Copper and zinc mediated oligomerisation of Aß peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.

Requirements for ICAM-1 immunogene therapy of lymphoma.

Intercellular cell adhesion molecule-1 (ICAM-1) is a cell-surface glycoprotein capable of eliciting bidirectional signals that activate signalling pathways in leukocytes, endothelial, and smooth muscle cells. Gene transfer of xenogeneic ICAM-1 into EL-4 lymphomas causes complete tumor rejection; however, it is unknown whether the mechanism responsible involves the "foreignness" of the ICAM-1 transgene, bidirectional signalling events, ICAM-1-receptor interaction, or a combination of the latter. To begin to address this question, we constructed four different therapeutic expression vectors encoding full-length ICAM-1, and forms in which the N-terminal ligand-binding domains and cytoplasmic tail had been deleted. Mouse EL-4 tumors (0.5 cm in diameter), which actively suppress the immune response, were significantly inhibited in their growth following injection of expression plasmids encoding either full-length xenogenic (human) ICAM-1, or a functional cytoplasmic domain-deficient form that retains ligand-binding activity. Efficacy of ICAM-1-mediated antitumor immunity was significantly augmented by administration of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), which suppressed blood supply to the tumor, leading to enhanced leukocyte infiltration, and complete tumor eradication in a gene dosage and CD8(+) T cell and NK cell-dependent fashion. Generation of potent cytotoxic T cell (CTL)-mediated antitumor immunity was reflected by ICAM-1-facilitated apoptosis of tumor cells in situ. In contrast, nonfunctional ICAM-1 lacking the N-terminal ligand-binding Ig domain failed to generate antitumor immunity, even in the presence of DMXAA. These studies demonstrate that ICAM-1-stimulated antitumor immunity can overcome tumor-mediated immunosuppression, particularly when employed in combination with an attack on the tumor vasculature. The ligand-binding domain of ICAM-1 is essential for generating antitumor immunity, whereas the cytoplasmic domain and bidirectional activation of tumor signalling pathways are not essential.

Family tries

My younger brother , you know, was the best one of us. Yeah, he frigging was, so no one say a word about him and what he done, right. H e got onto an A-grade Rugby League club team and we used to see him real as life on the telly on Sunday. Yep, there he was in all his glory in all our glory. H e even got into the papers too. Got a great shot of him still. There he lies stretched out full length along the ground, the soles of his boots showing, his arms fully out though with the elbows bent just a little to ease his fall, but you know what, his effing face is turned to the camera. H e knows that that was good. It was too, the try of the week. Great picture, that's why I keep it, as does mum and dad. H e was bloody brilliant, but with his fame came the girls from better suburbs than ours can ever hope to be. Finally, got himself hitched to a fancy bitch whose folks had money in the bank and possibly owned the bloody thing too. Well, it was like that, but he should've stuck with his own kind, though they ain't much better. I should know, because of what lately happened to me. By the way, my name is Rose, and I've got thorns as more than one has found out to his grief...

Ambient thing loop

Ambient Thing is an uplifting, melodic and motivational track featuring synths, drums and light vocals. Atmospheric and reflective, yet incredibly inspiring, it expresses positivity, strong emotional connection and success. Upbeat and positive, it is suitable for your business/corporate projects, videos, websites, films, creative pursuits and more.

Ambient thing loop 2

Ambient Thing is an uplifting, melodic and motivational track featuring synths, piano,drums. Atmospheric and reflective, yet incredibly inspiring, it expresses positivity, strong emotional connection and success. Upbeat and positive, it is suitable for your business/corporate projects, videos, websites, films, creative pursuits and more.

Ambient thing loop 3

Ambient Thing is an uplifting, melodic and motivational track featuring synths, piano,drums. Atmospheric and reflective, yet incredibly inspiring, it expresses positivity, strong emotional connection and success. Upbeat and positive, it is suitable for your business/corporate projects, videos, websites, films, creative pursuits and more.

A probabilistic framework for extracting narrative act boundaries and semantics in motion pictures

This work constitutes the first attempt to extract the important narrative structure, the 3-Act storytelling paradigm in film. Widely prevalent in the domain of film, it forms the foundation and framework in which a film can be made to function as an effective tool for story telling, and its extraction is a vital step in automatic content management for film data. The identification of act boundaries allows for structuralizing film at a level far higher than existing segmentation frameworks, which include shot detection and scene identification, and provides a basis for inferences about the semantic content of dramatic events in film. A novel act boundary likelihood function for Act 1 and 2 is derived using a Bayesian formulation under guidance from film grammar, tested under many configurations and the results are reported for experiments involving 25 full-length movies. The result proves to be a useful tool in both the automatic and semi-interactive setting for semantic analysis of film, with potential application to analogues occuring in many other domains, including news, training video, sitcoms.

Toward automatic extraction of expressive elements from motion pictures : tempo

This paper addresses the challenge of bridging the semantic gap that exists between the simplicity of features that can be currently computed in automated content indexing systems and the richness of semantics in user queries posed for media search and retrieval. It proposes a unique computational approach to extraction of expressive elements of motion pictures for deriving high-level semantics of stories portrayed, thus enabling rich video annotation and interpretation. This approach, motivated and directed by the existing cinematic conventions known as film grammar, as a first step toward demonstrating its effectiveness, uses the attributes of motion and shot length to define and compute a novel measure of tempo of a movie. Tempo flow plots are defined and derived for a number of full-length movies and edge analysis is performed leading to the extraction of dramatic story sections and events signaled by their unique tempo. The results confirm tempo as a useful high-level semantic construct in its own right and a promising component of others such as rhythm, tone or mood of a film. In addition to the development of this computable tempo measure, a study is conducted as to the usefulness of biasing it toward either of its constituents, namely, motion or shot length. Finally, a refinement is made to the shot length normalizing mechanism, driven by the peculiar characteristics of shot length distribution exhibited by movies. Results of these additional studies, and possible applications and limitations are discussed.

Novel approach to determining tempo and dramatic story sections in motion pictures

This paper presents an original computational approach to extraction of movie tempo for deriving story sections and events that convey high level semantics of stories portrayed in motion pictures, thus enabling better video annotation and interpretation systems. This approach, inspired by the existing cinematic conventions known as film grammar, uses the attributes of motion and shot length to define and compute a novel continuous measure of tempo of a movie. Tempo flow plots are derived for several full-length motion pictures and edge detection is performed to extract dramatic story sections and events occurring in the movie, underlined by their unique tempo. The results confirm reliable detection of actual distinct tempo changes and serve as useful index into the dramatic development and narration of the story in motion pictures.

Towards automatic extraction of expressive elements from motion pictures : tempo

This paper proposes a unique computational approach to extraction of expressive elements of motion pictures for deriving high level semantics of stories portrayed, thus enabling better video annotation and interpretation systems. This approach, motivated and directed by the existing cinematic conventions known as film grammar, as a first step towards demonstrating its effectiveness, uses the attributes of motion and shot length to define and compute a novel measure of tempo of a movie. Tempo flow plots are defined and derived for four full-length movies and edge analysis is performed leading to the extraction of dramatic story sections and events signaled by their unique tempo. The results confirm tempo as a useful attribute in its own right and a promising component of semantic constructs such as tone or mood of a film.

Neighborhood coherence and edge based approaches to film scene extraction

In order to enable high-level semantics-based video annotation and interpretation, we tackle the problem of automatic decomposition of motion pictures into meaningful story units, namely scenes. Since a scene is a complicated and subjective concept, we first propose guidelines from film production to determine when a scene change occurs in film. We examine different rules and conventions followed as part of Film Grammar to guide and shape our algorithmic solution for determining a scene boundary. Two different techniques are proposed as new solutions in this paper. Our experimental results on 10 full-length movies show that our technique based on shot sequence coherence performs well and reasonably better than the color edges-based approach.

Indexing narrative structure and semantics in motion pictures with a probabilistic framework

This work constitutes the first attempt to extract an important narrative structure, the 3-Act story telling paradigm, in film. This narrative structure is prevalent in the domain of film as it forms the foundation and framework in which the film can be made to function as an effective tool for story telling, and its extraction is a vital step in automatic content management for film data. A novel act boundary likelihood function for Act 1 is derived using a Bayesian formulation under guidance from film grammar, tested under many configurations and the results are reported for experiments involving 25 full length movies. The formulation is shown to be a useful tool in both the automatic and semi-interactive setting for semantic analysis of film.

Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).

Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic RNA dimer stability

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.

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

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.