9) occurred in different continents and in various geo-tectonic setting (e.g. Athens earthquake, 7 September 1999; Abruzzo earthquake, 6 April 2009, etc.).The RST approach gives a statistically - based definition of "TIR anomalies" and offers a suitable method for their identification even in very different local (e.g. related to atmosphere and/or surface) and observational (e.g. related to time/season, but also to solar and satellite zenithal angles) conditions. It has been always carried out by using a validation/confutation approach, to verify the presence/absence of anomalous space-time TIR transients in the presence/absence of seismic activity.In this paper, the RST approach is extensively implemented on 10 years of TIR satellite records collected by the geostationary satellite sensor MSG/SEVIRI over the Greece region. The results of the analysis performed to investigate possible correlations (within predefined space-time windows) of anomalous TIR transients with time and place of occurrence of earthquakes with M>4 will be discussed in terms of reliability and effectiveness also in the perspective of a time-Dependent Assessment of Seismic Hazard (t-DASH) system.

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

Science.gov (United States)

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Deprotonated imidodiphosphate in AMPPNP-containing protein structures

International Nuclear Information System (INIS)

Dauter, Miroslawa; Dauter, Zbigniew

2011-01-01

In certain AMPPNP-containing protein structures, the nitrogen bridging the two terminal phosphate groups can be deprotonated. Many different proteins utilize the chemical energy provided by the cofactor adenosine triphosphate (ATP) for their proper function. A number of structures in the Protein Data Bank (PDB) contain adenosine 5′-(β,γ-imido)triphosphate (AMPPNP), a nonhydrolysable analog of ATP in which the bridging O atom between the two terminal phosphate groups is substituted by the imido function. Under mild conditions imides do not have acidic properties and thus the imide nitrogen should be protonated. However, an analysis of protein structures containing AMPPNP reveals that the imide group is deprotonated in certain complexes if the negative charges of the phosphate moieties in AMPPNP are in part neutralized by coordinating divalent metals or a guanidinium group of an arginine

Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum

DEFF Research Database (Denmark)

Jakobsen, P H; Hviid, L; Theander, T G

1993-01-01

The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living i...... by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides.......The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living...

An initial assessment of a SMAP soil moisture disaggregation scheme using TIR surface evaporation data over the continental United States

Science.gov (United States)

Mishra, Vikalp; Ellenburg, W. Lee; Griffin, Robert E.; Mecikalski, John R.; Cruise, James F.; Hain, Christopher R.; Anderson, Martha C.

2018-06-01

The Soil Moisture Active Passive (SMAP) mission is dedicated toward global soil moisture mapping. Typically, an L-band microwave radiometer has spatial resolution on the order of 36-40 km, which is too coarse for many specific hydro-meteorological and agricultural applications. With the failure of the SMAP active radar within three months of becoming operational, an intermediate (9-km) and finer (3-km) scale soil moisture product solely from the SMAP mission is no longer possible. Therefore, the focus of this study is a disaggregation of the 36-km resolution SMAP passive-only surface soil moisture (SSM) using the Soil Evaporative Efficiency (SEE) approach to spatial scales of 3-km and 9-km. The SEE was computed using thermal-infrared (TIR) estimation of surface evaporation over Continental U.S. (CONUS). The disaggregation results were compared with the 3 months of SMAP-Active (SMAP-A) and Active/Passive (AP) products, while comparisons with SMAP-Enhanced (SMAP-E), SMAP-Passive (SMAP-P), as well as with more than 180 Soil Climate Analysis Network (SCAN) stations across CONUS were performed for a 19 month period. At the 9-km spatial scale, the TIR-Downscaled data correlated strongly with the SMAP-E SSM both spatially (r = 0.90) and temporally (r = 0.87). In comparison with SCAN observations, overall correlations of 0.49 and 0.47; bias of -0.022 and -0.019 and unbiased RMSD of 0.105 and 0.100 were found for SMAP-E and TIR-Downscaled SSM across the Continental U.S., respectively. At 3-km scale, TIR-Downscaled and SMAP-A had a mean temporal correlation of only 0.27. In terms of gain statistics, the highest percentage of SCAN sites with positive gains (>55%) was observed with the TIR-Downscaled SSM at 9-km. Overall, the TIR-based downscaled SSM showed strong correspondence with SMAP-E; compared to SCAN, and overall both SMAP-E and TIR-Downscaled performed similarly, however, gain statistics show that TIR-Downscaled SSM slightly outperformed SMAP-E.

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

International Nuclear Information System (INIS)

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

2011-01-01

Highlights: ► APPL1 regulates the protein level of EGFR in response to EGF stimulation. ► Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. ► Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

Protein: MPA1 [TP Atlas

Lifescience Database Archive (English)

Full Text Available MPA1 TLR signaling molecules Rsad2 Vig1 Radical S-adenosyl methionine domain-containing pr...otein 2 Viperin, Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible 10090 Mus musculus 58185 Q8CBB9 21435586 ...

G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

DEFF Research Database (Denmark)

Dou, Q P; Zhao, S; Levin, A H

1994-01-01

report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes...... complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex...

Misexpression of AtTX12 encoding a Toll/interleukin-1 receptor domain induces growth defects and expression of defense-related genes partially independently of EDS1 in Arabidopsis.

Science.gov (United States)

Song, Sang-Kee

2016-12-01

In this study, a tissue-specific GAL4/UAS activation tagging system was used for the characterization of genes which could induce lethality when ubiquitously expressed. A dominant mutant exhibiting stunted growth was isolated and named defective root development 1-D (drd1-D). The T-DNA tag was located within the promoter region of AtTX12, which is predicted to encode a truncated nucleotide-binding leucinerich repeat (NLR) protein, containing a Toll/interleukin-1 receptor (TIR) domain. The transcript levels of AtTX12 and defense-related genes were elevated in drd1-D, and the misexpression of AtTX12 recapitulated the drd1-D phenotypes. In the presence of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), a key transducer of signals triggered by TIR-type NLRs, a low-level of AtTX12 misexpression induced strong defective phenotypes including seedling lethality whereas, in the absence of EDS1, a high-level of AtTX12 misexpression induced weak growth defects like dwarfism, suggesting that AtTX12 might function mainly in an EDS1-dependent and partially in an EDS1-independent manner. [BMB Reports 2016; 49(12): 693-698].

Impact of line parameter database, continuum absorption, full grind configuration, and L1B update on GOSAT TIR methane retrieval

Science.gov (United States)

Yamada, A.; Saitoh, N.; Nonogaki, R.; Imasu, R.; Shiomi, K.; Kuze, A.

2016-12-01

The thermal infrared (TIR) band of Thermal and Near-infrared Sensor for Carbon Observation Fourier Transform Spectrometer (TANSO-FTS) onboard Greenhouse Gases Observing Satellite (GOSAT) observes CH4 profile at wavenumber range from 1210 cm-1 to 1360 cm-1 including CH4 ν4 band. The current retrieval algorithm (V1.0) uses LBLRTM V12.1 with AER V3.1 line database to calculate optical depth. LBLRTM V12.1 include MT_CKD 2.5.2 model to calculate continuum absorption. The continuum absorption has large uncertainty, especially temperature dependent coefficient, between BPS model and MT_CKD model in the wavenumber region of 1210-1250 cm-1(Paynter and Ramaswamy, 2014). The purpose of this study is to assess the impact on CH4 retrieval from the line parameter databases and the uncertainty of continuum absorption. We used AER v1.0 database, HITRAN2004 database, HITRAN2008 database, AER V3.2 database, and HITRAN2012 database (Rothman et al. 2005, 2009, and 2013. Clough et al., 2005). AER V1.0 database is based on HITRAN2000. The CH4 line parameters of AER V3.1 and V3.2 databases are developed from HITRAN2008 including updates until May 2009 with line mixing parameters. We compared the retrieved CH4 with the HIPPO CH4 observation (Wofsy et al., 2012). The difference of AER V3.2 was the smallest and 24.1 ± 45.9 ppbv. The differences of AER V1.0, HITRAN2004, HITRAN2008, and HITRAN2012 were 35.6 ± 46.5 ppbv, 37.6 ± 46.3 ppbv, 32.1 ± 46.1 ppbv, and 35.2 ± 46.0 ppbv, respectively. Compare AER V3.2 case to HITRAN2008 case, the line coupling effect reduced difference by 8.0 ppbv. Median values of Residual difference from HITRAN2008 to AER V1.0, HITRAN2004, AER V3.2, and HITRAN2012 were 0.6 K, 0.1 K, -0.08 K, and 0.08 K, respectively, while median values of transmittance difference were less than 0.0003 and transmittance differences have small wavenumber dependence. We also discuss the retrieval error from the uncertainty of the continuum absorption, the test of full grid

Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy.

Science.gov (United States)

Bohannon, Kevin P; Holz, Ronald W; Axelrod, Daniel

2017-10-01

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors1[W][OA

Science.gov (United States)

Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-01-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors. PMID:22535423

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

Science.gov (United States)

Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

2017-01-01

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

Erwinia amylovora Enfeksiyonu Sonrası Elma, Armut ve Ayva Çeşitlerinde Konukçu Protein Miktarlarının Belirlenmesi

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Şerife Çetin

2014-11-01

Full Text Available Ateş yanıklığı hastalığına neden olan Erwinia amylovora, Rosaceae familyasından başta armut, elma ve ayvalarda zararlı bakteriyel bir patojendir. Bu çalışmada, E. amylovora’ nın 2 virülent izolatının (Ea234-1 ve Ea240-3 farklı elma (Braeburn, Fuji, Gala ve Golden Delicious, armut (Santa Maria ve Williams ve ayva (Eşme ve Ekmek çeşitlerinde enfeksiyonu sonucu zamana bağlı olarak toplam protein miktarlarının belirlenmesi amaçlanmıştır. E. amylovora (108 hücre ml-1 ile yaprak inokulasyonundan sonraki 24, 36 ve 72. saatlerde örnekler alınmıştır. Enfeksiyonların doğrulanması için, bakteri inokule edilen bitkilerden re-izolasyonlar yapılmış ve etmen biyokimyasal ve moleküler testlerle E. amylovora olarak tanılanmıştır. Toplam protein miktarlarının belirlenmesinde Bradford yöntemi ve SDS-PAGE analizleri için Laemmli yöntemleri kullanılmış, protein ekstraktlarının 595 nm’ de absorbans değerleri elde edilmiştir. Elde edilen bulgulara göre, elma çeşitlerinde E. amylovora enfeksiyonu sonrası kontrole kıyasla 24. saatte toplam protein miktarının arttığı, 36. ve 72. saatte miktarın azaldığı, Braeburn çeşidinin en yüksek protein miktarına sahip olduğu belirlenmiştir. Armut çeşitlerinde, 24. ve 36. saatlerde toplam protein miktarı artarken, 72. saatte protein miktarının azaldığı ve Santa Maria çeşidinin en yüksek protein miktarına sahip olduğu tespit edilmiştir. Ayva çeşitlerinde, toplam protein miktarının 72. saatte arttığı ve Eşme çeşidinin en yüksek protein miktarına sahip olduğu belirlenmiştir. SDS-PAGE analizi sonucunda, farklı molekül ağırlıklarına sahip protein fraksiyonları elde edilmiştir. Elma ve ayva çeşitlerinde sırasıyla yaklaşık 55-70 ve 35-55 kDa, armut çeşitlerinde ise yaklaşık 55-70 kDa molekül ağırlığında protein bantları belirlenmiştir.

The group A streptococcal collagen-like protein 1, Scl1, mediates biofilm formation by targeting the EDA-containing variant of cellular fibronectin expressed in wounded tissue

Science.gov (United States)

Oliver-Kozup, Heaven; Martin, Karen H.; Schwegler-Berry, Diane; Green, Brett J.; Betts, Courtney; Shinde, Arti V.; Van De Water, Livingston; Lukomski, Slawomir

2012-01-01

Summary Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C′ loop region recognized by the α9β1 integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization. PMID:23217101

Use of protein containing magnetic microparticles in radioassays

International Nuclear Information System (INIS)

Ithakissios, D.S.; Kubiatowicz, D.O.

1977-01-01

We describe a radioassay method that involves the use of magnetic protein microparticles composed of a water-insoluble protein matrix containing magnetically responsive material. We define two different types of particles according to the mechanism of action: The substrate is sorbed nonspecifically by the protein matrix of the particle or by a second substance such as charcoal or ion-exchange resin incorporated within the protein matrix of the particle. These particles are useful for separating free from bound substrate. Examples of these are albumin magnetic microparticles for use in a total thyroxine radioassay and triiodothyronine uptake test, or albumin magnetic microparticles containing charcoal for use in a vitamin B 12 radioassay. The substrate is sorbed specifically by a binding protein incorporated within the matrix of the particles. The binding protein can include antibodies or other specific nonimmune proteins. Particles of this type are useful in solid-phase radioassays. These particles are exemplified by albumin magnetic microparticles containing sockeye salmon serum, used in a solid-phase B 12 radioassay. We discuss the methods for the preparation of both types of magnetic microparticles and their use in radioassays. We describe a unique inexpensive magnetic separation rack, which provides simple, fast, and reproducible separation of the magnetic microparticles from their suspending medium during the assay

Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma.

Science.gov (United States)

Marsh, Erica E; Chibber, Shani; Wu, Ju; Siegersma, Kendra; Kim, Julie; Bulun, Serdar

2016-04-01

To determine the presence, differential expression, and regulation of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Laboratory in vivo and in vitro study with the use of human leiomyoma and myometrial tissue and primary cells. Academic medical center. Leiomyoma and myometrial tissue samples and cultured cells. 5-Aza-2'-deoxycytidine (5-aza-dC) treatment. Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. In vivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression in vitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 in vitro. The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both in vivo and in vitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Modular organization of proteins containing C1q-like globular domain.

Science.gov (United States)

Kishore, U; Reid, K B

1999-05-01

The first step in the activation of the classical pathway of complement cascade by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of immunoglobulin G (IgG) or immunoglobulin M (IgM). The globular heads of C1q are located C-terminal to the six triple-helical stalks present in the molecule, each head is considered to be composed of the C-terminal halves (3 x 135 residues) of one A-, one B- and one C-chain. It is not known if the C-terminal globular regions, present in each of the three types of chains, are independently folded modules (with each chain having distinct binding properties towards immunoglobulins) or whether the different binding functions of C1q are dependent upon a globular structure which relies on contributions from all three chains. Recent reports of recombinant production and characterisation of soluble globular head regions of all the three chains indicate that the globular regions of C1q may adopt a modular organization, i.e., each globular head of C1q may be composed of three, structurally and functionally, independent domains, thus retaining multivalency in the form of a heterotrimer. Modules of the same type as the C1q C-terminal module are also found in a variety of noncomplement proteins that include the C-terminal regions of the human type VIII and type X collagens, precerebellin, the chipmunk hibernation proteins, the human endothelial cell protein, multimerin, the serum protein, Acrp-30 which is secreted from mouse adipocytes, and the sunfish inner-ear specific structural protein. The C1q molecule is the only one of these proteins for which, to date, a function has been ascribed to the module. The existence of a shared structural region between C1q and certain collagens may suggest an evolutionarily common ancestral precursor. Various structural and biochemical data suggest that these modules may be responsible for multimerisation through patches of aromatic residues within them.

The expression of the new epididymal luminal protein of PDZ domain containing 1 is decreased in asthenozoospermia

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A-Juan Liang

2018-01-01

Full Text Available Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ. Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 (Pdzk1, also named Na+/H+ exchange regulatory cofactor 3 (NHERF3, which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzk1 was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzk1 protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzk1 may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Energy Technology Data Exchange (ETDEWEB)

Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

International Nuclear Information System (INIS)

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

2010-01-01

Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

TAIL1: an isthmin-like gene, containing type 1 thrombospondin-repeat and AMOP domain, mapped to ARVD1 critical region.

Science.gov (United States)

Rossi, Valeria; Beffagna, Giorgia; Rampazzo, Alessandra; Bauce, Barbara; Danieli, Gian Antonio

2004-06-23

Isthmins represent a novel family of vertebrate secreted proteins containing one copy of the thrombospondin type 1 repeat (TSR), which in mammals is shared by several proteins with diverse biological functions, including cell adhesion, angiogenesis, and patterning of developing nervous system. We have determined the genomic organization of human TAIL1 (thrombospondin and AMOP containing isthmin-like 1), a novel isthmin-like gene encoding a protein that contains a TSR and a C-terminal AMOP domain (adhesion-associated domain in MUC4 and other proteins), characteristic of extracellular proteins involved in adhesion processes. TAIL1 gene encompasses more than 24.4 kb. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. Several consensus binding sites for the transcription factors Sp1 and MZF-1 were identified in this promoter region. In humans, TAIL1 gene is located on chromosome 14q24.3 within ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region; preliminary evidence suggests that it is expressed in several tissues, showing multiple alternative splicing.

Bactericidal/Permeability-increasing protein fold-containing family member A1 in airway host protection and respiratory disease.

Science.gov (United States)

Britto, Clemente J; Cohn, Lauren

2015-05-01

Bactericidal/permeability-increasing protein fold-containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease.

Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

Science.gov (United States)

Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

1997-07-01

Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

Simulació d'un tir parabòlic amb un applet de física

OpenAIRE

Fàbregas Cuadrada, Josep Manel

2016-01-01

Aquest projecte té un vessant clarament pedagògica, en un intent d'apropar l'ensenyament a l'aula a partir de la informàtica en les seves infinites aplicacions. En particular l'assignatura que es pretén portar a l'aula des de la informàtica és la Física. I de forma més concreta un tema anomenat cinemàtica, i amb més precisió el 'tir parabòlic'. L'objectiu principal ha estat crear un applet de simulació d'un tir parabòlic, per tal de poder ser observat i descrit a classe. I per facilitar als a...

Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development.

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Lian Jin

2016-09-01

Full Text Available The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB protein and an auxin/indole-3-acetic acid (Aux/IAA protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV, a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis.

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava.

Science.gov (United States)

López, C E; Zuluaga, A P; Cooke, R; Delseny, M; Tohme, J; Verdier, V

2003-08-01

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava (Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.

Defective Proteasome Delivery of Polyubiquitinated Proteins by Ubiquilin-2 Proteins Containing ALS Mutations.

Directory of Open Access Journals (Sweden)

Lydia Chang

Full Text Available Ubiquilin proteins facilitate delivery of ubiquitinated proteins to the proteasome for degradation. Interest in the proteins has been heightened by the discovery that gene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS. However, the mechanisms by which the mutations cause ALS are not known. Here we report on the underlying defect of ubiquilin-2 proteins containing ALS-linked mutations in affecting proteasome-mediated degradation. We found that overexpression of ubiquilin-2 proteins containing any one of five different ALS mutations slow degradation of Myc, a prototypic proteasome substrate. Examination of coprecipitating proteins indicated that the mutant proteins are generally capable of binding polyubiquitinated proteins, but defective in binding the proteasome. GST-pulldown studies revealed that many of the mutants bind weaker to the S5a subunit of the proteasome, compared with wild type (WT ubiquilin-2 protein. The results suggest the mutant proteins are unable to deliver their captured cargo to the proteasome for degradation, which presumably leads to toxicity. Quantification of cell death is consistent with this idea. Measurement of protein turnover further indicated the mutant proteins have longer half-lives than WT ubiquilin-2. Our studies provide novel insight into the mechanism by which ALS-linked mutations in UBQLN2 interfere with protein degradation.

PDZ-containing proteins: alternative splicing as a source of functional diversity.

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Sierralta, Jimena; Mendoza, Carolina

2004-12-01

Scaffold proteins allow specific protein complexes to be assembled in particular regions of the cell at which they organize subcellular structures and signal transduction complexes. This characteristic is especially important for neurons, which are highly polarized cells. Among the domains contained by scaffold proteins, the PSD-95, Discs-large, ZO-1 (PDZ) domains are of particular relevance in signal transduction processes and maintenance of neuronal and epithelial polarity. These domains are specialized in the binding of the carboxyl termini of proteins allowing membrane proteins to be localized by the anchoring to the cytoskeleton mediated by PDZ-containing scaffold proteins. In vivo studies carried out in Drosophila have taught that the role of many scaffold proteins is not limited to a single process; thus, in many cases the same genes are expressed in different tissues and participate in apparently very diverse processes. In addition to the differential expression of interactors of scaffold proteins, the expression of variants of these molecular scaffolds as the result of the alternative processing of the genes that encode them is proving to be a very important source of variability and complexity on a main theme. Alternative splicing in the nervous system is well documented, where specific isoforms play roles in neurotransmission, ion channel function, neuronal cell recognition, and are developmentally regulated making it a major mechanism of functional diversity. Here we review the current state of knowledge about the diversity and the known function of PDZ-containing proteins in Drosophila with emphasis in the role played by alternatively processed forms in the diversity of functions attributed to this family of proteins.

Modulation of IL-33/ST2-TIR and TLR signalling pathway by fingolimod and analogues in immune cells.

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Rüger, K; Ottenlinger, F; Schröder, M; Zivković, A; Stark, H; Pfeilschifter, J M; Radeke, H H

2014-12-01

For the immune modulatory drug fingolimod (FTY720), lymphocyte sequestration has been extensively studied and accepted as mode of action. Further, direct effects on immune cell signalling are incompletely understood. Herein, we used the parent drug and newly synthesized analogues to investigate their effects on dendritic cell (DC) calcium signalling and on Th1, Th2 and Th17 responses. DC calcium signalling was determined with a single cell-based confocal assay and IL-33/ST2-TIR Th2-like response with ST2-transduced EL4-6.1 thymoma cells. The Th1/Th17 responses were examined with a LPS/TLR-enhanced antigen presentation assay with OVA-TCRtg CD4 and CD8 spleen cells. Our results revealed a comparable influence of fingolimod and S1P on intracellular calcium level in DC, while an oxy-derivative of fingolimod exhibited an EC50 of 3.3 nm, being 14 times more potent than FTY720-P. The IL-33/ST2-TIR Th2-like response in ST2-EL4 cells was inhibited by fingolimod and analogues at varying degrees. Using the OVA-TCRtg LPS/TLR-enhanced spleen cell assay, we found that fingolimod inhibited both IL-17 and IFN-γ production. In contrast, fingolimod phosphate failed to decrease Th1 cytokines. Interestingly, the effects of the parent compound fingolimod were modulated by the PP2A inhibitor okadaic acid, thus suggesting PP2A as relevant intracellular target. These studies describe detailed immune-modulating properties of fingolimod, including interference with a prototypical Th2 response via IL-33/ST2-TIR. Moreover, differential effects of fingolimod versus its phosphorylated derivative on TLR-activated and antigen-dependent Th1 activation suggest PP2A as an additional target of fingolimod immune therapy. Together with the analogues tested, these data may guide the development of more specific fingolimod derivatives. © 2014 John Wiley & Sons Ltd.

A highly sensitive method for detection of molybdenum-containing proteins

International Nuclear Information System (INIS)

Kalakutskii, K.L.; Shvetsov, A.A.; Bursakov, S.A.; Letarov, A.V.; Zabolotnyi, A.I.; L'vov, N.P.

1992-01-01

A highly sensitive method for detection of molybdenum-containing proteins in gels after electrophoresis has been developed. The method involves in vitro labeling of the proteins with the radioactive isotope 185 W. The method used to detect molybdenum-accumulating proteins in lupine seeds, xanthine dehydrogenase and another molybdenum-containing protein in wheat, barley, and pea seedlings, and nitrate reductase and xanthine dehydrogenase in bacteroides from lupine nodules. Nitrogenase could not be detected by the method. 16 refs., 5 figs

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

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Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Spectroscopic and thermodynamic study of the complexation of Cm(III) and Eu(III) with hydrophilic bis-triazinyl pyridines

International Nuclear Information System (INIS)

Ruff, Christian

2013-01-01

In the present work the complexation of Cm(III) and Eu(III) with a hydrophilic 2,6-bis-(1,2,4-triazinyl)-pyridine (aq-BTP) is studied. Aq-BTP complexes actinides(III) selectively over lanthanides(III) in nitric acid solution. The object of this work is the identification and the spectroscopic and thermodynamic characterization of the Cm(III) and Eu(III) complex species present in solution. The results should contribute to a better fundamental understanding of the driving force behind BTPs selectivity towards trivalent actinides on a molecular level. Time-resolved laser fluorescence spectroscopy (TRLFS), luminescence and UV/Vis spectroscopy are applied. Information on the structure of M(III)-aq-BTP complex species is obtained from density functional theory. Three different M(III) complex species containing one, two or three aq-BTP ligands are identified in H 2 O at pH 3.0. Relative fluorescence intensity factors are determined for each of the [M(aq-BTP) n ] complexes (M = Cm(III)/Eu(III), n = 1 - 3). These factors are required to quantify the complexes. The stability constant logβ 3 of the [Cm(aq-BTP) 3 ] complex (which is the one relevant to extraction processes) is two orders of magnitude higher than that of the corresponding Eu(III) complex. This difference is in agreement with the separation factor (SF Am(III)/Eu(III) = 150) determined experimentally by liquid-liquid extraction. The difference in the stability constants originates from the different reaction enthalpies for the formation of the [M(aq-BTP) 3 ] complexes. These results represent the thermodynamic driving force for the aq-BTPs selectivity towards trivalent actinides over lanthanides. Comparing the stability constants of the [M(aq-BTP) n ] species (M = Cm(III)/Eu(III), n = 1 - 3) shows an increasing selectivity with increasing number of coordinated aq-BTP ligands. Hence, high selectivity is achieved if the f-element ions are fully coordinated by nine N-donor atoms (three aq-BTP ligands). A less

Nitrogen Metabolism in Lactating Goats Fed with Diets Containing Different Protein Sources

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A. B. Santos

2014-05-01

Full Text Available This study aimed to evaluate urea excretion, nitrogen balance and microbial protein synthesis in lactating goats fed with diets containing different protein sources in the concentrate (soybean meal, cottonseed meal, aerial part of cassava hay and leucaena hay. Four Alpine goats whose mean body weight was 42.6±6.1 kg at the beginning of the experiment, a mean lactation period of 94.0±9.0 days and a production of 1.7±0.4 kg of milk were distributed in a 4×4 Latin square with four periods of 15 days. Diets were formulated to be isonitrogenous, containing 103.0 g/kg of CP, 400 g/kg of Tifton 85 hay and 600 g/kg of concentrate. Diet containing cottonseed meal provided (p<0.05 increased excretion of urea and urea nitrogen in the urine (g/d and mg/kg of BW when compared with leucaena hay. The diets affected the concentrations of urea nitrogen in plasma (p<0.05 and excretion of urea nitrogen in milk, being that soybean meal and cottonseed meal showed (p<0.05 higher than the average aerial part of the cassava hay. The use of diets with cottonseed meal as protein source in the concentrate in feeding of lactating goats provides greater nitrogen excretion in urine and negative nitrogen balance, while the concentrate with leucaena hay as a source of protein, provides greater ruminal microbial protein synthesis.

Synthesis and characterisation of nitrogen-containing poly heterocyclic molecules for the complexation of polluting metal cations; Synthese et caracterisation de molecules polyheterocycliques azotees pour la complexation de cations metalliques polluants

Energy Technology Data Exchange (ETDEWEB)

Leconte, N

2007-12-15

The recovery of actinides(III) from the mixture actinides(III)/lanthanides(III) is a great deal of the nuclear waste management. Experiences have shown that derivatives of 2,6- bis(1,2,4-triazine-3-yl)pyridine (BTP) are able to extract selectively actinides(III) from lanthanides(III). But the properties of these molecules need to be reinforced to exhibit best extraction performances and to resist to the harsh conditions of the extracting processes. Originally functionalized ligands were synthesized and tested in a radioactive medium. In a second part, this work focuses on the control and the detection of actinides(III). This could be done thanks to the use of a chemical sensor such as quartz crystal microbalance. The study of this apparatus required the preliminary synthesis of original complexing BTP-derived structures. The last part of this work deals with the study of new synthetic ways to afford the BTP moiety. The palladium-catalyzed cross-coupling reaction of 3-methylthio-1,2,4-triazine with various 2-stannylated pyridines have been explored. Our investigations have also allowed to develop a method for synthesizing unsymmetrical 1,2,4,5-tetrazine from the cross-coupling reaction between 3-methylthio-6-(morpholine-N-yl)-1,2,4,5-tetrazine and organoboron / organo-stannane derivatives. (author)

The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module

DEFF Research Database (Denmark)

Rosenthal, J A; Chen, H; Slepnev, V I

1999-01-01

Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. E...... fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.......Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries...

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

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Kasimsetty, Sashi G; Shigeoka, Alana A; Scheinok, Andrew A; Gavin, Amanda L; Ulevitch, Richard J; McKay, Dianne B

2017-08-01

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2 -/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2 -/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2 -/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation. Copyright © 2017 by The American Association of Immunologists, Inc.

CUB-domain-containing protein 1 overexpression in solid cancers promotes cancer cell growth by activating Src family kinases.

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Leroy, C; Shen, Q; Strande, V; Meyer, R; McLaughlin, M E; Lezan, E; Bentires-Alj, M; Voshol, H; Bonenfant, D; Alex Gaither, L

2015-10-29

The transmembrane glycoprotein, CUB (complement C1r/C1s, Uegf, Bmp1) domain-containing protein 1 (CDCP1) is overexpressed in several cancer types and is a predictor of poor prognosis for patients on standard of care therapies. Phosphorylation of CDCP1 tyrosine sites is induced upon loss of cell adhesion and is thought to be linked to metastatic potential of tumor cells. Using a tyrosine-phosphoproteomics screening approach, we characterized the phosphorylation state of CDCP1 across a panel of breast cancer cell lines. We focused on two phospho-tyrosine pTyr peptides of CDCP1, containing Tyr707 and Tyr806, which were identified in all six lines, with the human epidermal growth factor 2-positive HCC1954 cells showing a particularly high phosphorylation level. Pharmacological modulation of tyrosine phosphorylation indicated that, the Src family kinases (SFKs) were found to phosphorylate CDCP1 at Tyr707 and Tyr806 and play a critical role in CDCP1 activity. We demonstrated that CDCP1 overexpression in HEK293 cells increases global phosphotyrosine content, promotes anchorage-independent cell growth and activates several SFK members. Conversely, CDCP1 downregulation in multiple solid cancer cell lines decreased both cell growth and SFK activation. Analysis of primary human tumor samples demonstrated a correlation between CDCP1 expression, SFK and protein kinase C (PKC) activity. Taken together, our results suggest that CDCP1 overexpression could be an interesting therapeutic target in multiple solid cancers and a good biomarker to stratify patients who could benefit from an anti-SFK-targeted therapy. Our data also show that multiple tyrosine phosphorylation sites of CDCP1 are important for the functional regulation of SFKs in several tumor types.

Freeform TIR collimators for the removal of angular color variation in white LED spotlights

NARCIS (Netherlands)

Prins, C.R.; Schneider, C.; IJzerman, W.L.; Tukker, T.W.; Thije Boonkkamp, ten J.H.M.; Winston, R.; Gordon, J.

2013-01-01

Angular color variation in white, phosphor-converted LEDs causes undesirable yellow rings in the beams of spotlights. We developed an inverse method to design TIR collimators that remove the angular color variation for point light sources and significantly reduce color variation for extended light

Water binding of proteins in the processing frankfurter-type sausages. Part. 1. Water-binding ability of freeze-dried meat fractions containing myofibrillar and stromal proteins.

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Heinevetter, L; Gassmann, B; Kroll, J

1987-01-01

As soon as possible and 48 h after slaughter respectively, from both blade-bone muscle groups of cattle and pig carcasses the "thick pieces" were excised, extracted, and fractionated. Residues and precipitates from water and salt extracts resulted were freeze-dried, and an improved Baumann capillary suction apparatus was used to measure their water binding capacity (WBC) with and without addition of 2% sodium chloride and/or heating to 80 degrees C. With one exception the WBC results followed a relative pattern demonstrating the final residues (stromal proteins and leavings of myofibrillar proteins) binding the highest amount of added water, precipitates of dialysis (mainly containing myofibrillar proteins) a remarkable amount and powdered meats the least. As scanning electron micrographs confirmed, there were no fibrous structures in the precipitates resulted from dialysis of salt solutions (1.0 mol/1). Heating decreased the spontaneous water uptake of all fractions. Addition of sodium chloride had only a noticeable capillary-suction and swelling effect on unheated samples. Hence swelling of undissolved protein structures (extraction of myosin and possibly of actomyosin) is therefore not the only way for water binding in frankfurter-type sausages.

Plasma levels of selenium-containing proteins in Inuit adults from Nunavik.

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Achouba, Adel; Dumas, Pierre; Ouellet, Nathalie; Lemire, Mélanie; Ayotte, Pierre

2016-11-01

Selenium (Se) is highly abundant in marine foods traditionally consumed by Inuit of Nunavik (Northern Quebec, Canada) and accordingly, their Se intake is among the highest in the world. However, little is known regarding the biological implications of this high Se status in this Arctic indigenous population. We used a method combining affinity chromatography and inductively coupled plasma-mass spectrometry with quantification by post-column isotope dilution to determine total Se levels and concentrations of Se-containing proteins in archived plasma samples of Inuit adults who participated to the 2004 Nunavik Inuit Health Survey (N = 852). Amounts of mercury (Hg) associated with Se-containing proteins were also quantified. Results show that glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and selenoalbumin (SeAlb) represented respectively 25%, 52% and 23% of total plasma Se concentrations. In addition, small amounts of Hg co-eluted with each Se-containing protein and up to 50% of plasma Hg was associated to SelP. Total plasma Se concentrations (median = 139 μg L− 1; interquartile range (IQR) = 22.7 μg L− 1) were markedly lower and less variable than whole blood Se concentration (median = 261 μg L− 1, IQR = 166 μg L− 1). A non linear relation was observed between whole blood Se and plasma Se levels, with plasma Se concentrations leveling off at approximately 200 μg L− 1, whereas 16% and 3% of individuals exhibited whole blood concentrations higher than 500 μg L− 1 and 1000 μg L− 1, respectively. In contrast, a linear relationship was previously reported in communities consuming Brazil nuts which are rich Se, mainly present as selenomethionine. This suggests that a different selenocompound, possibly selenoneine, is present in the Arctic marine food chain and accumulates in the blood cellular fraction of Inuit.

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

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Bjoern von Einem

Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

The proteome of neurofilament-containing protein aggregates in blood

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Rocco Adiutori

2018-07-01

Full Text Available Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf, the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP, for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS. Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins.

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Schiera, Gabriella; Di Liegro, Carlo Maria; Puleo, Veronica; Colletta, Oriana; Fricano, Anna; Cancemi, Patrizia; Di Cara, Gianluca; Di Liegro, Italia

2016-11-01

Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2.

Experiences from occupational exposure limits set on aerosols containing allergenic proteins

DEFF Research Database (Denmark)

Nielsen, Gunnar; Larsen, Søren; Hansen, Jitka S

2012-01-01

Occupational exposure limits (OELs) together with determined airborne exposures are used in risk assessment based managements of occupational exposures to prevent occupational diseases. In most countries, OELs have only been set for few protein-containing aerosols causing IgE-mediated allergies. ...... is available for setting OELs for proteins and protein-containing aerosols where the critical effect is IgE sensitization and IgE-mediated airway diseases.......Occupational exposure limits (OELs) together with determined airborne exposures are used in risk assessment based managements of occupational exposures to prevent occupational diseases. In most countries, OELs have only been set for few protein-containing aerosols causing IgE-mediated allergies...... for setting OELs. Our aim is to analyse prerequisites for setting OELs for the allergenic protein-containing aerosols. Opposite to the key effect of toxicological reactions, two thresholds, one for the sensitization phase and one for elicitation of IgE-mediated symptoms in sensitized individuals, are used...

Influence of casein as a percentage of true protein and protein level on color and texture of milks containing 1 and 2% fat.

Science.gov (United States)

Misawa, Noriko; Barbano, David M; Drake, MaryAnne

2016-07-01

Combinations of fresh liquid microfiltration retentate of skim milk, ultrafiltered retentate and permeate produced from microfiltration permeate, cream, and dried lactose monohydrate were used to produce a matrix of 20 milks. The milks contained 5 levels of casein as a percentage of true protein of about 5, 25, 50, 75, and 80% and 4 levels of true protein of 3.0, 3.76, 4.34, and 5.0% with constant lactose percentage of 5%. The experiment was replicated twice and repeated for both 1 and 2% fat content. Hunter color measurements, relative viscosity, and fat globule size distribution were measured, and a trained panel documented appearance and texture attributes on all milks. Overall, casein as a percentage of true protein had stronger effects than level of true protein on Hunter L, a, b values, relative viscosity, and fat globule size when using fresh liquid micellar casein concentrates and milk serum protein concentrates produced by a combination of microfiltration and ultrafiltration. As casein as a percentage of true protein increased, the milks became more white (higher L value), less green (lower negative a value), and less yellow (lower b value). Relative viscosity increased and d(0.9) generally decreased with increasing casein as a percentage of true protein. Panelists perceived milks with increasing casein as a percentage of true protein as more white, more opaque, and less yellow. Panelists were able to detect increased throat cling and mouthcoating with increased casein as a percentage of true protein in 2% milks, even when differences in appearance among milks were masked. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

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Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular dynamics simulations of ter-pyridine and bis-triazinyl-pyridine complexes with lanthanide cations; Etude de dynamique moleculaire de complexes de la bis-triazinyl-pyridine (BTP) et de la terpyridine avec des lanthanides(3)

Energy Technology Data Exchange (ETDEWEB)

Guilbaud, Ph. [CEA Valrho, (DCC/DRRV/SEMP), 30 - Marcoule (France)

2000-07-01

The search for ligands which specifically separate actinides(III) from lanthanides(III) by liquid-liquid extraction has prompted considerable research in the Process Design and Modeling Department ('Service d'Etude et de Modelisation des Procedes'- SEMP). Ligands with soft donor atoms AS) that are able to perform this separation have already been investigated and research is currently under way to improve their performance for high acidic feeds. Theoretical chemistry research is conducted in the Theoretical and Structural Chemistry Laboratory ('Laboratoire de Chimie Theorique et Structurale') to improve our understanding of the complexation and extraction of these cations with such ligands. Theoretical studies were first carried out for the ter-pyridine (TPY) and bis-triazinyl-pyridine (BTP) ligands that display fairly good ability to separate and extract actinide(III) from lanthanide(III) ions. Molecular dynamics simulations were performed on ter-pyridine and bis-triazinyl-pyridine complexes with three lanthanide cations (La{sup 3+}, Eu{sup 3+} and Lu{sup 3+}) for vacuum and for water solutions. These calculations were carried out without counter-ions, with three nitrate (NO{sub 3}{sup -}) ions, and, in the case of ter-pyridine, with three {alpha}-bromo-caprate anions that are likely to be used experimentally as synergistic agents for the separation and extraction of An(III) from Ln(III). Molecular dynamics simulations were first performed for vacuum to evaluate the distances between nitrogen and lanthanide atoms (Ln{sup 3+},N) and intrinsic interaction energies to poly-nitrogenous ligands with or without NO{sub 3} ions, and for both ligands. The (Ln{sup 3+},N) distances decrease and the cation/ligand interaction energies increase along the La{sup 3+}, Eu{sup 3+}, Lu{sup 3+} series, with decreasing Ln(III) ion radii. The introduction of nitrate counter-ions makes the (Ln{sup 3+},N) distances slightly higher, and the TPY/Ln{sup 3+} and BTP

Structure and Function of SET and MYND Domain-Containing Proteins

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Nicholas Spellmon

2015-01-01

Full Text Available SET (Suppressor of variegation, Enhancer of Zeste, Trithorax and MYND (Myeloid-Nervy-DEAF1 domain-containing proteins (SMYD have been found to methylate a variety of histone and non-histone targets which contribute to their various roles in cell regulation including chromatin remodeling, transcription, signal transduction, and cell cycle control. During early development, SMYD proteins are believed to act as an epigenetic regulator for myogenesis and cardiomyocyte differentiation as they are abundantly expressed in cardiac and skeletal muscle. SMYD proteins are also of therapeutic interest due to the growing list of carcinomas and cardiovascular diseases linked to SMYD overexpression or dysfunction making them a putative target for drug intervention. This review will examine the biological relevance and gather all of the current structural data of SMYD proteins.

eEF1A Mediates the Nuclear Export of SNAG-Containing Proteins via the Exportin5-Aminoacyl-tRNA Complex

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José Manuel Mingot

2013-11-01

Full Text Available Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors.

Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

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Yeon Jeong Kim

Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

DEFF Research Database (Denmark)

Wilkinson, C R; Seeger, M; Hartmann-Petersen, R

2001-01-01

The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA do...

Phosphorescent light-emitting iridium complexes serve as a hypoxia-sensing probe for tumor imaging in living animals

Science.gov (United States)

Takeuchi, Toshiyuki; Zhang, Shaojuan; Negishi, Kazuya; Yoshihara, Toshitada; Hosaka, Masahiro; Tobita, Seiji

2010-02-01

Iridium complex, a promising organic light-emitting diode material for next generation television and computer displays, emits phosphorescence. Phosphorescence is quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting iridium complex Ir(btp)2(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor (HIF)-1 expression. BTP was further applied to imaging five nude mouse-transplanted tumors. All tumors presented a bright BTP-emitting image as early as 5 min after the injection. The BTP-dependent tumor image peaked at 1 to 2 h after the injection, and was then removed from tumors within 24 h. The minimal BTP image recognition size was at least 2 mm in diameter. By morphological examination and phosphorescence lifetime measurement, BTP is presumed to localize to the tumor cells, not to stay in the tumor microvessels by binding to albumin. The primary problem on suse of luminescent probe for tumor imaging is its weak penetrance to deep tissues from the skin surface. Since BTP is easily modifiable, we made BTP analogues with a longer excitation/emission wavelength to improve the tissue penetrance. One of them, BTPHSA, displayed 560/720 wavelength, and depicted its clear imaging from tumors transplanted over 6-7 mm deep from the skin surface. We suggest that BTP analogues have a vast potential for imaging hypoxic lesions such as tumor tissues.

Bulletins Aperçu sur l'adaptation : enseignements tirés de la ...

International Development Research Centre (IDRC) Digital Library (Canada)

21 avr. 2016 ... Les bulletins Aperçu sur l'adaptation sont neuf études de cas issues de sept projets soutenus par le programme Adaptation aux changements climatiques en Afrique. Chaque bulletin présente les enseignements tirés de travaux de recherche exécutés avec la participation active de collectivités menacées ...

Nanoparticles for Protein Sensing in Primary Containers: Interaction Analysis and Application.

Science.gov (United States)

Pérez Medina Martínez, Víctor; Espinosa-de la Garza, Carlos E; Méndez-Silva, Diego A; Bolívar-Vichido, Mariana; Flores-Ortiz, Luis F; Pérez, Néstor O

2018-05-01

Silver nanoparticles (AgNPs) are known to interact with proteins, leading to modifications of the plasmonic absorption that can be used to monitor this interaction, entailing a promising application for sensing adsorption of therapeutic proteins in primary containers. First, transmission electron microscopy in combination with plasmonic absorption and light scattering responses were used to characterize AgNPs and protein-AgNP complexes, including its concentration dependence, using two therapeutic molecules as models: a monoclonal antibody (mAb) and a synthetic copolymer (SC). Upon interaction, a protein corona was formed around AgNPs with the consequent shifting and broadening of their characteristic surface plasmon resonance (SPR) band (400 nm) to 410 nm and longer wavelenghts. Additional studies revealed secondary and three-dimensional structure modifications of model proteins upon interaction with AgNPs by circular dichroism and fluorescence techniques, respectively. Based on the modification of the SPR condition of AgNPs upon interaction with proteins, we developed a novel protein-sensing application of AgNPs in primary containers. This strategy was used to conduct a compatibility assessment of model proteins towards five commercially available prefillable glass syringe (PFS) models. mAb- and SC-exposed PFSs showed that 74 and 94% of cases were positive for protein adsorption, respectively. Interestingly, protein adsorption on 15% of total tested PFSs was negligible (below the nanogram level). Our results highlight the need of a case-by-case compatibility assessment of therapeutic proteins and their primary containers. This strategy has the potential to be easily applied on other containers and implemented during early-stage product development by pharmaceutical companies and for routine use during batch release by packaging manufacturers.

Displacement affinity chromatography of protein phosphatase one (PP1 complexes

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Gourlay Robert

2008-11-01

Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

Discovering protein-ligand chalcogen bonding in the protein data bank using endocyclic sulfur-containing heterocycles as ligand search subsets.

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Mitchell, Miguel O

2017-09-24

The chalcogen bond, the noncovalent, electrostatic attraction between covalently bonded atoms in group 16 and Lewis bases, is present in protein-ligand interactions based on X-ray structures deposited in the Protein Data Bank (PDB). Discovering protein-ligand chalcogen bonding in the PDB employed a strategy that focused on searching the database for protein complexes of five-membered, heterocyclic ligands containing endocyclic sulfur with endo electron-withdrawing groups (isothiazoles; thiazoles; 1,2,3-, 1,2.4-, 1,2,5-, 1,3,4-thiadiazoles) and thiophenes with exo electron-withdrawing groups, e.g., 2-chloro, 2-bromo, 2-amino, 2-alkylthio. Out of 930 ligands investigated, 33 or 3.5% have protein-ligand S---O interactions of which 31 are chalcogen bonds and two appear to be S---HO hydrogen bonds. The bond angles for some of the chalcogen bonds found in the PDB are less than 90°, and an electrostatic model is proposed to explain this phenomenon.

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

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Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Brown pigment formation in heated sugar-protein mixed suspensions containing unmodified and peptically modified whey protein concentrates.

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Rongsirikul, Narumol; Hongsprabhas, Parichat

2016-01-01

Commercial whey protein concentrate (WPC) was modified by heating the acidified protein suspensions (pH 2.0) at 80 °C for 30 min and treating with pepsin at 37 °C for 60 min. Prior to spray-drying, such modification did not change the molecular weights (MWs) of whey proteins determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After spray-drying the modified whey protein concentrate with trehalose excipient (MWPC-TH), it was found that the α-lactalbumin (α-La) was the major protein that was further hydrolyzed the most. The reconstituted MWPC-TH contained β-lactoglobulin (β-Lg) as the major protein and small molecular weight (MW) peptides of less than 6.5 kDa. The reconstituted MWPC-TH had higher NH2 group, Trolox equivalent antioxidant capacity (TEAC), lower exposed aromatic ring and thiol (SH) contents than did the commercial WPC. Kinetic studies revealed that the addition of MWPC-TH in fructose-glycine solution was able to reduce brown pigment formation in the mixtures heated at 80 to 95 °C by increasing the activation energy (Ea) of brown pigment formation due to the retardation of fluoresced advanced glycation end product (AGEs) formation. The addition of MWPC to reducing sugar-glycine/commercial WPC was also able to lower brown pigment formation in the sterilized (121 °C, 15 min) mixed suspensions containing 0.1 M reducing sugar and 0.5-1.0 % glycine and/or commercial (P < 0.05). It was demonstrated that the modification investigated in this study selectively hydrolyzed α-La and retained β-Lg for the production of antibrowning whey protein concentrate.

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus

DEFF Research Database (Denmark)

Hansen, T S; Andreasen, P H; Dreisig, H

1991-01-01

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 ...... by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.......We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63...... nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified...

UBA domain containing proteins in fission yeast

DEFF Research Database (Denmark)

Hartmann-Petersen, Rasmus; Semple, Colin A M; Ponting, Chris P

2003-01-01

characterised on both the functional and structural levels. One example of a widespread ubiquitin binding module is the ubiquitin associated (UBA) domain. Here, we discuss the approximately 15 UBA domain containing proteins encoded in the relatively small genome of the fission yeast Schizosaccharomyces pombe...

Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1) levels in patients with overt and subclinical hyperthyroidism: effects of treatment.

Science.gov (United States)

Erem, Cihangir; Civan, Nadim; Coskun, Hulya; Mentese, Ahmet; Suleyman, Akile Karacin; Altay, Diler Us; Akgul, Zeynep; Deger, Orhan

2016-06-01

Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1) has been shown to increase in parallel with platelet activation in acute ischaemic and thrombotic diseases. There has been no study evaluating SCUBE1 levels in patients with overt hyperthyroidism (OHyper) and subclinical hyperthyroidism (SHyper), conditions which are known to show impairment of both endothelial and platelet function. This study sought to evaluate SCUBE1 concentrations in patients with SHyper and OHyper, and assessed the effects of antithyroid drug (ATD) therapy on circulating SCUBE1 levels. Forty-five untreated patients with OHyper, 20 untreated patients with SHyper and 30 age- and sex-matched healthy controls were prospectively included in the study. Biochemical and hormonal parameters were evaluated in all patients before and after treatment. Compared with the control subjects, SCUBE1 levels were significantly increased in patients with SHyper and OHyper (P hyperthyroidism, especially in the subclinical period. © 2015 John Wiley & Sons Ltd.

Two-dimensional gel electrophoresis of selenized yeast and autoradiography of 75Se-containing proteins

International Nuclear Information System (INIS)

Chery, C.C.; Dumont, E.; Cornelis, R.; Moens, L.

2001-01-01

Two-dimensional high-resolution gel electrophoresis (2DE) has been applied to the fractionation of 75 Se-containing proteins in yeast, grown in 75 Se-containing medium, and autoradiography was used for detection of the 75 Se-containing proteins. Gel filtration and ultrafiltration were used to check whether the selenium side-chains were stable in the presence of the chemicals used for lysis and 2DE. The mass distribution of the selenium-containing proteins was estimated by use of gel filtration and the results were compared with the distribution obtained by 2DE. A 2DE map of selenium-containing proteins in yeast is presented, and compared with a total protein map of yeast. (orig.)

Viral Interactions with PDZ Domain-Containing Proteins-An Oncogenic Trait?

Science.gov (United States)

James, Claire D; Roberts, Sally

2016-01-18

Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

Effects of a diet containing genetically modified rice expressing the Cry1Ab/1Ac protein (Bacillus thuringiensis toxin) on broiler chickens.

Science.gov (United States)

Li, Zeyang; Gao, Yang; Zhang, Minhong; Feng, Jinghai; Xiong, Yandan

2015-01-01

The aim of this study was to evaluate the effect of feeding Bacillus thuringiensis (Bt) rice expressing the Cry1Ab/1Ac protein on broiler chicken. The genetically modified (GM) Bt rice was compared with the corresponding non-GM rice regarding performance of feeding groups, their health status, relative organ weights, biochemical serum parameters and occurrence of Cry1Ab/1Ac gene fragments. One hundred and eighty day-old Arbor Acres female broilers with the same health condition were randomly allocated to the two treatments (6 replicate cages with 15 broilers in each cage per treatment). They received diets containing GM rice (GM group) or its parental non-GM rice (non-GM group) at 52-57% of the air-dried diet for 42 days. The results show that the transgenic rice had a similar nutrient composition as the non-GM rice and had no adverse effects on chicken growth, biochemical serum parameters and necropsy during the 42-day feeding period. In birds fed the GM rice, no transgenic gene fragments were detected in the samples of blood, liver, kidneys, spleen, jejunum, ileum, duodenum and muscle tissue. In conclusion, the results suggest that Bt rice expressing Cry1Ab/1Ac protein has no adverse effects on broiler chicken. Therefore, it can be considered as safe and used as feed source for broiler chicken.

Fas-associated factor 1 is a scaffold protein that promotes β-transducin repeat-containing protein (β-TrCP)-mediated β-catenin ubiquitination and degradation.

Science.gov (United States)

Zhang, Long; Zhou, Fangfang; Li, Yihao; Drabsch, Yvette; Zhang, Juan; van Dam, Hans; ten Dijke, Peter

2012-08-31

FAS-associated factor 1 (FAF1) antagonizes Wnt signaling by stimulating β-catenin degradation. However, the molecular mechanism underlying this effect is unknown. Here, we demonstrate that the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) is required for FAF1 to suppress Wnt signaling and that FAF1 specifically associates with the SCF (Skp1-Cul1-F-box protein)-β-TrCP complex. Depletion of β-TrCP reduced FAF1-mediated β-catenin polyubiquitination and impaired FAF1 in antagonizing Wnt/β-catenin signaling. FAF1 was shown to act as a scaffold for β-catenin and β-TrCP and thereby to potentiate β-TrCP-mediated β-catenin ubiquitination and degradation. Data mining revealed that FAF1 expression is statistically down-regulated in human breast carcinoma compared with normal breast tissue. Consistent with this, FAF1 expression is higher in epithelial-like MCF7 than mesenchymal-like MDA-MB-231 human breast cancer cells. Depletion of FAF1 in MCF7 cells resulted in increased β-catenin accumulation and signaling. Importantly, FAF1 knockdown promoted a decrease in epithelial E-cadherin and an increase in mesenchymal vimentin expression, indicative for an epithelial to mesenchymal transition. Moreover, ectopic FAF1 expression reduces breast cancer cell migration in vitro and invasion/metastasis in vivo. Thus, our studies strengthen a tumor-suppressive function for FAF1.

Immunogenicity of therapeutic proteins. Part 2: impact of container closures.

Science.gov (United States)

Sharma, Basant

2007-01-01

Immunogenicity as a potential consequence of therapeutic protein administration is increasingly being scrutinized in the biopharmaceuticals industry, particularly with the imminent introduction of biosimilar products. Immunogenicity is an important safety aspect requiring rigorous investigation to fully appreciate its impact. Factors involved in product handling, such as storage temperature, light exposure, and shaking, have been implicated in immunogenicity, while container closure systems are no less important. Intended to provide a stable environment for the dosage form, container closures may also interact with a product, affecting performance and potentially enhancing immunogenicity. Glass surfaces, air-liquid interfaces, and lubricants can mediate protein denaturation, while phthalates in plastics and latex rubber are sources of extractables and leachates that may contaminate a product, causing allergic reactions and increasing immunogenicity. The manufacture of therapeutic proteins therefore requires rigorous safety evaluations not just in the context of the product, but also product containment.

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

Directory of Open Access Journals (Sweden)

Aleksander F Sikorski

2007-01-01

Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs

DEFF Research Database (Denmark)

Krogh, T N; Bachmann, E; Teisner, B

1997-01-01

By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine...... residues arranged in six epidermal-growth-factor-like domains. The purification of several C-terminal peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were present on all C-terminal peptides...... at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N-linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary complex-type and small amounts (less than 5...

The roles of USH1 proteins and PDZ domain-containing USH proteins in USH2 complex integrity in cochlear hair cells.

Science.gov (United States)

Zou, Junhuang; Chen, Qian; Almishaal, Ali; Mathur, Pranav Dinesh; Zheng, Tihua; Tian, Cong; Zheng, Qing Y; Yang, Jun

2017-02-01

Usher syndrome (USH) is the most common cause of inherited deaf-blindness, manifested as USH1, USH2 and USH3 clinical types. The protein products of USH2 causative and modifier genes, USH2A, ADGRV1, WHRN and PDZD7, interact to assemble a multiprotein complex at the ankle link region of the mechanosensitive stereociliary bundle in hair cells. Defects in this complex cause stereociliary bundle disorganization and hearing loss. The four USH2 proteins also interact in vitro with USH1 proteins including myosin VIIa, USH1G (SANS), CIB2 and harmonin. However, it is unclear whether the interactions between USH1 and USH2 proteins occur in vivo and whether USH1 proteins play a role in USH2 complex assembly in hair cells. In this study, we identified a novel interaction between myosin VIIa and PDZD7 by FLAG pull-down assay. We further investigated the role of the above-mentioned four USH1 proteins in the cochlear USH2 complex assembly using USH1 mutant mice. We showed that only myosin VIIa is indispensable for USH2 complex assembly at ankle links, indicating the potential transport and/or anchoring role of myosin VIIa for USH2 proteins in hair cells. However, myosin VIIa is not required for USH2 complex assembly in photoreceptors. We further showed that, while PDZ protein harmonin is not involved, its paralogous USH2 proteins, PDZD7 and whirlin, function synergistically in USH2 complex assembly in cochlear hair cells. In summary, our studies provide novel insight into the functional relationship between USH1 and USH2 proteins in the cochlea and the retina as well as the disease mechanisms underlying USH1 and USH2. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

Science.gov (United States)

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

Directory of Open Access Journals (Sweden)

In Sil Jeong

Full Text Available Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD PHOSPHATASE-LIKE 1 (CPL1 regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds RNA binding motifs (dsRBMs at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3 as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain

DEFF Research Database (Denmark)

Pedersen, Lotte Bang; Birkelund, S; Holm, A

1996-01-01

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may......-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel...... retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect...

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

OpenAIRE

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-01-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly ...

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata.

Science.gov (United States)

Li, Shu; Sun, Penglin; Williams, Justin Stephen; Kao, Teh-hui

2014-03-01

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S2-SLF1 (SLF1 of S 2-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP-MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.

Adaptor proteins intersectin 1 and 2 bind similar proline-rich ligands but are differentially recognized by SH2 domain-containing proteins.

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Olga Novokhatska

Full Text Available BACKGROUND: Scaffolding proteins of the intersectin (ITSN family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs. METHODOLOGY/PRINCIPAL FINDINGS: We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform. CONCLUSIONS/SIGNIFICANCE: Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.

Rheological properties of soybean protein isolate gels containing emulsion droplets

NARCIS (Netherlands)

Kim, K.H.; Renkema, J.M.S.; Vliet, van T.

2001-01-01

Rheological properties of soybean protein gels containing various volume fractions oil droplets have been studied at small and large deformations. Dynamic viscoelastic properties of soybean protein isolate gels were determined as a function of the volume fraction of oil droplets stabilised by the

Performance of juvenile mojarra supplied with feed containing varying levels of crude protein

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Ricardo Henrique Bastos de Souza

2016-04-01

Full Text Available ABSTRACT The growth of the Brazilian aquaculture has stimulated the development of the productive chain of native species, including marine environment. The objective of this study was to evaluate the growth performance of juvenile mojarra fish (Diapterus rhombeus fed diets containing different concentrations of crude protein (32, 36, 40 and 44 g 100 g-1. The 80 juvenile mojarra (7.2±1.5 g were kept in 16 circular tanks (150 L. The study design used was completely randomized with four treatments and four repetitions. The fish were fed four times a day. At the end of the experiment (60 days the final weight, feed intake, weight gain (WG, feed:gain ratio (FGR, protein efficiency rate (PER, energy efficiency rate, specific growth, survival rate and, body composition were evaluated. It was verified significant effect of protein level on the WG, with the best value at the level of 38.20 g 100 g-1 of crude protein. For FGR, the best estimated value occurred with 38.06 g 100 g-1 of crude protein, similar to that reported for the PER (38.91 g 100 g-1. The other performance parameters and body composition were not influenced by crude protein levels. Diet crude protein concentrations between 38.06 and 38.91 g 100 g-1 provide the best performance indices for juvenile mojarra.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C{sub 2}H{sub 2} zinc finger protein

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Han, D.; Zhang, C. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); Fan, W.J. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); The Second Affiliated Hospital, University of South China, Hengyang City, Hunan Province (China); Pan, W.J.; Feng, D.M.; Qu, S.L.; Jiang, Z.S. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China)

2014-10-31

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C{sub 2}H{sub 2} motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

ERG induces epigenetic activation of Tudor domain-containing protein 1 (TDRD1) in ERG rearrangement-positive prostate cancer.

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Kacprzyk, Lukasz A; Laible, Mark; Andrasiuk, Tatjana; Brase, Jan C; Börno, Stefan T; Fälth, Maria; Kuner, Ruprecht; Lehrach, Hans; Schweiger, Michal R; Sültmann, Holger

2013-01-01

Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell's gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors. Gene expression measurements by real-time quantitative PCR revealed a remarkable co-expression of TDRD1 and ERG (r(2) = 0.77) but not ETV1 (r(2)prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (ρ = -0.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERG-negative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression. We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures.

RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

International Nuclear Information System (INIS)

Reynolds, P.; Weber, S.; Prakash, L.

1985-01-01

The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures

Protein Esaslı Yağ İkame Maddesi Kullanımının Yağsız Yoğurdun Kalitesi Üzerine Etkisi

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Fatma Sezen

2015-02-01

Full Text Available Bu çalışmada, protein esaslı yağ ikame maddesi olan Dairy-Lo™ 'nun %1 ve %2 gibi iki farklı oranı kullanılarak, rekonstitüe sütten üretilen, yağsız set tipi yoğurtların fiziksel, kimyasal, tekstürel ve duyusal özellikleri incelenmiştir. Yoğurt örneklerinde depolamanın 1., 7. ve 15. günlerinde titrasyon asitliği, pH, asetaldehit, laktik asit, serum ayrılması, viskozite, konsistens değerleri saptanmıştır. Ayrıca yoğurt örneklerinde duyusal değerlendirmeler de yapılmıştır.  Bu çalışma sonucu elde edilen bulgular, protein esaslı yağ ikame maddesi Dairy-Lo™ ilavesinin yağsız yoğurdun konsistens ve viskozitesini artırıp, serum ayrılmasını azalttığını göstermiştir. Dairy-Lo™ 'nun %2 oranında kullanımının yağsız set tip yoğurdun kalitesini iyileştirdiği belirlenmiştir.

Auxin: Harnessing a loose cannon

NARCIS (Netherlands)

Weijers, D.

2015-01-01

The auxin receptor TIR1 is an F-box protein functioning in a ubiquitin ligase complex to target repressors for degradation. It is itself an unstable protein, but newly identified mutations protect both TIR1 and its substrates from degradation. These mutations could help in identifying the substrates

Study on expression of SH2 domain-containing protein tyrosine phosphatase SHP-1 and SHP-2 in γ-ray irradiation-induced thymus lymphoma in mice

International Nuclear Information System (INIS)

Huang Dingde; Chen Qi; Han Ling; Cai Jianming; Li Bailong; Huang Yuecheng; Gao Jianguo; Sun Suping

2003-01-01

Objective: To investigate the expression of SH2 domain containing-protein tyrosine phosphatase SHP-1 and SHP-2 in γ-ray irradiation-induced thymus lymphoma in mice. Methods: Altogether 338 BALB/c mice were randomly divided into irradiation groups and controls. Irradiation groups which were irradiated with γ-rays included canceration groups confirmed with histology and uncanceration groups. The controls were fed synchronistically with irradiation groups. The expression of SHP-1 and SHP-2 was detected with Western blot in thymus cells. Results: The expression of SHP-1 in canceration groups was much higher than that in uncanceration groups and controls significantly, while the expression of SHP-2 in canceration groups was higher than that in uncanceration groups and controls. When authors detected the expression of SHP-2 with Western blot, the authors found another protein with a molecular weight of 55x10 3 , which expression in canceration groups was higher than that in uncanceration groups and controls. Conclusion: The expression of SH2 domain-containing protein tyrosine phosphatase SHP-1 and SHP-2 is significantly increased in canceration groups, suggesting that SHP-1 and SHP-2 may be related with γ-ray induced thymus lymphoma in mice. Further research is expected on the relationship between development of cancer and SHP-1 and SHP-2

Speciation of iodine-containing proteins in Nori seaweed by gel electrophoresis laser ablation ICP-MS.

Science.gov (United States)

Romarís-Hortas, V; Bianga, J; Moreda-Piñeiro, A; Bermejo-Barrera, P; Szpunar, J

2014-09-01

An analytical approach providing an insight into speciation of iodine in water insoluble fraction of edible seaweed (Nori) was developed. The seaweed, harvested in the Galician coast (Northwestern Spain), contained 67.7±1.3 μg g(-1) iodine of which 25% was water soluble and could be identifies as iodide. Extraction conditions of water insoluble residue using urea, NaOH, SDS and Triton X-100 were investigated. The protein pellets obtained in optimized conditions (after precipitation of urea extracts with acetone), were digested with trypsin and protease XIV. Size exclusion chromatography-ICP-MS of both enzymatic digests demonstrated the occurrence of iodoaminoacids putatively present in proteins. Intact proteins could be separated by gel electrophoresis after an additional extraction of the protein extract with phenol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with laser ablation ICP-MS detection of (127)I indicated the presence of iodine in protein bands corresponding to molecular masses of 110 kDa, 40 kDa, 27 kDa, 20 kDa and 10 kDa. 2D IEF-SDS PAGE with laser ablation ICP-MS (127)I imaging allowed the detection of 5 iodine containing protein spots in the alkaline pI range. Copyright © 2014 Elsevier B.V. All rights reserved.

The two capsid proteins of maize rayado fino virus contain common peptide sequences.

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Falk, B W; Tsai, J H

1986-01-01

Virions of maize rayado fino virus (MRFV) were purified and two major capsid proteins (ca. Mr 29,000 and 22,000) were resolved by SDS-PAGE. When the two major capsid proteins were isolated from gels and compared by one-dimensional peptide mapping after digestion with Staphylococcus aureus V-8 protease, indistinguishable peptide maps were obtained, suggesting that these two proteins contain common peptide sequences. Some preparations also showed minor protein components that were intermediate between the Mr 22,000 and Mr 29,000 capsid proteins. One of the minor proteins, ca. Mr 27,000, gave a peptide map indistinguishable from the major capsid proteins. In vitro ageing of partially purified preparations or virion treatment with proteolytic enzymes failed to show conversion of the Mr 29,000 protein to a Mr 22,000. Protease inhibitors added to the buffers used for virion purification did not affect the apparent 1:3 ratio of 29,000 to 22,000 proteins in the purified preparations.

A TALE-inspired computational screen for proteins that contain approximate tandem repeats.

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Perycz, Malgorzata; Krwawicz, Joanna; Bochtler, Matthias

2017-01-01

TAL (transcription activator-like) effectors (TALEs) are bacterial proteins that are secreted from bacteria to plant cells to act as transcriptional activators. TALEs and related proteins (RipTALs, BurrH, MOrTL1 and MOrTL2) contain approximate tandem repeats that differ in conserved positions that define specificity. Using PERL, we screened ~47 million protein sequences for TALE-like architecture characterized by approximate tandem repeats (between 30 and 43 amino acids in length) and sequence variability in conserved positions, without requiring sequence similarity to TALEs. Candidate proteins were scored according to their propensity for nuclear localization, secondary structure, repeat sequence complexity, as well as covariation and predicted structural proximity of variable residues. Biological context was tentatively inferred from co-occurrence of other domains and interactome predictions. Approximate repeats with TALE-like features that merit experimental characterization were found in a protein of chestnut blight fungus, a eukaryotic plant pathogen.

Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX

International Nuclear Information System (INIS)

Zakhartchouk, Alexander; Connors, Wayne; Van Kessel, Andrew; Tikoo, Suresh Kumar

2004-01-01

Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins. We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX. Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids. Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins. Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion. Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication. These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides

Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion

International Nuclear Information System (INIS)

Kim, Eui Tae; Kim, Kyeong Kyu; Matunis, Mike J.; Ahn, Jin-Hyun

2009-01-01

Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.

Direct protein-protein interaction between PLCγ1 and the bradykinin B2 receptor-Importance of growth conditions

International Nuclear Information System (INIS)

Duchene, Johan; Chauhan, Sharmila D.; Lopez, Frederic; Pecher, Christiane; Esteve, Jean-Pierre; Girolami, Jean-Pierre; Bascands, Jean-Loup; Schanstra, Joost P.

2005-01-01

Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells

Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

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Gao, Lan; Li, Hao-Ming

2017-08-01

Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

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B.M. Ribeiro

1998-06-01

Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Localization of CORO1A in the Macrophages Containing Mycobacterium leprae

International Nuclear Information System (INIS)

Suzuki, Koichi; Takeshita, Fumihiko; Nakata, Noboru; Ishii, Norihisa; Makino, Masahiko

2006-01-01

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A

Lactobacillus bulgaricus Fajlarının Restriksiyon Fragment Uzunluk Polimorfizmi, Protein Profilleri ve Konakçı Özgüllüklerine Göre Tanımlanması

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Esra Acar Soykut

2015-02-01

Full Text Available Lactobacillus delbrueckii subsp. bulgaricus suşlarına etkili 19 adet faj, restriksiyon fragment uzunluk polimorfizmi (RFLP, yapısal protein şablonları ve konakçı spektrumlarına göre tanımlanmış ve sınıflandırılmıştır. Fajlar, farklı süt ürünlerinden L. bulgaricus starter kültürleri (Y4, V1 ve V2 ile izole edilmiştir. Konakçı spektrumunun belirlenmesi için; doğal ve ticari L. bulgaricus suşlarının L. bulgaricus V1 ve V2 fajları ile S. thermophilus B3 fajlarına duyarlılıkları test edilmiştir. Doğal L. bulgaricus suşlarının tamamının, L. bulgaricus fajlarının hepsine duyarlılık gösterdiği tespit edilmiştir. Bununla birlikte bazı doğal L. bulgaricus suşlarının, S. thermophilus fajlarına duyarlı olduğu ve heterolog konakçı görevi gördüğü anlaşılmıştır. Çalışılan bu L. bulgaricus fajlarının yapısal protein profilleri ortaya çıkarılmış, fajların iki veya üç major (29.6, 29.2, 26.2 kDa ve ortak olan üç minor (100.8, 65, 50 kDa yapısal proteine sahip oldukları tespit edilmiştir. Bu fajlara ait DNA’lar Eco RI, Pvu II, HindIII, Hha I ve Bam HI restriksiyon enzimleri ile kesime alınmıştır. Oluşan fragmentler doğrultusunda fajlar gruplara ayrılmıştır. Kesim sonucu oluşan fragmentlerin toplamından faj genom büyüklüklerinin 29–34 kb arasında olduğu tespit edilmiştir.

RFX Transcription Factor DAF-19 Regulates 5-HT and Innate Immune Responses to Pathogenic Bacteria in Caenorhabditis elegans

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Choi, Sunju; Xu, Lu; Sze, Ji Ying

2013-01-01

In Caenorhabditis elegans the Toll-interleukin receptor domain adaptor protein TIR-1 via a conserved mitogen-activated protein kinase (MAPK) signaling cascade induces innate immunity and upregulates serotonin (5-HT) biosynthesis gene tph-1 in a pair of ADF chemosensory neurons in response to infection. Here, we identify transcription factors downstream of the TIR-1 signaling pathway. We show that common transcription factors control the innate immunity and 5-HT biosynthesis. We demonstrate that a cysteine to tyrosine substitution in an ARM motif of the HEAT/Arm repeat region of the TIR-1 protein confers TIR-1 hyperactivation, leading to constitutive tph-1 upregulation in the ADF neurons, increased expression of intestinal antimicrobial genes, and enhanced resistance to killing by the human opportunistic pathogen Pseudomonas aeruginosa PA14. A forward genetic screen for suppressors of the hyperactive TIR-1 led to the identification of DAF-19, an ortholog of regulatory factor X (RFX) transcription factors that are required for human adaptive immunity. We show that DAF-19 concerts with ATF-7, a member of the activating transcription factor (ATF)/cAMP response element-binding B (CREB) family of transcription factors, to regulate tph-1 and antimicrobial genes, reminiscent of RFX-CREB interaction in human immune cells. daf-19 mutants display heightened susceptibility to killing by PA14. Remarkably, whereas the TIR-1-MAPK-DAF-19/ATF-7 pathway in the intestinal immunity is regulated by DKF-2/protein kinase D, we found that the regulation of tph-1 expression is independent of DKF-2 but requires UNC-43/Ca2+/calmodulin-dependent protein kinase (CaMK) II. Our results suggest that pathogenic cues trigger a common core-signaling pathway via tissue-specific mechanisms and demonstrate a novel role for RFX factors in neuronal and innate immune responses to infection. PMID:23505381

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus.

Science.gov (United States)

Hansen, T S; Andreasen, P H; Dreisig, H; Højrup, P; Nielsen, H; Engberg, J; Kristiansen, K

1991-09-15

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.

Vector coherent state representations of SO5 contains SU2 + SU2 contains U1 + U1 and SO5 contains U1 + U1

International Nuclear Information System (INIS)

Pan Feng

1991-01-01

VCS representations of SO 5 contains SU 2 + SU 2 contains U 1 + U 1 and SO 5 contains U 1 + U 1 are discussed. Reduced matrix elements for SO 5 contains SU 2 + SU 2 are derived. The multiplicity of a weight for SO 5 is determined by using the K-matrix technique

Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

International Nuclear Information System (INIS)

Hayakawa, Hiroshi; Fujikane, Aya; Ito, Riyoko; Matsumoto, Masaki; Nakayama, Keiichi I.; Sekiguchi, Mutsuo

2010-01-01

Research highlights: → We performed comprehensive survey for proteins that bind to oxidized RNA. → HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. → Knockdown of HNRNPD/C expression caused increased sensitivity to H 2 O 2 . → Amounts of HNRNPD protein rapidly decreased when cells were exposed to H 2 O 2 . -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins

International Nuclear Information System (INIS)

Krawczyk, P. M.; Stap, C.; Van Oven, C.; Hoebe, R.; Aten, J. A.

2006-01-01

For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains. (authors)

Transfer buffer containing methanol can be reused multiple times in protein electrotransfer.

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Pettegrew, Colin J; Jayini, Renuka; Islam, M Rafiq

2009-04-01

We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to PVDF membrane. Repeated use of the transfer buffer containing methanol will significantly reduce the volume of hazardous waste generated and its disposal cost as well as its adverse effect on environment.

Topology of transmembrane channel-like gene 1 protein.

Science.gov (United States)

Labay, Valentina; Weichert, Rachel M; Makishima, Tomoko; Griffith, Andrew J

2010-10-05

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.

Thermodynamics of complexation of lanthanides with 2,6-bis(5,6-diethyl-1,2,4-triazin-3-yl) pyridine

Energy Technology Data Exchange (ETDEWEB)

Rawat, N.; Bhattacharyya, A.; Tomar, B.S. [Bhabha Atomic Research Centre, Trombay, Mumbai (India). Radiochemistry Div.; Ghosh, S.K.; Gadly, T. [Bhabha Atomic Research Centre, Trombay, Mumbai (India). Bioorganic Div.

2011-07-01

Solvent extraction studies on separation of trivalent actinides from lanthanides using 2,6-bis(5,6-dialkyl-1,2,4-triazin-3-yl) pyridines have shown promising results with respect to separation factor and efficiency in acidic medium. In order to understand their complexation behavior, the stability constant (log {beta}) of trivalent lanthanides (La, Nd, Eu, Tb, Ho, Tm, Lu) with 2,6-bis(5,6-diethyl-1,2,4-triazin-3-yl)pyridine (ethyl-BTP) have been determined in methanol medium (ionic strength 0.01 M) using spectrophotometric titrations. The stoichiometry of the complexes is found to vary with the ionic size of lanthanide ion. The variation in log {beta} across the lanthanide series is attributed to variation in solvation characteristics of the metal ion. Comparison of log {beta} for Ln(III)-ethyl-BTP complexes with other alkyl derivatives showed increase in the stability with increasing length of the alkyl group due to hydrophobic interaction. In the case of Eu(III), the speciation was also corroborated by time resolved fluorescence spectroscopy. The thermodynamic parameters ({delta} G, {delta} H, {delta} S) for complexation of Eu(III) with ethyl-BTP, were determined by microcalorimetry, which revealed strong metal ion-ligand interaction with the reactions driven mainly by enthalpy. (orig.)

Fragile X mental retardation protein recognizes a G quadruplex structure within the survival motor neuron domain containing 1 mRNA 5'-UTR.

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McAninch, Damian S; Heinaman, Ashley M; Lang, Cara N; Moss, Kathryn R; Bassell, Gary J; Rita Mihailescu, Mihaela; Evans, Timothy L

2017-07-25

G quadruplex structures have been predicted by bioinformatics to form in the 5'- and 3'-untranslated regions (UTRs) of several thousand mature mRNAs and are believed to play a role in translation regulation. Elucidation of these roles has primarily been focused on the 3'-UTR, with limited focus on characterizing the G quadruplex structures and functions in the 5'-UTR. Investigation of the affinity and specificity of RNA binding proteins for 5'-UTR G quadruplexes and the resulting regulatory effects have also been limited. Among the mRNAs predicted to form a G quadruplex structure within the 5'-UTR is the survival motor neuron domain containing 1 (SMNDC1) mRNA, encoding a protein that is critical to the spliceosome. Additionally, this mRNA has been identified as a potential target of the fragile X mental retardation protein (FMRP), whose loss of expression leads to fragile X syndrome. FMRP is an RNA binding protein involved in translation regulation that has been shown to bind mRNA targets that form G quadruplex structures. In this study we have used biophysical methods to investigate G quadruplex formation in the 5'-UTR of SMNDC1 mRNA and analyzed its interactions with FMRP. Our results show that SMNDC1 mRNA 5'-UTR forms an intramolecular, parallel G quadruplex structure comprised of three G quartet planes, which is bound specifically by FMRP both in vitro and in mouse brain lysates. These findings suggest a model by which FMRP might regulate the translation of a subset of its mRNA targets by recognizing the G quadruplex structure present in their 5'-UTR, and affecting their accessibility by the protein synthesis machinery.

Prefunctionalized Porous Organic Polymers: Effective Supports of Surface Palladium Nanoparticles for the Enhancement of Catalytic Performances in Dehalogenation.

Science.gov (United States)

Zhong, Hong; Liu, Caiping; Zhou, Hanghui; Wang, Yangxin; Wang, Ruihu

2016-08-22

Three porous organic polymers (POPs) containing H, COOMe, and COO(-) groups at 2,6-bis(1,2,3-triazol-4-yl)pyridyl (BTP) units (i.e., POP-1, POP-2, and POP-3, respectively) were prepared for the immobilization of metal nanoparticles (NPs). The ultrafine palladium NPs are uniformly encapsulated in the interior pores of POP-1, whereas uniform- and dual-distributed palladium NPs are located on the external surface of POP-2 and POP-3, respectively. The presence of carboxylate groups not only endows POP-3 an outstanding dispersibility in H2 O/EtOH, but also enables the palladium NPs at the surface to show the highest catalytic activity, stability, and recyclability in dehalogenation reactions of chlorobenzene at 25 °C. The palladium NPs on the external surface are effectively stabilized by the functionalized POPs containing BTP units and carboxylate groups, which provides a new insight for highly efficient catalytic systems based on surface metal NPs of porous materials. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

Science.gov (United States)

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea

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Wenjun Zhu

2017-09-01

Full Text Available We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.

Understanding the role of BAR and SH3 domain-containing proteins in fungi

NARCIS (Netherlands)

Gkourtsa, A.

2017-01-01

This thesis addresses the role of SH3 and BAR domain-containing proteins in different fungal species. SH3 domains are small modules that mediate protein-protein interactions and BAR domains are dimerization domains with membrane binding and bending properties. It is known that the ScRvs167 protein

Growth of plasmodium falciparum in human erythrocytes containing abnormal membrane proteins

International Nuclear Information System (INIS)

Schulman, S.; Roth, E.F. Jr.; Cheng, B.; Rybicki, A.C.; Sussman, I.I.; Wong, M.; Nagel, R.L.; Schwartz, R.S.; Wang, W.; Ranney, H.M.

1990-01-01

To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, the authors have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin [HS(Sp + )], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1 0 )], HE due to a spectrin αI domain structural variant that results in increased content of spectrin dimers [HE(Spα I/65 )], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [ 3 H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp + ) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. The extent of decreased parasite growth in HS(Sp + ) RBCs closely correlated with the extent of RBC spectrin deficiency. Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth and development

Decaffeinated green and black tea polyphenols decrease weight gain and alter microbiome populations and function in diet-induced obese mice.

Science.gov (United States)

Henning, Susanne M; Yang, Jieping; Hsu, Mark; Lee, Ru-Po; Grojean, Emma M; Ly, Austin; Tseng, Chi-Hong; Heber, David; Li, Zhaoping

2017-09-30

Decaffeinated green tea (GT) and black tea (BT) polyphenols inhibit weight gain in mice fed an obesogenic diet. Since the intestinal microflora is an important contributor to obesity, it was the objective of this study to determine whether the intestinal microflora plays a role in the anti-obesogenic effect of GT and BT. C57BL/6J mice were fed a high-fat/high-sucrose diet (HF/HS, 32% energy from fat; 25% energy from sucrose) or the same diet supplemented with 0.25% GTP or BTP or a low-fat/high-sucrose (LF/HS, 10.6% energy from fat, 25% energy from sucrose) diet for 4 weeks. Bacterial composition was assessed by MiSeq sequencing of the 16S rRNA gene. GTP and BTP diets resulted in a decrease of cecum Firmicutes and increase in Bacteroidetes. The relative proportions of Blautia, Bryantella, Collinsella, Lactobacillus, Marvinbryantia, Turicibacter, Barnesiella, and Parabacteroides were significantly correlated with weight loss induced by tea extracts. BTP increased the relative proportion of Pseudobutyrivibrio and intestinal formation of short-chain fatty acids (SCFA) analyzed by gas chromatography. Cecum propionic acid content was significantly correlated with the relative proportion of Pseudobutyrivibrio. GTP and BTP induced a significant increase in hepatic 5'adenosylmonophosphate-activated protein kinase (AMPK) phosphorylation by 70 and 289%, respectively (P < 0.05) determined by Western blot. In summary, both BTP and GTP induced weight loss in association with alteration of the microbiota and increased hepatic AMPK phosphorylation. We hypothesize that BTP increased pAMPK through increased intestinal SCFA production, while GTPs increased hepatic AMPK through GTP present in the liver.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Sepiella japonica.

Science.gov (United States)

Huo, Liping; Bao, Miaomiao; Lv, Zhenming; Chi, Changfeng; Wang, Tianming; Liu, Huihui

2018-05-01

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-κB (NF-κB). In this study a novel isoform of MyD88 in Sepiella japonica (SjMyD88) was cloned and functionally characterized (GenBank accession no. AQY56781.1). The complete cDNA sequence of SjMyD88 was 1912 bp and contained a 1017 bp open reading frame encoding 338 amino acid residues, which was similar to its mollusk orthologues in the length. BLASTp analysis suggested the deduced amino acids sequence of SjMyD88 shared high identity to the known MyD88, for instance, 64% identity with Octopus bimaculoides. Sequence analysis revealed two conserved domains, the N-terminal DD and the C-terminal TIR domain appeared in SjMyD88, which was consistent with MyD88 proteins from other species. The fusion expression of SjMyD88 and green fluorescent protein (EGFP) in HEK293 cells was conducted and cytoplasm localization was detected. Meanwhile, the TIR-pmCherry fusion protein showed red fluorescence and mainly distributed in the cytoplasm. After cotransfection MyD88-EGFP and TIR-pmCherry red obviously overlapped and changed to yellowish green. The results suggested that there was the interaction between homologous TIR-pmcherry and MyD88-EGFP. Tissues expression profiles analysis showed that SjMyD88 ubiquitously expressed in all tested tissues with the highest expression in the gills and livers except reproductive related tissue, and it was significantly induced in livers under LPS stress. These data provide insight into the roles of SjMyD88 in the TLR signaling pathway of S. japonica in response to pathogenic bacteria. Copyright © 2018. Published by Elsevier Ltd.

Beyond the traditional definition of breakthrough pain: an observational study.

Science.gov (United States)

Gatti, Antonio; Gentili, Marta; Iorno, Vittorio; Mammucari, Massimo; Tufaro, Giuseppe; Lazzari, Marzia; Sabato, Alessandro Fabrizio

2013-03-01

Breakthrough pain (BTP) is traditionally defined as a transitory pain flare in opioid-treated patients with chronic background pain. This definition has, however, been challenged in recent years. This study aimed to analyze BTP prevalence in different pain conditions. This was a prospective, non-interventional, observational study conducted from June to September 2011 in two Italian pain treatment reference centres. Consecutive patients aged >18 years with oncological or non-oncological pain were eligible for this study; background pain was acute/ subacute (3 months). The characteristics of pain were evaluated by means of a structured interview by physicians, and patients were asked to complete a dedicated clinical study form. The following outcomes were assessed: chronic pain duration (in patients with chronic pain), BTP prevalence, and number and severity of daily BTP episodes. All outcomes were assessed in four populations of patients with: (a) chronic oncological pain; (b) chronic non-oncological pain; (c) non-chronic oncological pain; (d) non-chronic non-oncological pain. The correlation between BTP and gender was also investigated. Of 1,270 patients with chronic pain, 1,086 had non-oncological pain (85.5%). Most patients (68.6%) with non-oncological pain were female (P = 0.001). Pain duration was significantly longer in non-oncological pain versus oncological pain groups (P = 0.002). BTP prevalence was lower in non-oncological patients (P definition. BTP frequency and severity is similar in oncological and non-oncological pain.

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

Science.gov (United States)

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-08-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Both nuclear and cytoplasmic components are defective in oocytes of the B6.Y(TIR) sex-reversed female mouse.

Science.gov (United States)

Amleh, A; Smith, L; Chen, H; Taketo, T

2000-03-15

In the mammalian gonadal primordium, activation of the Sry gene on the Y chromosome initiates a cascade of genetic events leading to testicular organization whereas its absence results in ovarian differentiation. An exception occurs when the Y chromosome of Mus musculus domesticus from Tirano, Italy (Y(TIR)), is placed on the C57BL/6J (B6) genetic background. The B6.Y(TIR) progeny develop only ovaries or ovotestes despite Sry transcription in fetal life. Consequently, the XY offspring with bilateral ovaries develop into apparently normal females, but their eggs fail to develop after fertilization. Our previous studies have shown that the primary cause of infertility can be attributed to oocytes rather than their surrounding somatic cells in the XY ovary. This study attempted to identify the defects in oocytes from the B6.Y(TIR) female mouse. We examined the developmental potential of embryos from XY and XX females after exchanging their nuclear components by microsurgery following in vitro maturation and fertilization. The results suggest that both nuclear and cytoplasmic components are defective in oocytes from XY females. In the XY fetal ovary, most germ cells entered meiosis and their autosomes appeared to synapse normally while the X and Y chromosomes remained unpaired during meiotic prophase. This lack of X-Y pairing probably caused aneuploidy in some secondary oocytes following in vitro maturation. However, normal numbers of chromosomes in the rest of the secondary oocytes indicate that aneuploidy alone can not explain the nuclear defect in oocytes. Copyright 2000 Academic Press.

An Assessment of Polynomial Regression Techniques for the Relative Radiometric Normalization (RRN of High-Resolution Multi-Temporal Airborne Thermal Infrared (TIR Imagery

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Mir Mustafizur Rahman

2014-11-01

Full Text Available Thermal Infrared (TIR remote sensing images of urban environments are increasingly available from airborne and satellite platforms. However, limited access to high-spatial resolution (H-res: ~1 m TIR satellite images requires the use of TIR airborne sensors for mapping large complex urban surfaces, especially at micro-scales. A critical limitation of such H-res mapping is the need to acquire a large scene composed of multiple flight lines and mosaic them together. This results in the same scene components (e.g., roads, buildings, green space and water exhibiting different temperatures in different flight lines. To mitigate these effects, linear relative radiometric normalization (RRN techniques are often applied. However, the Earth’s surface is composed of features whose thermal behaviour is characterized by complexity and non-linearity. Therefore, we hypothesize that non-linear RRN techniques should demonstrate increased radiometric agreement over similar linear techniques. To test this hypothesis, this paper evaluates four (linear and non-linear RRN techniques, including: (i histogram matching (HM; (ii pseudo-invariant feature-based polynomial regression (PIF_Poly; (iii no-change stratified random sample-based linear regression (NCSRS_Lin; and (iv no-change stratified random sample-based polynomial regression (NCSRS_Poly; two of which (ii and iv are newly proposed non-linear techniques. When applied over two adjacent flight lines (~70 km2 of TABI-1800 airborne data, visual and statistical results show that both new non-linear techniques improved radiometric agreement over the previously evaluated linear techniques, with the new fully-automated method, NCSRS-based polynomial regression, providing the highest improvement in radiometric agreement between the master and the slave images, at ~56%. This is ~5% higher than the best previously evaluated linear technique (NCSRS-based linear regression.

Assessment of water vapor content from MIVIS TIR data

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V. Tramutoli

2006-06-01

Full Text Available The main objective of land remotely sensed images is to derive biological, chemical and physical parameters by inverting sample sets of spectral data. For the above aim hyperspectral scanners on airborne platform are a powerful remote sensing instrument for both research and environmental applications because of their spectral resolution and the high operability of the platform. Fine spectral information by MIVIS (airborne hyperspectral scanner operating in 102 channels ranging from VIS to TIR allows researchers to characterize atmospheric parameters and their effects on measured data which produce undesirable features on surface spectral signatures. These effects can be estimated (and remotely sensed radiances corrected if atmospheric spectral transmittance is known at each image pixel. Usually ground-based punctual observations (atmospheric sounding balloons, sun photometers, etc. are used to estimate the main physical parameters (like water vapor and temperature profiles which permit us to estimate atmospheric spectral transmittance by using suitable radiative transfer model and a specific (often too strong assumption which enable atmospheric properties measured only in very few points to be extended to the whole image. Several atmospheric gases produce observable absorption features, but only water vapor strongly varies in time and space. In this work the authors customize a self-sufficient «split-window technique» to derive (at each image pixel atmospheric total columnar water vapor content (TWVC using only MIVIS data collected by the fourth MIVIS spectrometer (Thermal Infrared band. MIVIS radiances have been simulated by means of MODTRAN4 radiative transfer code and the coefficients of linear regression to estimate TWVC from «split-windows» MIVIS radiances, based on 450 atmospheric water vapor profiles obtained by radiosonde data provided by NOAANESDIS. The method has been applied to produce maps describing the spatial variability of

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

Science.gov (United States)

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.

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Ricardo Núñez Miguel

2007-08-01

Full Text Available The Toll-like receptor 4 (TLR4 is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3 and nuclear factor kappaB (NFkappaB respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

OpenAIRE

Matsuda, M; Mayer, B J; Hanafusa, H

1991-01-01

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion o...

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway.

Science.gov (United States)

Mittal, Yash; Pavlova, Yelena; Garcia-Marcos, Mikel; Ghosh, Pradipta

2011-09-16

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.

Development, validation and psychometric properties of a diagnostic/prognostic tool for breakthrough pain in mixed chronic-pain patients.

Science.gov (United States)

Samolsky Dekel, Boaz Gedaliahu; Remondini, Francesca; Gori, Alberto; Vasarri, Alessio; Di Nino, GianFranco; Melotti, Rita Maria

2016-02-01

Breakthrough pain (BTP) shows variable prevalence in different clinical contexts of cancer and non-cancer patients. BTP diagnostic tools with demonstrated reliability, validation and prognostic capability are lacking. We report the development, psychometric and validation properties of a diagnostic/prognostic tool, the IQ-BTP, for BTP recognition, its likelihood and clinical features among chronic-pain (CP) patients. n=120 consecutive mixed cancer/non-cancer CP in/outpatients. Development, psychometric analyses and formal validation included: Face/Content validity (by 'experts' opinion and assessing the relationship between the IQ-BTP classes and criteria derived from BTP operational-case-definition); Construct validity, by Principle Component Analysis (PCA); and the strength of Spearman correlation between IQ-BTP classes and the Brief Pain Inventory (BPI) items; Reliability, by Cronbach's alpha statistics. Associations with clinical/demographic moderators were assessed applying χ(2) analysis. Potential-BTP was found in 36.7% of patients (38.4% of non-cancer and 32.4% of cancer patients). Among these the likelihood for BTP diagnosis was 'high' in 25%, 'intermediate' in 41% and, 'low' 34% of patients. Analyses showed significant differences between IQ-BTP classes and between the latter BPI pain-item scores. Correlation between IQ-BTP classes and BPI items was moderate. PCA and scree test identified 3 components accounting for 62.3% of the variance. Cronbach's alpha was 0.71. The IQ-BTP showed satisfactory psychometric and validation properties. With adequate feasibility it enabled the allocating of cancer/non-cancer CP patients in three prognostic classes. Results are sufficient to warrant a subsequent impact study of the IQ-BTP as prognostic model and screening tool for BTP in both CP populations. Copyright © 2016 Elsevier B.V. All rights reserved.

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs.

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Arieti, Fabiana; Gabus, Caroline; Tambalo, Margherita; Huet, Tiphaine; Round, Adam; Thore, Stéphane

2014-06-01

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs-the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

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Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

2012-03-15

The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

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Hidenori Ohnishi

Full Text Available MyD88, a Toll/interleukin-1 receptor homology (TIR domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

Room temperature phosphorescence study on the structural flexibility of single tryptophan containing proteins

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Kowalska-Baron, Agnieszka; Gałęcki, Krystian; Wysocki, Stanisław

2015-01-01

In this study, we have undertaken efforts to find correlation between phosphorescence lifetimes of single tryptophan containing proteins and some structural indicators of protein flexibility/rigidity, such as the degree of tryptophan burial or its exposure to solvent, protein secondary and tertiary structure of the region of localization of tryptophan as well as B factors for tryptophan residue and its immediate surroundings. Bearing in mind that, apart from effective local viscosity of the protein/solvent matrix, the other factor that concur in determining room temperature tryptophan phosphorescence (RTTP) lifetime in proteins is the extent of intramolecular quenching by His, Cys, Tyr and Trp side chains, the crystallographic structures derived from the Brookhaven Protein Data Bank were also analyzed concentrating on the presence of potentially quenching amino acid side chains in the close proximity of the indole chromophore. The obtained results indicated that, in most cases, the phosphorescence lifetimes of tryptophan containing proteins studied tend to correlate with the above mentioned structural indicators of protein rigidity/flexibility. This correlation is expected to provide guidelines for the future development of phosphorescence lifetime-based method for the prediction of structural flexibility of proteins, which is directly linked to their biological function.

Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors.

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Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-06-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

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Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

2010-01-01

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Deleted in Malignant Brain Tumors-1 Protein (DMBT1: A Pattern Recognition Receptor with Multiple Binding Sites

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Enno C. I. Veerman

2010-12-01

Full Text Available Deleted in Malignant Brain Tumors-1 protein (DMBT1, salivary agglutinin (DMBT1SAG, and lung glycoprotein-340 (DMBT1GP340 are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW. Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

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Cândida F Pereira

Full Text Available Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1 structural proteins (matrix, capsid and nucleocapsid, enzymes (protease, reverse transcriptase, RNAse H and integrase and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

Ternary WD40 repeat-containing protein complexes: evolution, composition and roles in plant immunity

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Jimi C. Miller

2016-01-01

Full Text Available Plants, like mammals, rely on their innate immune system to perceive and discriminate among the majority of their microbial pathogens. Unlike mammals, plants respond to this molecular dialogue by unleashing a complex chemical arsenal of defense metabolites to resist or evade pathogen infection. In basal or non-host resistance, plants utilize signal transduction pathways to detect non-self, damaged-self and altered-self-associated molecular patterns and translate these danger signals into largely inducible chemical defenses. The WD40 repeat (WDR-containing proteins Gβ and TTG1 are constituents of two independent ternary protein complexes functioning at opposite ends of a plant immune signaling pathway. Gβ and TTG1 are also encoded by single-copy genes that are ubiquitous in higher plants, implying the limited diversity and functional conservation of their respective complexes. In this review, we summarize what is currently known about the evolutionary history of these WDR-containing ternary complexes, their repertoire and combinatorial interactions, and their downstream effectors and pathways in plant defense.

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

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Wieczorek Andrew S

2012-12-01

Full Text Available Abstract Background The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. Results We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. Conclusions The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

DEFF Research Database (Denmark)

Reddy, Madhava C; Christensen, Jesper; Vasquez, Karen M

2005-01-01

-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA......), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so...

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7.

Science.gov (United States)

Roe, Andrew J; Naylor, Stuart W; Spears, Kevin J; Yull, Helen M; Dransfield, Tracy A; Oxford, Matthew; McKendrick, Iain J; Porter, Megan; Woodward, Martin J; Smith, David G E; Gally, David L

2004-10-01

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Orai1 mediates exacerbated Ca(2+ entry in dystrophic skeletal muscle.

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Xiaoli Zhao

Full Text Available There is substantial evidence indicating that disruption of Ca(2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD. However, the exact nature of the Ca(2+ deregulation and the Ca(2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca(2+ entry (SOCE for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca(2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca(2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca(2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca(2+ store contributes to the disrupted Ca(2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of

Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity

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Simon, Joseph R.; Carroll, Nick J.; Rubinstein, Michael; Chilkoti, Ashutosh; López, Gabriel P.

2017-06-01

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

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Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

2015-08-28

The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

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Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors

DEFF Research Database (Denmark)

Le Douarin, B; You, J; Nielsen, Anders Lade

1998-01-01

Ligand-induced gene activation by nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs), that interact with their ligand-dependent AF-2 activating domain. Included in the group of the putative AF-2 TIFs identified so far is TIF1alpha, a member of a new...... family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein......-protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP...

Co-localisation of advanced glycation end products and D-β-aspartic acid-containing proteins in gelatinous drop-like corneal dystrophy.

Science.gov (United States)

Kaji, Yuichi; Oshika, Tetsuro; Takazawa, Yutaka; Fukayama, Masashi; Fujii, Noriko

2012-08-01

Gelatinous drop-like corneal dystrophy (GDLD), also known as familial subepithelial corneal amyloidosis, is an autosomal recessive disorder that causes progressive corneal opacity due to accumulation of amyloid fibrils in the corneal stroma. Genetic analyses have revealed that a mutation in membrane component chromosome 1 surface marker 1 gene is responsible for GDLD. However, the mechanism of amyloid formation in the corneal stroma remains unclear. The present study attempted to reveal the role of advanced glycation end products (AGE) and d-amino acids in amyloid formation in GDLD. Informed consent was obtained from five patients with GDLD, three patients with bullous keratopathy and three patients with interstitial keratitis and all the specimens were analysed. Localisation of amyloid fibrils was analysed using Congo-red and thioflavin T staining. In addition, the localisation of AGE (N(ε)-carboxy(methyl)-L-lysine, pyrraline and pentosidine) and D-β-aspartic acid-containing proteins, a major form of d-amino acid-containing proteins, was analysed immunohistochemically. In all GDLD specimens, strong immunoreactivity to AGE and D-β-aspartic acid-containing proteins was detected in the subepithelial amyloid-rich region. In contrast, amyloid fibrils, AGE, or D-amino acid-containing proteins were slightly detected in the corneal stroma of patients with bullous keratopathy and interstitial keratitis. Abnormally accumulated proteins rich in AGE and D-β-aspartic acid co-localise in the amyloid lesions in GDLD. These results indicate that non-enzymatic post-translational modifications of proteins, including AGE formation and isomerisation of aspartyl residues, will be the cause as well as the result of amyloid fibril formations in GDLD.

Repeat-containing protein effectors of plant-associated organisms

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Carl H. Mesarich

2015-10-01

Full Text Available Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Lactobacillus bulgaricus Fajlarının Restriksiyon Fragment Uzunluk Polimorfizmi, Protein Profilleri ve Konakçı Özgüllüklerine Göre Tanımlanması

OpenAIRE

Soykut, Esra Acar; Tunail, Nezihe

2014-01-01

Lactobacillus delbrueckii subsp. bulgaricus suşlarına etkili 19 adet faj, restriksiyon fragment uzunluk polimorfizmi (RFLP), yapısal protein şablonları ve konakçı spektrumlarına göre tanımlanmış ve sınıflandırılmıştır. Fajlar, farklı süt ürünlerinden L. bulgaricus starter kültürleri (Y4, V1 ve V2) ile izole edilmiştir. Konakçı spektrumunun belirlenmesi için; doğal ve ticari L. bulgaricus suşlarının L. bulgaricus V1 ve V2 fajları ile S. thermophilus B3 fajlarına duyarlılıkları test edilmiştir....

TRDistiller: a rapid filter for enrichment of sequence datasets with proteins containing tandem repeats.

Science.gov (United States)

Richard, François D; Kajava, Andrey V

2014-06-01

The dramatic growth of sequencing data evokes an urgent need to improve bioinformatics tools for large-scale proteome analysis. Over the last two decades, the foremost efforts of computer scientists were devoted to proteins with aperiodic sequences having globular 3D structures. However, a large portion of proteins contain periodic sequences representing arrays of repeats that are directly adjacent to each other (so called tandem repeats or TRs). These proteins frequently fold into elongated fibrous structures carrying different fundamental functions. Algorithms specific to the analysis of these regions are urgently required since the conventional approaches developed for globular domains have had limited success when applied to the TR regions. The protein TRs are frequently not perfect, containing a number of mutations, and some of them cannot be easily identified. To detect such "hidden" repeats several algorithms have been developed. However, the most sensitive among them are time-consuming and, therefore, inappropriate for large scale proteome analysis. To speed up the TR detection we developed a rapid filter that is based on the comparison of composition and order of short strings in the adjacent sequence motifs. Tests show that our filter discards up to 22.5% of proteins which are known to be without TRs while keeping almost all (99.2%) TR-containing sequences. Thus, we are able to decrease the size of the initial sequence dataset enriching it with TR-containing proteins which allows a faster subsequent TR detection by other methods. The program is available upon request. Copyright © 2014 Elsevier Inc. All rights reserved.

The contribution of Landsat 8 TIRS sensor data to the identification of plastic covered vineyards

Science.gov (United States)

Novelli, Antonio; Tarantino, Eufemia

2015-06-01

Plastic covering is a common practice in agricultural fields. From an agronomic point of view, plastic coverings offer many advantages against unfavourable growing conditions. This explains their widespread utilization with consequent positive impact on local economy. On the other hand, plasticulture raises both environmental and landscape issues. In the Apulia Region (Italy) the wide implementation of such practice generally relates to vineyard cultivation. Continuous vineyard protection has resulted in negative effects on the hydrogeological balance of soils, causing a deep modification of the traditional rural landscape and therefore affecting its quality. To guarantee both the protection of local economy as well as the preservation of local environment and landscape features, a detailed site mapping of the areas involved is necessary. Indeed, the quantification of this phenomenon is essential in the periodic updating of the existing land use database and in the development of local policies. In this study we evaluate the potential of the novel Thermal Infrared Sensor bands (TIRS) provided by the LANDSAT 8 mission in plasticulture discrimination. Using the evident anomaly retrieved in the study area on the Quality Assessment (QA) band, a fast procedure involving TIRS data was developed, proposing a new index (Plastic Surface Index- PSI) able to emphasize plasticulture. For the aim of this study, two different acquisition dates on a test area in the Apulia region (Italy) were analyzed, one in the growing season with high plastic covering density and one in the post-harvest period with low plastic cover density.

BRCA1 interacts directly with the Fanconi anemia protein FANCA.

Science.gov (United States)

Folias, Alexandra; Matkovic, Mara; Bruun, Donald; Reid, Sonja; Hejna, James; Grompe, Markus; D'Andrea, Alan; Moses, Robb

2002-10-01

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.

Muscle and liver protein synthesis in growing rats fed diets containing raw legumes as the main source of protein

International Nuclear Information System (INIS)

Goena, M.; Santidrian, S.; Cuevillas, F.; Larralde, J.

1986-01-01

Although legumes are widely used as protein sources, their effects on protein metabolism remain quite unexplored. The authors have measured the rates of gastrocnemius muscle and liver protein synthesis in growing rats fed ad libitum over periods of 12 days on diets containing raw field bean (Vicia faba L.), raw kidney bean (Phaseolus vulgaris L.), and raw bitter vetch (Vicia ervilia L.) as the major sources of protein. Diets were isocaloric and contained about 12% protein. Protein synthesis was evaluated by the constant-intravenous-infusion method, using L-/ 14 C/-tyrosine, as well as by the determination of the RNA-activity (g of newly synthesized protein/day/g RNA). Results showed that, as compared to well-fed control animals, those fed the raw legume diets exhibited a marked reduction in the rate of growth with no changes in the amount of food intake (per 100 g b.wt.). These changes were accompanied by a significant reduction in the rate of muscle protein synthesis in all legume-treated rats, being this reduction greater in the animals fed the Ph. vulgaris and V. ervilia diets. Liver protein synthesis was slightly higher in the rats fed the V. faba and V. ervilia diets, and smaller in the Ph. vulgaris-fed rats. It is suggested that both sulfur amino acid deficiency and the presence of different anti-nutritive factors in raw legumes may account for these effects

Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells▿

Science.gov (United States)

Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-01-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions. PMID:18684830

Different types of nsP3-containing protein complexes in Sindbis virus-infected cells.

Science.gov (United States)

Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-10-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

International Nuclear Information System (INIS)

López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

2014-01-01

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export

Comparative in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and selected polynuclear aromatic hydrocarbons on cyp1a1 gene transcription in cells which contain or are deficient in the 4S binding protein

International Nuclear Information System (INIS)

Kamps, C.; Safe, S.

1990-01-01

Using [ 3 H]-benzo[a]pyrene as the radioligand, several cell culture lines have been screened for the presence (or absence) of the 4S binding protein. Murine Hepa 1c1c7 cells contained both the 4S binding protein and the 9S (Ah) receptor whereas only the 9S receptor was detected in rat hepatoma H-4-II E cells in culture. The effects of a series of polynuclear aromatic hydrocarbons (PAHs) which included benzo[e]pyrene, benzo[ghi]perylene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and their interactive effects on CYP1A1 gene transcription was determined by Northern analysis in both cell lines. The results showed that the PAHs which exhibited high affinity for the 4S binding protein were inactive as inducers in both cell lines; TCDD was active in both cell lines and the interactive effects between the PAHs and TCDD did not significantly modulate TCDD-mediated CYP1A1 gene transcription. The results suggest that the 4S binding protein does not regulate CYP1A1 gene transcription

Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

Science.gov (United States)

Beilin, Orit; Karussis, Dimitrios M; Korczyn, Amos D; Gurwitz, David; Aronovich, Ramona; Mizrachi-Kol, Rachel; Chapman, Joab

2007-04-16

Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1.

Science.gov (United States)

Brown, James R; Conn, Kristen L; Wasson, Peter; Charman, Matthew; Tong, Lily; Grant, Kyle; McFarlane, Steven; Boutell, Chris

2016-07-01

Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML

Understanding overpressure in the FAA aerosol can test by C3H2F3Br (2-BTP).

Science.gov (United States)

Linteris, Gregory Thomas; Babushok, Valeri Ivan; Pagliaro, John Leonard; Burgess, Donald Raymond; Manion, Jeffrey Alan; Takahashi, Fumiaki; Katta, Viswanath Reddy; Baker, Patrick Thomas

2016-05-01

Thermodynamic equilibrium calculations, as well as perfectly-stirred reactor (PSR) simulations with detailed reaction kinetics, are performed for a potential halon replacement, C 3 H 2 F 3 Br (2-BTP, C 3 H 2 F 3 Br, 2-Bromo-3,3,3-trifluoropropene), to understand the reasons for the unexpected enhanced combustion rather than suppression in a mandated FAA test. The high pressure rise with added agent is shown to depend on the amount of agent, and is well-predicted by an equilibrium model corresponding to stoichiometric reaction of fuel, oxygen, and agent. A kinetic model for the reaction of C 3 H 2 F 3 Br in hydrocarbon-air flames has been applied to understand differences in the chemical suppression behavior of C 3 H 2 F 3 Br vs. CF 3 Br in the FAA test. Stirred-reactor simulations predict that in the conditions of the FAA test, the inhibition effectiveness of C 3 H 2 F 3 Br at high agent loadings is relatively insensitive to the overall stoichiometry (for fuel-lean conditions), and the marginal inhibitory effect of the agent is greatly reduced, so that the mixture remains flammable over a wide range of conditions. Most important, the flammability of the agent-air mixtures themselves (when compressively preheated), can support low-strain flames which are much more difficult to extinguish than the easy-to extinguish, high-strain primary fireball from the impulsively released fuel mixture. Hence, the exothermic reaction of halogenated hydrocarbons in air should be considered in other situations with strong ignition sources and low strain flows, especially at preheated conditions.

Safety evaluation of an IPP tripeptide-containing milk protein hydrolysate

NARCIS (Netherlands)

Ponstein-Simarro Doorten, A.Y.; Wiel, J.A.G. van de; Jonker, D.

2009-01-01

Tensguard™ is a milk protein hydrolysate containing the lactotripeptide IPP. It is derived from cow's milk, which is present in the human diet and has a safe history of consumption. The final Tensguard™ product, a supplement or a functional food ingredient, is intended for use by people who want to

Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi

Directory of Open Access Journals (Sweden)

N. Leulliot

2013-01-01

Full Text Available Pyrococcus abyssi virus 1 (PAV1 was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23 that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137 at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

The mycobacterial DNA-binding protein 1 (MDP1 from Mycobacterium bovis BCG influences various growth characteristics

Directory of Open Access Journals (Sweden)

Maurischat Sven

2008-06-01

Full Text Available Abstract Background Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1 has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene. Results The expression rate of the MDP1 protein in the recombinant M. bovis BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain M. bovis BCG containing the empty vector. In comparison to this reference strain, the recombinant M. bovis BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain. Conclusion The MDP1 protein has a major impact on various growth characteristics of M. bovis BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.

ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein-protein interactions with HPRT1.

Science.gov (United States)

Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S; Jackson, Brian C; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A; Johnson, Richard J; Koppaka, Vindhya; Thompson, David C

2013-02-25

Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

UCH-L1-containing exosomes mediate chemotherapeutic resistance transfer in breast cancer.

Science.gov (United States)

Ning, Kuan; Wang, Teng; Sun, Xu; Zhang, Pengfei; Chen, Yun; Jin, Jian; Hua, Dong

2017-06-01

Chemotherapy resistance has become a serious challenge in the treatment of breast cancer. Previous studies showed cells can transfer proteins, including those responsible for drug resistance to adjacent cells via exosomes. The switches of drug resistance via exosomes transfer were assessed by CellTiter-Blue Viability assay, flow cytometry, and immunostaining analysis. Relative protein levels of Ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1), P-glycoprotein (P-gp), extracellular-signal regulated protein kinase1/2 (ERK1/2), and phospho-extracellular-signal regulated protein kinase1/2 (p-ERK1/2) were measured by Western blot. Immunohistochemistry was performed on 93 breast cancer samples to assess the associations of UCH-L1 levels with immunofluorescence value of UCH-L1 in circulating exosomes. The Adriamycin-resistant human breast cancer cells (MCF7/ADM) secreted exosomes carrying UCH-L1 and P-gp proteins into the extracellular microenvironment then integrated into Adriamycin-sensitive human breast cancer cells (MCF7/WT) in a time-dependent manner, transferring the chemoresistance phenotype. Notably, in blood samples from patients with breast cancer, the level of exosomes carrying UCH-L1 before chemotherapy was significantly negatively correlated with prognosis. Our study demonstrated that UCH-L1-containing exosomes can transfer chemoresistance to recipient cells and these exosomes may be useful as non-invasive diagnostic biomarkers for detection of chemoresitance in breast cancer patients, achieving more effective and individualized chemotherapy. © 2017 Wiley Periodicals, Inc.

Status of waste form testing

International Nuclear Information System (INIS)

Lawroski, H.

1984-01-01

The promulgation of the amendment of 10 CFR Part 61 by the Nuclear Regulatory Commission of December 27, 1982 by Federal Register Notice with an effective date of December 27, 1983 established the criteria for licensing requirements, paragraph 60.56, contained the description to provide adequate stability of the site through the use of suitable waste forms. In May, 1983, the NRC published a final Branch Technical Position (BTP) paper on waste form. The position taken by the BTP was considerably more severe than indicated in 10 CFR Part 61. An extensive and expensive testing program was started in 1983. As an interim measure, the presently utilized solidification processes such as cement, Dow binder, Envirostone and bitumen, and the presently qualified High Integrity containers (HICs) were considered acceptable with the caveat that acceptable process control programs were being utilized. The NRC requested that topical reports for licenses be submitted. The topical reports were to contain test results to substantiate the acceptability of the waste forms. The test results to date show that the volume of wastes will have to increase to meet the position taken by the NRC in the BTP. This position will cause more waste to be generated which is contrary to the emphasis by states and others to reduce the volume of waste. The details of testing will be discussed in the paper to be presented

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain.

Science.gov (United States)

Rust, K; Grosso, L; Zhang, V; Chang, D; Persson, A; Longmore, W; Cai, G Z; Crouch, E

1991-10-01

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.

Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300).

OpenAIRE

Shetty, S; Takahashi, T; Matsui, H; Ayengar, R; Raghow, R

1999-01-01

The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We de...

The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

Science.gov (United States)

Chen, Kelan; Dobson, Renwick C J; Lucet, Isabelle S; Young, Samuel N; Pearce, F Grant; Blewitt, Marnie E; Murphy, James M

2016-06-15

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

Energy Technology Data Exchange (ETDEWEB)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

2014-06-15

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

International Nuclear Information System (INIS)

Lalime, Erin N.; Pekosz, Andrew

2014-01-01

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

Science.gov (United States)

Nishimura, Akiko; Yamamoto, Katsuyoshi; Oyama, Masaaki; Kozuka-Hata, Hiroko

2016-01-01

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1+ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1. PMID:26787842

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export*

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Port, Sarah A.; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H.

2016-01-01

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. PMID:27613868

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

Science.gov (United States)

Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

2016-10-28

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) + RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) + RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

DEFF Research Database (Denmark)

Juncker, Agnieszka; Larsen, Mette Voldby; Weinhold, Nils

2009-01-01

Background: Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable. Methodology/Principal Finding: In the present large......-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly...

Monocyte chemoattractant protein-1 (MCP-1 regulates macrophage cytotoxicity in abdominal aortic aneurysm.

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Qiwei Wang

Full Text Available AIMS: In abdominal aortic aneurysm (AAA, macrophages are detected in the proximity of aortic smooth muscle cells (SMCs. We have previously demonstrated in a murine model of AAA that apoptotic SMCs attract monocytes and other leukocytes by producing MCP-1. Here we tested whether infiltrating macrophages also directly contribute to SMC apoptosis. METHODS AND RESULTS: Using a SMC/RAW264.7 macrophage co-culture system, we demonstrated that MCP-1-primed RAWs caused a significantly higher level of apoptosis in SMCs as compared to control macrophages. Next, we detected an enhanced Fas ligand (FasL mRNA level and membrane FasL protein expression in MCP-1-primed RAWs. Neutralizing FasL blocked SMC apoptosis in the co-culture. In situ proximity ligation assay showed that SMCs exposed to primed macrophages contained higher levels of receptor interacting protein-1 (RIP1/Caspase 8 containing cell death complexes. Silencing RIP1 conferred apoptosis resistance to SMCs. In the mouse elastase injury model of aneurysm, aneurysm induction increased the level of RIP1/Caspase 8 containing complexes in medial SMCs. Moreover, TUNEL-positive SMCs in aneurysmal tissues were frequently surrounded by CD68(+/FasL(+ macrophages. Conversely, elastase-treated arteries from MCP-1 knockout mice display a reduction of both macrophage infiltration and FasL expression, which was accompanied by diminished apoptosis of SMCs. CONCLUSION: Our data suggest that MCP-1-primed macrophages are more cytotoxic. MCP-1 appears to modulate macrophage cytotoxicity by increasing the level of membrane bound FasL. Thus, we showed that MCP-1-primed macrophages kill SMCs through a FasL/Fas-Caspase8-RIP1 mediated mechanism.

Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

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Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

2010-01-01

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

An endoplasmic reticulum-localized Coffea arabica BURP domain-containing protein affects the response of transgenic Arabidopsis plants to diverse abiotic stresses.

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Dinh, Sy Nguyen; Kang, Hunseung

2017-11-01

The Coffea arabica BURP domain-containing gene plays an important role in the response of transgenic Arabidopsis plants to abiotic stresses via regulating the level of diverse proteins. Although the functions of plant-specific BURP domain-containing proteins (BDP) have been determined for a few plants, their roles in the growth, development, and stress responses of most plant species, including coffee plant (Coffea arabica), are largely unknown. In this study, the function of a C. arabica BDP, designated CaBDP1, was investigated in transgenic Arabidopsis plants. The expression of CaBDP1 was highly modulated in coffee plants subjected to drought, cold, salt, or ABA. Confocal analysis of CaBDP1-GFP fusion proteins revealed that CaBDP1 is localized in the endoplasmic reticulum. The ectopic expression of CaBDP1 in Arabidopsis resulted in delayed germination of the transgenic plants under abiotic stress and in the presence of ABA. Cotyledon greening and seedling growth of the transgenic plants were inhibited in the presence of ABA due to the upregulation of ABA signaling-related genes like ABI3, ABI4, and ABI5. Proteome analysis revealed that the levels of several proteins are modulated in CaBDP1-expressing transgenic plants. The results of this study underscore the importance of BURP domain proteins in plant responses to diverse abiotic stresses.

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

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Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

Analysis of Thioester-Containing Proteins during the Innate Immune Response of Drosophila melanogaster

Science.gov (United States)

Bou Aoun, Richard; Hetru, Charles; Troxler, Laurent; Doucet, Daniel; Ferrandon, Dominique; Matt, Nicolas

2010-01-01

Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1–Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila. PMID:21063077

α/β-hydrolase domain containing protein 15 (ABHD15--an adipogenic protein protecting from apoptosis.

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Evelyn Walenta

Full Text Available Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15 is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ, the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

Identification of a novel protein complex containing ASIC1a and GABAA receptors and their interregulation.

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Dongbo Zhao

Full Text Available Acid-sensing ion channels (ASICs belong to the family of the epithelial sodium channel/degenerin (ENaC/DEG and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASICs were modified by the activation of γ-aminobutyric acid receptors (GABAA, a ligand-gated chloride channels, in hippocampal neurons. In contrast, the activity of GABAA receptors were also modulated by extracellular pH. However so far, the mechanisms underlying this intermodulation remain obscure. We hypothesized that these two receptors-GABAA receptors and ASICs channels might form a novel protein complex and functionally interact with each other. In the study reported here, we found that ASICs were modified by the activation of GABAA receptors either in HEK293 cells following transient co-transfection of GABAA and ASIC1a or in primary cultured dorsal root ganglia (DRG neurons. Conversely, activation of ASIC1a also modifies the GABAA receptor-channel kinetics. Immunoassays showed that both GABAA and ASIC1a proteins were co-immunoprecipitated mutually either in HEK293 cells co-transfected with GABAA and ASIC1a or in primary cultured DRG neurons. Our results indicate that putative GABAA and ASIC1a channels functionally interact with each other, possibly via an inter-molecular association by forming a novel protein complex.

TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1

International Nuclear Information System (INIS)

Wang Yuequn; Li Yongqing; Qi Xinzhu; Yuan Wuzhou; Ai Jianping; Zhu Chuanbing; Cao Lei; Yang Hong; Liu Fang; Wu Xiushan; Liu Mingyao

2004-01-01

The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted α-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway

The Effect of Temperature on Protein and Amino Acid Composition of Spirulina platensis.

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Leyla Hızarcı Uslu

2015-12-01

Full Text Available Sıcaklığın Spirulina platensis’in protein ve amino asit kompozisyonu üzerine etkisi. Bu çalışma, mevsime bağlı iklim değişikliğinin yaz ve kış mevsimlerinde kültüre alınan Spirulina platensis’in protein içeriği ve amino asit kompozisyonuna etkilerini incelemek amacıyla yürütülmüştür. Çalışma boyunca ışık yoğunluğu, pH ve tuzluluk günlük olarak ölçülmüş, sıcaklık ve çözünmüş oksijen gün boyunca ve gece ölçülmüştür. Yaz ve kış mevsiminde ortalama gündüz sıcaklığı sırasıyla 33,9±0,4 ºC ve 18,6±0,5 ºC olarak belirlenirken, gece sıcaklığı 29,9±0,2 °C ve 14.4±0.2°C olarak belirlenmiştir. Ortalama ışık yoğunluğu yaz mevsiminde 848,3 µmol m-2s-1 iken kışın 506,26±48 µmol m-2s-1 olmuştur. Yaz büyüme döneminde protein miktarı (72,9±03 % ve amino asit konsantrasyonu kış dönemine göre daha yüksek bulunmuştur. Amino asitlerden Prolin, Sistin ve Arjinin sadece kış mevsiminde gözlenmiştir

Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity

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Bolhaar Suzanne THP

2008-11-01

Full Text Available Abstract Background Mal d 1 is a major apple allergen causing food allergic symptoms of the oral allergy syndrome (OAS in birch-pollen sensitised patients. The Mal d 1 gene family is known to have at least 7 intron-containing and 11 intronless members that have been mapped in clusters on three linkage groups. In this study, the allelic diversity of the seven intron-containing Mal d 1 genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. Protein variant constitutions were subsequently compared with Skin Prick Test (SPT responses to study the association of deduced protein variants with allergenicity in a set of 14 cultivars. Results From the seven intron-containing Mal d 1 genes investigated, Mal d 1.01 and Mal d 1.02 were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. Mal d 1.04, Mal d 1.05 and Mal d 1.06 A, B and C were more variable, coding for three to six different protein variants. Comparison of Mal d 1 allelic composition between the high-allergenic cultivar Golden Delicious and the low-allergenic cultivars Santana and Priscilla, which are linked in pedigree, showed an association between the protein variants coded by the Mal d 1.04 and -1.06A genes (both located on linkage group 16 with allergenicity. This association was confirmed in 10 other cultivars. In addition, Mal d 1.06A allele dosage effects associated with the degree of allergenicity based on prick to prick testing. Conversely, no associations were observed for the protein variants coded by the Mal d 1.01 (on linkage group 13, -1.02, -1.06B, -1.06C genes (all on linkage group 16, nor by the Mal d 1.05 gene (on linkage group 6. Conclusion Protein variant compositions of Mal d 1.04 and -1.06A and, in case of Mal d 1.06A, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. This information

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

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Ana Crnković

2018-02-01

Full Text Available Synthesis of proteins with noncanonical amino acids (ncAAs enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs and tRNAs (o-tRNAs. Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep. The incorporation of Sep into a green fluorescent protein (GFP in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

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Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

Understanding overpressure in the FAA aerosol can test by C3H2F3Br (2-BTP)✩

Science.gov (United States)

Linteris, Gregory Thomas; Babushok, Valeri Ivan; Pagliaro, John Leonard; Burgess, Donald Raymond; Manion, Jeffrey Alan; Takahashi, Fumiaki; Katta, Viswanath Reddy; Baker, Patrick Thomas

2018-01-01

Thermodynamic equilibrium calculations, as well as perfectly-stirred reactor (PSR) simulations with detailed reaction kinetics, are performed for a potential halon replacement, C3H2F3Br (2-BTP, C3H2F3Br, 2-Bromo-3,3,3-trifluoropropene), to understand the reasons for the unexpected enhanced combustion rather than suppression in a mandated FAA test. The high pressure rise with added agent is shown to depend on the amount of agent, and is well-predicted by an equilibrium model corresponding to stoichiometric reaction of fuel, oxygen, and agent. A kinetic model for the reaction of C3H2F3Br in hydrocarbon-air flames has been applied to understand differences in the chemical suppression behavior of C3H2F3Br vs. CF3Br in the FAA test. Stirred-reactor simulations predict that in the conditions of the FAA test, the inhibition effectiveness of C3H2F3Br at high agent loadings is relatively insensitive to the overall stoichiometry (for fuel-lean conditions), and the marginal inhibitory effect of the agent is greatly reduced, so that the mixture remains flammable over a wide range of conditions. Most important, the flammability of the agent-air mixtures themselves (when compressively preheated), can support low-strain flames which are much more difficult to extinguish than the easy-to extinguish, high-strain primary fireball from the impulsively released fuel mixture. Hence, the exothermic reaction of halogenated hydrocarbons in air should be considered in other situations with strong ignition sources and low strain flows, especially at preheated conditions. PMID:29628525

A Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site

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Hosseinzadeh, Parisa; Tian, Shiliang; Marshall, Nicholas M.; Hemp, James; Mullen, Timothy; Nilges, Mark J.; Gao, Yi-Gui; Robinson, Howard; Stahl, David A.; Gennis, Robert B.; Lu, Yi

2016-05-25

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M^–1 cm^–1) and 558 nm (2290 M^–1 cm^–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10^–4 cm^–1, and another more rhombic, with a smaller A∥ value of 69 × 10^–4 cm^–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

Science.gov (United States)

Doucet, Alain; Williams, Martin; Gagnon, Mylene C; Sasseville, Maxime; Beauregard, Marc

2002-01-02

Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.

Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.

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Silvia Dragoni

Full Text Available BACKGROUND: An increase in the frequency of circulating endothelial colony forming cells (ECFCs, the only subset of endothelial progenitor cells (EPCs truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF. Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs and subjects suffering from renal cellular carcinoma (RCC-ECFCs. SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1. METHODOLOGY/PRINCIPAL FINDINGS: We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs. SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA or physiological (i.e. ATP stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective. CONCLUSIONS: Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3

The nucleotide sequence of human transition protein 1 cDNA

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Luerssen, H; Hoyer-Fender, S; Engel, W [Universitaet Goettingen (West Germany)

1988-08-11

The authors have screened a human testis cDNA library with an oligonucleotide of 81 mer prepared according to a part of the published nucleotide sequence of the rat transition protein TP 1. They have isolated a cDNA clone with the length of 441 bp containing the coding region of 162 bp for human transition protein 1. There is about 84% homology in the coding region of the sequence compared to rat. The human cDNA-clone encodes a polypeptide of 54 amino acids of which 7 are different to that of rat.

Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

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Yamakoshi, Takako [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Ur Rehman, Mati; Yoshihisa, Yoko [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Sugimori, Michiya [Department of Integrative Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Shimizu, Tadamichi, E-mail: shimizut@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan)

2013-03-01

Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

International Nuclear Information System (INIS)

Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

2013-01-01

Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes

Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

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Liu, Feng; Roth, Richard A.

1995-10-01

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

Development of a cloud-based Bioinformatics Training Platform.

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Revote, Jerico; Watson-Haigh, Nathan S; Quenette, Steve; Bethwaite, Blair; McGrath, Annette; Shang, Catherine A

2017-05-01

The Bioinformatics Training Platform (BTP) has been developed to provide access to the computational infrastructure required to deliver sophisticated hands-on bioinformatics training courses. The BTP is a cloud-based solution that is in active use for delivering next-generation sequencing training to Australian researchers at geographically dispersed locations. The BTP was built to provide an easy, accessible, consistent and cost-effective approach to delivering workshops at host universities and organizations with a high demand for bioinformatics training but lacking the dedicated bioinformatics training suites required. To support broad uptake of the BTP, the platform has been made compatible with multiple cloud infrastructures. The BTP is an open-source and open-access resource. To date, 20 training workshops have been delivered to over 700 trainees at over 10 venues across Australia using the BTP. © The Author 2016. Published by Oxford University Press.

Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

International Nuclear Information System (INIS)

Mak, Paul; Jaggi, Meena; Syed, Viqar; Chauhan, Subhash C.; Hassan, Sazzad; Biswas, Helal; Balaji, K.C.

2008-01-01

Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells

The spliceosome-associated protein Mfap1 binds to VCP in Drosophila.

Directory of Open Access Journals (Sweden)

Sandra Rode

Full Text Available Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to vcp mutations, Mfap1 overexpression causes c4da neuron dendrite pruning defects and mislocalization of TDP-43 in these cells, but genetic analyses show that Mfap1 is not a crucial VCP target during dendrite pruning. Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity.

Virus-like particles vaccine containing Clonorchis sinensis tegumental protein induces partial protection against Clonorchis sinensis infection.

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Lee, Dong-Hun; Kim, Ah-Ra; Lee, Su-Hwa; Quan, Fu-Shi

2017-12-29

Human clonorchiasis, caused by the infection of Clonorchis sinensis, is one of the major health problems in Southeast Asia. However, vaccine efficacy against C. sinensis infection remains largely unknown. In this study, for the first time, we generated virus-like particles (VLPs) vaccine containing the C. sinensis tegumental protein 22.3 kDa (CsTP 22.3) and the influenza matrix protein (M1) as a core protein, and investigated the vaccine efficacy in Sprague-Dawley rats. Intranasal immunization of VLPs vaccine induced C. sinensis-specific IgG, IgG2a and IgG2c in the sera and IgA responses in the feces and intestines. Notably, upon challenge infection with C. sinensis metacercariae, significantly lower adult worm loads (70.2%) were measured in the liver of rats immunized with VLPs, compared to those of naïve rats. Furthermore, VLPs immunization induced antibody secreting cells (ASC) responses and CD4+/CD8+ T cell responses in the spleen. Our results indicated that VLPs vaccine containing C. sinensis CsTP 22.3 kDa provided partial protection against C. sisnensis infection. Thus, VLPs could be a potential vaccine candidate against C. sinensis.

Evolution of light-harvesting complex proteins from Chl c-containing algae

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Puerta M Virginia

2011-04-01

Full Text Available Abstract Background Light harvesting complex (LHC proteins function in photosynthesis by binding chlorophyll (Chl and carotenoid molecules that absorb light and transfer the energy to the reaction center Chl of the photosystem. Most research has focused on LHCs of plants and chlorophytes that bind Chl a and b and extensive work on these proteins has uncovered a diversity of biochemical functions, expression patterns and amino acid sequences. We focus here on a less-studied family of LHCs that typically bind Chl a and c, and that are widely distributed in Chl c-containing and other algae. Previous phylogenetic analyses of these proteins suggested that individual algal lineages possess proteins from one or two subfamilies, and that most subfamilies are characteristic of a particular algal lineage, but genome-scale datasets had revealed that some species have multiple different forms of the gene. Such observations also suggested that there might have been an important influence of endosymbiosis in the evolution of LHCs. Results We reconstruct a phylogeny of LHCs from Chl c-containing algae and related lineages using data from recent sequencing projects to give ~10-fold larger taxon sampling than previous studies. The phylogeny indicates that individual taxa possess proteins from multiple LHC subfamilies and that several LHC subfamilies are found in distantly related algal lineages. This phylogenetic pattern implies functional differentiation of the gene families, a hypothesis that is consistent with data on gene expression, carotenoid binding and physical associations with other LHCs. In all probability LHCs have undergone a complex history of evolution of function, gene transfer, and lineage-specific diversification. Conclusion The analysis provides a strikingly different picture of LHC diversity than previous analyses of LHC evolution. Individual algal lineages possess proteins from multiple LHC subfamilies. Evolutionary relationships showed

Valosin containing protein (VCP) interacts with macrolide antibiotics without mediating their anti-inflammatory activities.

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Nujić, Krunoslav; Smith, Marjorie; Lee, Michael; Belamarić, Daniela; Tomašković, Linda; Alihodžić, Sulejman; Malnar, Ivica; Polančec, Denis; Schneider, Klaus; Eraković Haber, Vesna

2012-02-29

In addition to antibacterial activity, some macrolide antibiotics, such as azithromycin and clarithromycin, also exhibit anti-inflammatory properties in vitro and in vivo, although the targets and mechanism(s) of action remain unknown. The aim of the present study was to identify protein targets of azithromycin and clarithromycin which could potentially explain their anti-inflammatory effects. Using chemical proteomics approach, based on compound-immobilized affinity chromatography, valosin containing protein (VCP) was identified as a potential target of the macrolides. Validation studies confirmed the interaction of macrolides and VCP and gave some structural characteristics of this interaction. Cell based assays however, including the use of gene silencing and the study of VCP specific cellular functions in J774.A1 (murine macrophage) and IB3-1 (human cystic fibrotic epithelial) cell lines, failed to confirm an association between the binding of the macrolides to VCP and anti-inflammatory effects. These findings suggest the absence of an abundant high affinity protein target and the potential involvement of other biological molecules in the anti-inflammatory activity of macrolides. Copyright © 2011 Elsevier B.V. All rights reserved.

Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in spike 1 domain and membrane protein of feline infectious peritonitis virus.

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Takano, Tomomi; Morioka, Hiroyuki; Gomi, Kohji; Tomizawa, Keisuke; Doki, Tomoyoshi; Hohdatsu, Tsutomu

2014-04-01

Feline infectious peritonitis virus (FIP virus: FIPV) causes a fatal disease in wild and domestic cats. The development of an FIP-preventive vaccine requires an antigen that does not induce antibody-dependent enhancement, and T helper (Th)1 activity plays an important role in protect against FIPV infection. In the present study, we identified synthetic peptides including Th1 and a linear immunodominant antibody-binding epitope in the S1 domain and M protein of FIPV. We also identified peptides that strongly induce Th1 activity from those derived from the structural proteins (S, M, and N proteins) of FIPV based on this and previous studies (Satoh et al. [19]). No Th1 epitope-containing peptide was identified in the peptides derived from the S1 domain of type I FIPV. In contrast, 7 Th1 epitope-containing peptides were identified in the S1 domain of type II FIPV, and no linear immunodominant antibody-binding epitope was contained in any of these peptides. Eleven Th1 epitope-containing peptides common to each serotype were identified in the M protein-derived peptides, and 2 peptides (M-11 and M-12) contained the linear immunodominant antibody-binding epitope. Of the peptides derived from the S, M, and N proteins of FIPV, those that induced significantly stronger Th1 activity than that of the FIPV antigen were rescreened, and 4 peptides were identified. When 3 of these peptides (M-9, I-S2-15, and II-S1-24) were selected and administered with CpG-ODNs to SPF cats, M-9 and II-S1-24 induced Th1 activity. Our results may provide important information for the development of a peptide-based vaccine against FIPV infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Thionin-D4E1 chimeric protein protects plants against bacterial infections

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Stover, Eddie W; Gupta, Goutam; Hao, Guixia

2017-08-08

The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

quatre-quart1 is an indispensable U12 intron-containing gene that plays a crucial role in Arabidopsis development.

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Kwak, Kyung Jin; Kim, Bo Mi; Lee, Kwanuk; Kang, Hunseung

2017-05-17

Despite increasing understanding of the importance of the splicing of U12-type introns in plant development, the key question of which U12 intron-containing genes are essential for plant development has not yet been explored. Here, we assessed the functional role of the quatre-quart1 (QQT1) gene, one of the ~230 U12 intron-containing genes in Arabidopsis thaliana. Expression of QQT1 in the U11/U12-31K small nuclear ribonucleoprotein mutant (31k) rescued the developmental-defect phenotypes of the 31k mutant, whereas the miRNA-mediated qqt1 knockdown mutants displayed severe defects in growth and development, including severely arrested stem growth, small size, and the formation of serrated leaves. The structures of the shoot apical meristems in the qqt1 mutants were abnormal and disordered. Identification of QQT1-interacting proteins via a yeast two-hybrid screening and a firefly luciferase complementation-imaging assay revealed that a variety of proteins, including many chloroplast-targeted proteins, interacted with QQT1. Importantly, the levels of chloroplast-targeted proteins in the chloroplast were reduced, and the chloroplast structure was abnormal in the qqt1 mutant. Collectively, these results provide clear evidence that QQT1 is an indispensable U12 intron-containing gene whose correct splicing is crucial for the normal development of Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

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Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

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Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

2014-10-01

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

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Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

2010-12-03

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Tir8/Sigirr prevents murine lupus by suppressing the immunostimulatory effects of lupus autoantigens

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Lech, Maciej; Kulkarni, Onkar P.; Pfeiffer, Stephanie; Savarese, Emina; Krug, Anne; Garlanda, Cecilia; Mantovani, Alberto; Anders, Hans-Joachim

2008-01-01

The Sigirr gene (also known as Tir8) encodes for an orphan receptor of the Toll-like receptor (TLR)/interleukin 1 receptor family that inhibits TLR-mediated pathogen recognition in dendritic cells. Here, we show that Sigirr also inhibits the activation of dendritic cells and B cells upon exposure to RNA and DNA lupus autoantigens. To evaluate the functional role of Sigirr in the pathogenesis of systemic lupus erythematosus (SLE), we generated Sigirr-deficient C57BL/6-lpr/lpr mice. These mice developed a progressive lymphoproliferative syndrome followed by severe autoimmune lung disease and lupus nephritis within 6 mo of age as compared with the minor abnormalities observed in C57BL/6-lpr/lpr mice. Lack of Sigirr was associated with enhanced activation of dendritic cells and increased expression of multiple proinflammatory and antiapoptotic mediators. In the absence of Sigirr, CD4 T cell numbers were increased and CD4+CD25+ T cell numbers were reduced. Furthermore, lack of Sigirr enhanced the activation and proliferation of B cells, including the production of autoantibodies against multiple nuclear lupus autoantigens. These data identify Sigirr as a novel SLE susceptibility gene in mice. PMID:18644972

GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

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Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-06-01

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

The Disordered C-Terminus of Yeast Hsf1 Contains a Cryptic Low-Complexity Amyloidogenic Region

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Jordi Pujols

2018-05-01

Full Text Available Response mechanisms to external stress rely on networks of proteins able to activate specific signaling pathways to ensure the maintenance of cell proteostasis. Many of the proteins mediating this kind of response contain intrinsically disordered regions, which lack a defined structure, but still are able to interact with a wide range of clients that modulate the protein function. Some of these interactions are mediated by specific short sequences embedded in the longer disordered regions. Because the physicochemical properties that promote functional and abnormal interactions are similar, it has been shown that, in globular proteins, aggregation-prone and binding regions tend to overlap. It could be that the same principle applies for disordered protein regions. In this context, we show here that a predicted low-complexity interacting region in the disordered C-terminus of the stress response master regulator heat shock factor 1 (Hsf1 protein corresponds to a cryptic amyloid region able to self-assemble into fibrillary structures resembling those found in neurodegenerative disorders.

The Disordered C-Terminus of Yeast Hsf1 Contains a Cryptic Low-Complexity Amyloidogenic Region.

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Pujols, Jordi; Santos, Jaime; Pallarès, Irantzu; Ventura, Salvador

2018-05-06

Response mechanisms to external stress rely on networks of proteins able to activate specific signaling pathways to ensure the maintenance of cell proteostasis. Many of the proteins mediating this kind of response contain intrinsically disordered regions, which lack a defined structure, but still are able to interact with a wide range of clients that modulate the protein function. Some of these interactions are mediated by specific short sequences embedded in the longer disordered regions. Because the physicochemical properties that promote functional and abnormal interactions are similar, it has been shown that, in globular proteins, aggregation-prone and binding regions tend to overlap. It could be that the same principle applies for disordered protein regions. In this context, we show here that a predicted low-complexity interacting region in the disordered C-terminus of the stress response master regulator heat shock factor 1 (Hsf1) protein corresponds to a cryptic amyloid region able to self-assemble into fibrillary structures resembling those found in neurodegenerative disorders.

Critical electrolyte concentration of spermatozoal chromatin containing histone H1 variants

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J.R.P. Falco

1999-06-01

Full Text Available The critical electrolyte concentrations (CEC of sperm chromatin from animal species known or suspected to contain histone H1 variants were compared by examining the affinity of their DNA-protein complexes for toluidine blue in the presence of Mg2+. Bullfrog, sea urchin, bee and bumblebee spermatozoa were studied. The CEC for Rana catesbeiana and two sea urchin species were similar to that of histone H5-containing chromatin from chicken erythrocytes, thus confirming the biochemical and structural similarities of these DNA-protein complexes. The CEC for bees and the bumblebee, Bombus atratus, showed no particular phylogenetic relationship. We concluded that the CEC of histone H1-containing sperm cell chromatin is a useful indicator of variability in DNA-protein complexes but is of little phylogenetic value.Valores de concentração crítica de eletrólitos (CEC da cromatina de espermatozóides de espécies conhecidas ou suspeitas de apresentarem variantes da histona H1 foram comparados entre si. O objetivo foi estabelecer semelhanças ou diferenças nos complexos DNA-proteína de espermatozóides dessas espécies em nível citoquímico. A afinidade por moléculas de azul de toluidina em condições de competição com íons Mg2+ foi investigada nos espermatozóides do sapo boi e de ouriços do mar, abelhas e mamangava. Uma íntima relação entre os valores de CEC de Rana catesbeiana e de duas espécies de ouriço do mar com os da cromatina de eritrócitos de frango, que contém a histona H5, foi vista estar de acordo com certas semelhanças bioquímicas e estruturais entre seus complexos DNA-proteína. Quanto aos dados para abelhas e para a mamangava Bombus atratus, não se pôde associar a variabilidade em valores de CEC com a posição das espécies na respectiva árvore filogenética. Conclui-se, portanto, que a CEC de cromatina de espermatozóides que contêm histona H1 é um indicador útil da influência de variantes de H1 na organiza

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

Science.gov (United States)

Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

2014-11-01

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia: Mollusca).

Science.gov (United States)

Cavanaugh, C M; Abbott, M S; Veenhuis, M

1988-10-01

The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP(2)Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of RbuP(2)Case from tobacco (Nicotiana tabacum) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of S. velum. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of Thiobacillus neapolitanus, a free-living sulfuroxidizing chemoautotroph whose RbuP(2)Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the RbuP(2)Case activity.

A diet containing whey protein, glutamine, and TGFbeta modulates gut protein metabolism during chemotherapy-induced mucositis in rats.

Science.gov (United States)

Boukhettala, Nabile; Ibrahim, Ayman; Claeyssens, Sophie; Faure, Magali; Le Pessot, Florence; Vuichoud, Jacques; Lavoinne, Alain; Breuillé, Denis; Déchelotte, Pierre; Coëffier, Moïse

2010-08-01

Mucositis, a common side effect of chemotherapy, is characterized by compromised digestive function, barrier integrity and immune competence. Our aim was to evaluate the impact of a specifically designed diet Clinutren Protect (CP), which contains whey proteins, TGFbeta-rich casein, and free glutamine, on mucositis in rats. Mucositis was induced by three consecutive injections (day 0, day 1, day 2) of methotrexate (2.5 mg/kg). Rats had free access to CP or placebo diets from days -7 to 9. In the placebo diet, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein and glutamine by alanine. Intestinal parameters were assessed at day 3 and 9. Values, expressed as mean +/- SEM, were compared using two-way ANOVA. At day 3, villus height was markedly decreased in the placebo (296 +/- 11 microm) and CP groups (360 +/- 10 microm) compared with controls (464 +/- 27 microm), but more markedly in the placebo as compared to CP group. The intestinal damage score was also reduced in the CP compared with the placebo group. Glutathione content increased in the CP compared with the placebo group (2.2 +/- 0.2 vs. 1.7 +/- 0.2 micromol/g tissue). Gut protein metabolism was more affected in the placebo than in the CP group. The fractional synthesis rate was decreased in the placebo group (93.8 +/- 4.9%/day) compared with controls (121.5 +/- 12.1, P < 0.05), but not in the CP group (106.0 +/- 13.1). In addition, at day 9, rats exhibited improved body weight and food intake recovery in the CP compared to the placebo group. Clinutren Protect feeding reduces intestinal injury in the acute phase of methotrexate-induced mucositis in rats and improves recovery.

Heat Shock Protein-Inducing Property of Diarylheptanoid Containing Chalcone Moiety from Alpinia katsumadai

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Joo-Won Nam

2017-10-01

Full Text Available A new diarylheptanoid containing a chalcone moiety, katsumain H (1, was isolated from the seeds of Alpinia katsumadai. The structure was elucidated using a combination of 1D/2D NMR spectroscopy and mass spectrometry data analysis. The absolute configurations of C-3, C-5, and C-7 in 1 were assigned based on its optical rotation and after comparing its NMR chemical shifts with those of its diastereoisomers, katsumain E and katsumain F, which were previously isolated from this plant and characterized. In this study, the stimulatory effects of compounds 1 and 2 were evaluated on heat shock factor 1 (HSF1, heat shock protein 27 (HSP27, and HSP70. Compounds 1 and 2 increased the expression of HSF1 (1.056- and 1.200-fold, respectively, HSP27 (1.312- and 1.242-fold, respectively, and HSP70 (1.234- and 1.271-fold, respectively, without increased cytotoxicity.

Isolation and characterization of an Aux/IAA gene (LaIAA2) from Larix

African Journals Online (AJOL)

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... an F-box protein, TIR1, which is a component of the SCFTIR1 ubiquitin ligase ... protein-protein interactions (Ulmasov et al., 1997b; Tiwari et al., 2003). In plant .... Statistical analysis .... main classes in the family (Abel and Theologis, 1995;.

The 'tubulin-like' S1 protein of Spirochaeta is a member of the hsp65 stress protein family

Science.gov (United States)

Munson, D.; Obar, R.; Tzertzinis, G.; Margulis, L.

1993-01-01

A 65-kDa protein (called S1) from Spirochaeta bajacaliforniensis was identified as 'tubulin-like' because it cross-reacted with at least four different antisera raised against tubulin and was isolated, with a co-polymerizing 45-kDa protein, by warm-cold cycling procedures used to purify tubulin from mammalian brain. Furthermore, at least three genera of non-cultivable symbiotic spirochetes (Pillotina, Diplocalyx, and Hollandina) that contain conspicuous 24-nm cytoplasmic tubules displayed a strong fluorescence in situ when treated with polyclonal antisera raised against tubulin. Here we summarize results that lead to the conclusion that this 65-kDa protein has no homology to tubulin. S1 is an hsp65 stress protein homologue. Hsp65 is a highly immunogenic family of hsp60 proteins which includes the 65-kDa antigens of Mycobacterium tuberculosis (an active component of Freund's complete adjuvant), Borrelia, Treponema, Chlamydia, Legionella, and Salmonella. The hsp60s, also known as chaperonins, include E. coli GroEL, mitochondrial and chloroplast chaperonins, the pea aphid 'symbionin' and many other proteins involved in protein folding and the stress response.

NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

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Gao, Jinmin; Kim, Hyun-Min; Elia, Andrew E; Elledge, Stephen J; Colaiácovo, Monica P

2015-03-01

The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

Farklı Hayvan Türlerine Ait Karkas Etlerinin ve Mekanik Sıyrılmış Etlerin, Kimyasal Yapılarına ve SDS-PAGE Yardımıyla Saptanan Protein Profillerine Göre Belirlenmesi

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Ayça Yaralı

2015-02-01

Full Text Available Bu çalışmada farklı hayvan karkas etleri ve farklı mekanik sıyrılmış etlerinin kimyasal bileşimi, atomik absorbsiyon spektrofotometresi ile mineral madde bileşimi ve SDS­PAGE ile protein bant dizilimi incelenmiştir. Bu yöntemler ile tür ayrımı ve MDM ayrımı yapılmıştır. Özellikle MDM örneklerinin protein, yağ ve kalsiyum içerikleri diğer karkas etlerinden önemli ölçüde farklı olarak bulunmuştur. Karkas örneklerinin sodyum miktarının MDM örnekleriyle karşılaştırıldığında yüksek olduğu gözlenmiştir. Ayrıca, deneye tabi tutulan makro elementler içinde magnezyum içeriğinin düşük değerlerde olduğu gözlenmiştir. SDS­PAGE bant diziliminde myofibriler proteinler ve sarkoplazmik proteinler incelenmiştir. Elektoroforez jellerinin görüntü analizi ve nicel analizi için densitometre cihazı (The Imager BL, Biolab UviTec software kullanılmıştır.

The C. elegans TPR Containing Protein, TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis.

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Samantha Hughes

Full Text Available Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam or differentiated (hypodermal or neuronal fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27. We show that trd-1(RNAi and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.

Prismatic TIR (total internal reflection) low-concentration PV (photovoltaics)-integrated façade for low latitudes

International Nuclear Information System (INIS)

Sabry, Mohamed

2016-01-01

Low-concentration Façade-integrated Photovoltaic system in the form of TIR (total internal reflection) prismatic segmented façade could play an effective role in reducing the direct component of solar radiation transmitting through buildings, hence reducing both cooling and artificial lighting load on such buildings. A prismatic segmented façade is capable of allowing diffused skylight to transmit through it to the building interior, while preventing most of the direct solar radiation and converting it into clean energy by means of the integrated PV (​photovoltaics) cells. A range of prismatic TIR segmented façades with different head angles has been designed based on the geographical latitude of the chosen location. Each façade configuration is simulated by ray-tracing technique and its performance is investigated against realistic direct solar radiation data in two clear sky days representing summer and winter of the targeted location. Ray tracing simulations revealed that all of the selected configurations could collect most of the direct solar radiation in summer. In contrary, larger head angle of the segmented façade could collect wider intervals around the noon time till reaching a head angle of 23° at which most of the incident direct solar radiation could be collected. - Highlights: • 5 different head angles of prismatic segmented PV-integrated Façade are ray-traced. • Transmitted and PV-collected solar radiation percentages are determined. • DNI daily profiles with associated solar altitudes and azimuth data are simulated. • Expected transmitted and PV collected solar radiation are calculated for the proposed segments.

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

Science.gov (United States)

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum.

Science.gov (United States)

VieBrock, Lauren; Evans, Sean M; Beyer, Andrea R; Larson, Charles L; Beare, Paul A; Ge, Hong; Singh, Smita; Rodino, Kyle G; Heinzen, Robert A; Richards, Allen L; Carlyon, Jason A

2014-01-01

Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks' C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

Science.gov (United States)

Fitzgerald, Kerry D; Semler, Bert L

2013-09-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

Science.gov (United States)

Fitzgerald, Kerry D.; Semler, Bert L.

2013-01-01

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

Science.gov (United States)

Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

2010-01-01

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

New kids on the block: The Popeye domain containing (POPDC) protein family acting as a novel class of cAMP effector proteins in striated muscle.

Science.gov (United States)

Brand, Thomas; Schindler, Roland

2017-12-01

The cyclic 3',5'-adenosine monophosphate (cAMP) signalling pathway constitutes an ancient signal transduction pathway present in prokaryotes and eukaryotes. Previously, it was thought that in eukaryotes three effector proteins mediate cAMP signalling, namely protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC) and the cyclic-nucleotide gated channels. However, recently a novel family of cAMP effector proteins emerged and was termed the Popeye domain containing (POPDC) family, which consists of three members POPDC1, POPDC2 and POPDC3. POPDC proteins are transmembrane proteins, which are abundantly present in striated and smooth muscle cells. POPDC proteins bind cAMP with high affinity comparable to PKA. Presently, their biochemical activity is poorly understood. However, mutational analysis in animal models as well as the disease phenotype observed in patients carrying missense mutations suggests that POPDC proteins are acting by modulating membrane trafficking of interacting proteins. In this review, we will describe the current knowledge about this gene family and also outline the apparent gaps in our understanding of their role in cAMP signalling and beyond. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88.

Science.gov (United States)

Dunne, Aisling; Ejdeback, Mikael; Ludidi, Phumzile L; O'Neill, Luke A J; Gay, Nicholas J

2003-10-17

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies.

Science.gov (United States)

Noguchi, Hiroki; Mylemans, Bram; De Zitter, Elke; Van Meervelt, Luc; Tame, Jeremy R H; Voet, Arnout

2018-03-18

β-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins

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Katherine S. Bridge

2017-07-01

Full Text Available As core components of the microRNA-induced silencing complex (miRISC, Argonaute (AGO proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390 on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

Acetylation-Dependent Chromatin Reorganization by BRDT, a Testis-Specific Bromodomain-Containing Protein

Science.gov (United States)

Pivot-Pajot, Christophe; Caron, Cécile; Govin, Jérôme; Vion, Alexandre; Rousseaux, Sophie; Khochbin, Saadi

2003-01-01

The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor. PMID:12861021

Modified Optimization Water Index (mowi) for LANDSAT-8 Oli/tirs

Science.gov (United States)

Moradi, M.; Sahebi, M.; Shokri, M.

2017-09-01

Water is one of the most important resources that essential need for human life. Due to population growth and increasing need of human to water, proper management of water resources will be one of the serious challenges of next decades. Remote sensing data is the best way to the management of water resources due time and cost effectiveness over a greater range of temporal and spatial scales. Between many kinds of satellite data, from SAR to optic or from high resolution to low resolution, Landsat imagery is more interesting data for water detection and management of earth surface water. Landsat8 OLI/TIRS is the newest version of Landsat satellite series. In this paper, we investigated the full spectral potential of Landsat8 for water detection. It is developed many kinds of methods for this purpose that index based methods have some advantages than other methods. Pervious indices just use a limited number of spectral band. In this paper, Modified Optimization Water Index (MOWI) defined by consideration of a linear combination of bands that each coefficient of bands calculated by particle swarm algorithm. The result shows that modified optimization water index (MOWI) has a proper performance on different condition like cloud, cloud shadow and mountain shadow.

Consumption of Bt Maize Pollen Containing Cry1Ie Does Not Negatively Affect Propylea japonica (Thunberg (Coleoptera: Coccinellidae

Directory of Open Access Journals (Sweden)

Yonghui Li

2017-03-01

Full Text Available Propylea japonica (Thunberg (Coleoptera: Coccinellidae are prevalent predators and pollen feeders in East Asian maize fields. They are therefore indirectly (via prey and directly (via pollen exposed to Cry proteins within Bt-transgenic maize fields. The effects of Cry1Ie-producing transgenic maize pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, survival, larval developmental time, adult fresh weight, and fecundity did not differ between ladybirds consuming Bt or non-Bt maize pollen. In the second experiment, none of the tested lethal and sublethal parameters of P. japonica were negatively affected when fed a rapeseed pollen-based diet containing Cry1Ie protein at 200 μg/g dry weight of diet. In contrast, the larval developmental time, adult fresh weight, and fecundity of P. japonica were significantly adversely affected when fed diet containing the positive control compound E-64. In both experiments, the bioactivity of the Cry1Ie protein in the food sources was confirmed by bioassays with a Cry1Ie-sensitive lepidopteran species. These results indicated that P. japonica are not affected by the consumption of Cry1Ie-expressing maize pollen and are not sensitive to the Cry1Ie protein, suggesting that the growing of Bt maize expressing Cry1Ie protein will pose a negligible risk to P. japonica.

VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

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Shih, Yu-Tzu; Hsueh, Yi-Ping

2016-03-17

Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

Functional analysis of a novel KRAB/C2H2 zinc finger protein Mipu1

International Nuclear Information System (INIS)

Jiang, Lei; Tang, Daolin; Wang, Kangkai; Zhang, Huali; Yuan, Can; Duan, Dayue; Xiao, Xianzhong

2007-01-01

A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C 2 H 2 zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor

Solution structure of GSP13 from Bacillus subtilis exhibits an S1 domain related to cold shock proteins

International Nuclear Information System (INIS)

Yu Wenyu; Hu Jicheng; Yu Bingke; Xia Wei; Jin Changwen; Xia Bin

2009-01-01

GSP13 encoded by gene yugI is a σ B -dependent general stress protein in Bacillus subtilis, which can be induced by heat shock, salt stress, ethanol stress, glucose starvation, oxidative stress and cold shock. Here we report the solution structure of GSP13 and it is the first structure of S1 domain containing protein in Bacillus subtilis. The structure of GSP13 mainly consists of a typical S1 domain along with a C-terminal 50-residue flexible tail, different from the other known S1 domain containing proteins. Comparison with other S1 domain structures reveals that GSP13 has a conserved RNA binding surface, and it may function similarly to cold shock proteins in response to cold stress

Combining Cystatin C and Creatinine Yields a Reliable Glomerular Filtration Rate Estimation in Older Adults in Contrast to β-Trace Protein and β2-Microglobulin.

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Werner, Karin; Pihlsgård, Mats; Elmståhl, Sölve; Legrand, Helen; Nyman, Ulf; Christensson, Anders

2017-01-01

The glomerular filtration rate (GFR) is the most important measure of kidney function and chronic kidney disease (CKD). This study aims to validate commonly used equations for estimated GFR (eGFR) based on creatinine (cr), cystatin C (cys), β-trace protein (BTP), and β2-microglobulin (B2M) in older adults. We conducted a validation study with 126 participants aged between 72 and 98 with a mean measured GFR (mGFR) by iohexol clearance of 54 mL/min/1.73 m2. The eGFR equations (CKD-Epidemiology collaboration [CKD-EPI], Berlin Initiative Study [BIS], Full Age Spectrum [FAS], Modification of Diet in Renal Disease [MDRD]cr, Caucasian-Asian-Pediatric-Adult [CAPA]cys, Lund-Malmö Revised [LM-REV]cr, and MEAN-LM-CAPAcr-cys), were assessed in terms of bias (median difference: eGFR-mGFR), precision (interquartile range of the differences), and accuracy (P30: percentage of estimates ±30% of mGFR). The equations were compared to a benchmark equation: CKD-EPIcr-cys. All cystatin C-based equations underestimated the GFR compared to mGFR, whereas bias was mixed for the equations based only on creatinine. Accuracy was the highest for CKD-EPIcr-cys (98%) and lowest for MDRD (82%). Below mGFR 45 mL/min/1.73 m2 only equations incorporating cystatin C reached P30 accuracy >90%. CKD-EPIcr-cys was not significantly more accurate than the other cystatin C-based equations. In contrast, CKD-EPIcr-cys was significantly more accurate than all creatinine-based equations except LM-REVcr. This study confirms that it is reasonable to use equations incorporating cystatin C and creatinine in older patients across a wide spectrum of GFR. However, the results call into question the use of creatinine alone below mGFR 45 mL/min/1.73 m2. B2M and BTP do not demonstrate additional value in eGFR determination in older adults. © 2017 S. Karger AG, Basel.

Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

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Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

2014-01-01

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults.

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Kim, Il-Young; Schutzler, Scott; Schrader, Amy; Spencer, Horace J; Azhar, Gohar; Ferrando, Arny A; Wolfe, Robert R

2016-01-01

We have determined whole body protein kinetics, i.e., protein synthesis (PS), breakdown (PB), and net balance (NB) in human subjects in the fasted state and following ingestion of ~40 g [moderate protein (MP)], which has been reported to maximize the protein synthetic response or ~70 g [higher protein (HP)] protein, more representative of the amount of protein in the dinner of an average American diet. Twenty-three healthy young adults who had performed prior resistance exercise (X-MP or X-HP) or time-matched resting (R-MP or R-HP) were studied during a primed continuous infusion of l-[(2)H5]phenylalanine and l-[(2)H2]tyrosine. Subjects were randomly assigned into an exercise (X, n = 12) or resting (R, n = 11) group, and each group was studied at the two levels of dietary protein intake in random order. PS, PB, and NB were expressed as increases above the basal, fasting values (mg·kg lean body mass(-1)·min(-1)). Exercise did not significantly affect protein kinetics and blood chemistry. Feeding resulted in positive NB at both levels of protein intake: NB was greater in response to the meal containing HP vs. MP (P < 0.00001). The greater NB with HP was achieved primarily through a greater reduction in PB and to a lesser extent stimulation of protein synthesis (for all, P < 0.0001). HP resulted in greater plasma essential amino acid responses (P < 0.01) vs. MP, with no differences in insulin and glucose responses. In conclusion, whole body net protein balance improves with greater protein intake above that previously suggested to maximally stimulating muscle protein synthesis because of a simultaneous reduction in protein breakdown. Copyright © 2016 the American Physiological Society.

HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus.

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Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

2016-05-01

The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

Energy Technology Data Exchange (ETDEWEB)

Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

2003-06-11

Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

Effects of high-protein diet containing isolated whey protein in rats submitted to resistance training of aquatic jumps.

Science.gov (United States)

Avila, Eudes Thiago Pereira; da Rosa Lima, Thiago; Tibana, Ramires Alsamir; de Almeida, Paula Caroline; Fraga, Géssica Alves; de Souza Sena, Mariana; Corona, Luiz Felipe Petusk; Navalta, James Wilfred; Rezaei, Sajjad; Ghayomzadeh, Morteza; Damazo, Amílcar Sabino; Prestes, Jonato; Voltarelli, Fabrício Azevedo

2018-02-13

Isolated whey protein (IWP) can decrease body fat compared with other protein sources. The present study verified the effects of high protein diet (HD) containing IWP on several parameters of rats subjected to resistance training (RT). Thirty-two male Wistar rats (60 days of age) were separated into four groups (n = 8/group): sedentary normoproteic (IWP 14%; SN); sedentary hyperproteic (IWP 35%; SH); trained normoproteic (IWP 14%; TN), and trained hyperproteic (WPI 35%; TH). Relative tissue/organ weight (g): perirenal and retroperitoneal adipose tissues were lower in SH and TH compared with SN (no difference to TN); omental and subcutaneous adipose tissues were higher in SN compared with SH. Epididymal adipose tissue was higher in SN compared with other groups. Heart weight was higher in TH compared with TN and SN, but not SH; kidney and liver higher in TH and SH compared with SN and TN; gastrocnemius lower in SN compared with other groups; soleus higher in SH in relation to other groups. The triglycerides levels (mg/dL) was reduced in the TH groups compared with SH, TN, and SN. There were no changes both in the concentrations of adiponectin and leptin and in the protein expression of GLUT-4 and p70 s6k . HD containing WPI improved body composition, increased the weight of the heart, kidneys, liver and gastrocnemius and soleus muscles; however, this diet maintained the normal histomorphology of muscle and liver and, when associated with RT, reduced the serum levels of triglycerides. Copyright © 2018 Elsevier Inc. All rights reserved.

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

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Emanuela Leonardi

Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

Biological role of copper and copper-containing proteins in human and animal organism

OpenAIRE

ANTONYAK H.L.; VAZHNENKO A.V.; PANAS N.E.

2011-01-01

Current scientific data related to copper metabolism and functional activity of Cu-containing proteins in human and animal cells are reviewed in the article. Important functional role of this essential element in human and animal organism is analyzed.

Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

Science.gov (United States)

Saeki, Ayumi; Suzuki, Toshihiko; Hasebe, Akira; Kamezaki, Ryousuke; Fujita, Mari; Nakazawa, Futoshi; Shibata, Ken-Ichiro

2017-03-01

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1β responsible for the development of the disease. However, the mechanism of IL-1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1β and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1β production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1β. The inhibitors for ATP receptor reduced IL-1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity. © 2016 John Wiley & Sons Ltd.

Statistical analysis of long term (2006-2016) TIR imagery based on Generalized Extreme Value estimator: an application at Pisciarelli volcanic area (Campi Flegrei, Italy).

Science.gov (United States)

Petrillo, Zaccaria; Vilardo, Giuseppe; Sansivero, Fabio; Mangiacapra, Annarita; Caliro, Stefano; Caputo, Teresa

2017-04-01

Quantifying and monitoring energy budgets at calderas, released in terms of heat output during unrest periods, is crucial to understand the state of activity, the system evolution and to draw a possible future eruptive scenario. Campi Flegrei, a restless caldera in Southern Italy, during the last years is experiencing clear signs of potential reawakening. Indeed, is now more important then ever to consider, analyse and monitor all the potential precursors, contributing to the caldera volcanic hazard assessment. We analysed the continuous long term (2006-2016) TIR images night-time collected at Pisciarelli site. This volcanic area, is located above a critical volume which recently showed an increase and clustering of earthquakes distribution and which shows the most impressive gas discharge (mainly H2O and CO2) at Campi Flegrei caldera. We treated in a statistical way the TIR images, defining an anomaly zone, which we compared to a background area. The pixel distributions, as function of the temperature, showed a generalized extreme value structure. The anomaly area, with a long tail toward high temperature values, showed a positive factor form ( f > 0, Frechet distribution). This value was constantly above zero and kept stable along the whole 2006-2016 period, while the scale factor was estimated with a decreasing trend (variance reduction). Pixels of the background TIR images, in contrast, showed a factor form between zero and a weakly negative value (f = 0 or f < 0) Gumbel or Weibull distribution). We used the location parameter as representative of the temperature distribution (which is very near the average temperature) and analysed its trend as function of time, removing the annual variation using a 365.25 days mobile average.

Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.

Directory of Open Access Journals (Sweden)

Christos Tzitzilonis

Full Text Available Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15N,(1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15N,(1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.

Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

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Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

2018-01-15

The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

DEFF Research Database (Denmark)

Spetzler, J C; Westphal, V; Winther, Jakob R.

1998-01-01

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

Science.gov (United States)

Brown, W C; Zhao, S; Woods, V M; Tripp, C A; Tetzlaff, C L; Heussler, V T; Dobbelaere, D A; Rice-Ficht, A C

1993-01-01

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1

NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

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Jinmin Gao

2015-03-01

Full Text Available The formation of DNA double-strand breaks (DSBs must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

Free-Standing Metal Oxide Nanoparticle Superlattices Constructed with Engineered Protein Containers Show in Crystallo Catalytic Activity.

Science.gov (United States)

Lach, Marcel; Künzle, Matthias; Beck, Tobias

2017-12-11

The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The C-terminal MIR-containing region in the Pmt1 O-mannosyltransferase restrains sporulation and is dispensable for virulence in Beauveria bassiana.

Science.gov (United States)

He, Zhangjiang; Luo, Linli; Keyhani, Nemat O; Yu, Xiaodong; Ying, Shenghua; Zhang, Yongjun

2017-02-01

Protein O-mannosyltransferases (Pmts) belong to a highly conserved protein family responsible for the initiation of O-glycosylation of many proteins. Pmts contain one dolichyl-phosphate-mannose-protein mannosyltransferases (PMT) domain and three MIR motifs (mannosyltransferase, inositol triphosphate, and ryanodine receptor) that are essential for activity in yeast. We report that in the insect fungal pathogen, Beauveria bassiana, deletion of the C-terminal Pmt1 MIR-containing region (Pmt1∆ 311-902 ) does not alter O-mannosyltransferase activity, but does increase total cell wall protein O-mannosylation levels and results in phenotypic changes in fungal development and cell wall stability. B. bassiana mutants harboring the Pmt1 ∆ 311-902 mutation displayed a significant increase in conidiation with up-regulation of conidiation-associated genes and an increase in biomass accumulation as compared to the wild-type parent. However, decreased vegetative growth and blastospore production was noted, and Pmt1 ∆ 311-902 mutants were altered in cell wall composition and cell surface features. Insect bioassays revealed little effect on virulence for the Pmt1 ∆ 311-902 strain via cuticle infection or intrahemocoel injection assays, although differences in hyphal body differentiation in the host hemolymph and up-regulation of virulence-associated genes were noted. These data suggest novel roles for Pmt1 in negatively regulating conidiation and demonstrate that the C-terminal Pmt1 MIR-containing region is dispensable for enzymatic activity and organismal virulence.

A Study on Spreading Direction of Coal-fire Based with TIR Remote Sensing in Wuda Coalfield from 2000 to 2006, Northern China

International Nuclear Information System (INIS)

Huo, H-Y; Jiang, X-G; Song, X-F; Liu, L; Ni, Z-Y; Gao, C-X; Zhang, Y-Z

2014-01-01

Coal fires are a common and serious problem in most coal producing countries. Coal fires could not only lead to a huge loss of non-renewable energy resources, but it also can cause many environmental problems such as GHG emission, land subsidence and increment of surface temperature. So it is very important to monitor the dynamic changes of coal fires. As far as large scale coal field, remote sensing provided researchers with a new and useful technique for coal fire detection. This paper developed a research over coal fire spreading direction using a multi-temporal TIR remote sensing approach. The results successfully showed that the direction of coal fire spreading and predicted the coal fire direction of development on a regional scale or on a whole coal field scale, and a quantitative analysis of coal fires was made in the research. The results showed that the coal fires had an average annual increase of 0.5 million square meters from 1999 to 2006, and the TIR remote sensing proved to be an available tool for coal fire mapping and prediction of coal fire development

Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to the stress-inducible yeast protein STI1

DEFF Research Database (Denmark)

Honoré, B; Leffers, H; Madsen, Peder

1992-01-01

in families of fungal proteins required for mitosis and RNA synthesis. In particular, the protein has 42% amino acid sequence identity to STI1, a stress-inducible mediator of the heat shock response in Saccharomyces cerevisiae. Northern blot analysis indicated that the 3521 mRNA is up-regulated in several...

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

International Nuclear Information System (INIS)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

2006-01-01

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions

Association of innate defense proteins BPIFA1 and BPIFB1 with disease severity in COPD

Science.gov (United States)

De Smet, Elise G; Seys, Leen JM; Verhamme, Fien M; Vanaudenaerde, Bart M; Brusselle, Guy G; Bingle, Colin D; Bracke, Ken R

2018-01-01

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal inflammatory response in the lungs caused by the inhalation of noxious particles and gases. The airway epithelium has a protective function against these harmful agents by maintaining a physical barrier and by secreting defensive proteins, such as bactericidal/permeability-increasing fold-containing (BPIF) proteins, BPIFA1 and BPIFB1. However, inconsistent data regarding BPIFA1 expression in smokers and COPD patients have been reported to date. Therefore, we investigated the expression of BPIFA1 and BPIFB1 in a large cohort of never-smokers and smokers with and without COPD, both on the messenger RNA (mRNA) level in lung tissue and on the protein level in airway epithelium. Furthermore, we examined the correlation between BPIFA1 and BPIFB1 levels, goblet cell hyperplasia, and lung function measurements. BPIFA1 and BPIFB1 mRNA expressions were significantly increased in stage III–IV COPD patients compared with stage II COPD patients and subjects without COPD. In addition, protein levels in COPD patients were significantly increased in comparison with subjects without COPD. BPIFA1 and BPIFB1 levels were inversely correlated with measurements of airflow limitation and positively correlated with goblet cell hyperplasia. In addition, by the use of immunofluorescence double staining, we demonstrated the expression of BPIFB1 in goblet cells. In conclusion, we show that BPIFA1 and BPIFB1 levels are elevated in COPD patients and correlate with disease severity. PMID:29296079

A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast.

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Javier Encinar del Dedo

2015-03-01

Full Text Available Iron is an essential cofactor, but it is also toxic at high levels. In Schizosaccharomyces pombe, the sensor glutaredoxin Grx4 guides the activity of the repressors Php4 and Fep1 to mediate a complex transcriptional response to iron deprivation: activation of Php4 and inactivation of Fep1 leads to inhibition of iron usage/storage, and to promotion of iron import, respectively. However, the molecular events ruling the activity of this double-branched pathway remained elusive. We show here that Grx4 incorporates a glutathione-containing iron-sulfur cluster, alone or forming a heterodimer with the BolA-like protein Fra2. Our genetic study demonstrates that Grx4-Fra2, but not Fep1 nor Php4, participates not only in iron starvation signaling but also in iron-related aerobic metabolism. Iron-containing Grx4 binds and inactivates the Php4 repressor; upon iron deprivation, the cluster in Grx4 is probably disassembled, the proteins dissociate, and Php4 accumulates at the nucleus and represses iron consumption genes. Fep1 is also an iron-containing protein, and the tightly bound iron is required for transcriptional repression. Our data suggest that the cluster-containing Grx4-Fra2 heterodimer constitutively binds to Fep1, and upon iron deprivation the disassembly of the iron cluster between Grx4 and Fra2 promotes reverse metal transfer from Fep1 to Grx4-Fra2, and de-repression of iron-import genes. Our genetic and biochemical study demonstrates that the glutaredoxin Grx4 independently governs the Php4 and Fep1 repressors through metal transfer. Whereas iron loss from Grx4 seems to be sufficient to release Php4 and allow its nuclear accumulation, total or partial disassembly of the Grx4-Fra2 cluster actively participates in iron-containing Fep1 activation by sequestering its iron and decreasing its interaction with promoters.

Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane

NARCIS (Netherlands)

Heijden, van der M.W.; Carette, J.E.; Reinhoud, P.J.; Haegi, A.; Bol, J.F.

2001-01-01

Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and

TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii.

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Aoife T Heaslip

Full Text Available The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.