Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor.

The mechanism under which the signal-reception amino-terminal portion (A domain) of the prokaryotic enhancer-binding protein XylR controls the activity of the regulator has been investigated through complementation tests in vivo, in which the various protein segments were produced as independent polypeptides. Separate expression of the A domain repressed the otherwise constitutive activity of a truncated derivative of XylR deleted of its A domain (XylR delta A). Such inhibition was not released by m-xylene, the natural inducer of the system. Repression caused by the A domain was specific for XylR because it did not affect activation of the sigma 54 promoter PnifH by a derivative of its cognate regulator, NifA, deleted of its own A domain. The A domain was also unable to repress the activity of a NifA-XylR hybrid protein resulting from fusing two-thirds of the central domain of NifA to the carboxyl-terminal third of XylR, which includes its DNA-binding domain. The inhibitory effect caused by the A domain of XylR on XylR delta A seems, therefore, to result from specific interactions in trans between the two truncated proteins and not from mere hindering of an activating surface.

The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.

Pex1, a pollen-specific gene with an extensin-like domain.

We report here the identification of a pollen-specific gene from Zea mays that contains multiple Ser-(Pro)n repeats, the motif found in the cell wall-associated extensins. Sequence analysis reveals that the encoded protein has a putative globular domain at the N terminus and an extensin-like domain at the C terminus. The Pex1 (pollen extensin-like) gene is expressed exclusively in pollen, not in vegetative or female tissues, and is not induced in leaves upon wounding. We propose that the encoded protein may have a role in reproduction, either as a structural element deposited in the pollen tube wall during its rapid growth or as a sexual recognition molecule that interacts with partner molecules in the pistil.

Development of prospective mathematics teachers’ professional noticing in a specific domain: proportional reasoning

The aim of this research is to identify aspects that support the development of prospective mathematics teachers’ professional noticing in a b-learning context. The study presented here investigates the extent to which prospective secondary mathematics teachers attend and interpret secondary school students’ proportional reasoning and decide how to respond. Results show that interactions in an on-line discussion improve prospective mathematics teachers’ ability to identify and interpret important aspects of secondary school students’ mathematical thinking.

A time-domain test for some types of nonlinearity

The bispectrum and third-order moment can be viewed as equivalent tools for testing for the presence of nonlinearity in stationary time series. This is because the bispectrum is the Fourier transform of the third-order moment. An advantage of the bispectrum is that its estimator comprises terms that are asymptotically independent at distinct bifrequencies under the null hypothesis of linearity. An advantage of the third-order moment is that its values in any subset of joint lags can be used in the test, whereas when using the bispectrum the entire (or truncated) third-order moment is required to construct the Fourier transform. In this paper, we propose a test for nonlinearity based upon the estimated third-order moment. We use the phase scrambling bootstrap method to give a nonparametric estimate of the variance of our test statistic under the null hypothesis. Using a simulation study, we demonstrate that the test obtains its target significance level, with large power, when compared to an existing standard parametric test that uses the bispectrum. Further we show how the proposed test can be used to identify the source of nonlinearity due to interactions at specific frequencies. We also investigate implications for heuristic diagnosis of nonstationarity.

Integration of semantically annotated data by the KnoFuss architecture

Most of the existing work on information integration in the Semantic Web concentrates on resolving schema-level problems. Specific issues of data-level integration (instance coreferencing, conflict resolution, handling uncertainty) are usually tackled by applying the same techniques as for ontology schema matching or by reusing the solutions produced in the database domain. However, data structured according to OWL ontologies has its specific features: e.g., the classes are organized into a hierarchy, the properties are inherited, data constraints differ from those defined by database schema. This paper describes how these features are exploited in our architecture KnoFuss, designed to support data-level integration of semantic annotations.

Representing Variability in Software Architecture

Software Architecture is a high level description of a software intensive system that enables architects to have a better intellectual control over the complete system. It is also used as a communication vehicle among the various system stakeholders. Variability in software-intensive systems is the ability of a software artefact (e.g., a system, subsystem, or component) to be extended, customised, or configured for deployment in a specific context. Although variability in software architecture is recognised as a challenge in multiple domains, there has been no formal consensus on how variability should be captured or represented. In this research, we addressed the problem of representing variability in software architecture through a three phase approach. First, we examined existing literature using the Systematic Literature Review (SLR) methodology, which helped us identify the gaps and challenges within the current body of knowledge. Equipped with the findings from the SLR, a set of design principles have been formulated that are used to introduce variability management capabilities to an existing Architecture Description Language (ADL). The chosen ADL was developed within our research group (ALI) and to which we have had complete access. Finally, we evaluated the new version of the ADL produced using two distinct case studies: one from the Information Systems domain, an Asset Management System (AMS); and another from the embedded systems domain, a Wheel Brake System (WBS). This thesis presents the main findings from the three phases of the research work, including a comprehensive study of the state-of-the-art; the complete specification of an ADL that is focused on managing variability; and the lessons learnt from the evaluation work of two distinct real-life case studies.

Digital 3D reconstruction as a research environment in art and architecture history: uncertainty classification and visualisation

The dissertation addresses the still not solved challenges concerned with the source-based digital 3D reconstruction, visualisation and documentation in the domain of archaeology, art and architecture history. The emerging BIM methodology and the exchange data format IFC are changing the way of collaboration, visualisation and documentation in the planning, construction and facility management process. The introduction and development of the Semantic Web (Web 3.0), spreading the idea of structured, formalised and linked data, offers semantically enriched human- and machine-readable data. In contrast to civil engineering and cultural heritage, academic object-oriented disciplines, like archaeology, art and architecture history, are acting as outside spectators. Since the 1990s, it has been argued that a 3D model is not likely to be considered a scientific reconstruction unless it is grounded on accurate documentation and visualisation. However, these standards are still missing and the validation of the outcomes is not fulfilled. Meanwhile, the digital research data remain ephemeral and continue to fill the growing digital cemeteries. This study focuses, therefore, on the evaluation of the source-based digital 3D reconstructions and, especially, on uncertainty assessment in the case of hypothetical reconstructions of destroyed or never built artefacts according to scientific principles, making the models shareable and reusable by a potentially wide audience. The work initially focuses on terminology and on the definition of a workflow especially related to the classification and visualisation of uncertainty. The workflow is then applied to specific cases of 3D models uploaded to the DFG repository of the AI Mainz. In this way, the available methods of documenting, visualising and communicating uncertainty are analysed. In the end, this process will lead to a validation or a correction of the workflow and the initial assumptions, but also (dealing with different hypotheses) to a better definition of the levels of uncertainty.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

The AdapLib Library for Execution of Adaptive Devices: Architecture and Use

In this paper it is presented the theoretical background, the architecture (using the ""4+1"" model), and the use of the library for execution of adaptive devices, AdapLib. This library was created seeking to be accurate to the adaptive devices theory, and to allow its easy extension considering the specific details of solutions that employ this kind of device. As an example, it is presented a case study in which the library was used to create a proof of concept to monitor and diagnose problems in an online news portal.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.