Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MR-F4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder (Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine. (c) 2006 Elsevier Inc. All rights reserved.

Inferring 3D Structure with a Statistical Image-Based Shape Model

We present an image-based approach to infer 3D structure parameters using a probabilistic "shape+structure'' model. The 3D shape of a class of objects may be represented by sets of contours from silhouette views simultaneously observed from multiple calibrated cameras. Bayesian reconstructions of new shapes can then be estimated using a prior density constructed with a mixture model and probabilistic principal components analysis. We augment the shape model to incorporate structural features of interest; novel examples with missing structure parameters may then be reconstructed to obtain estimates of these parameters. Model matching and parameter inference are done entirely in the image domain and require no explicit 3D construction. Our shape model enables accurate estimation of structure despite segmentation errors or missing views in the input silhouettes, and works even with only a single input view. Using a dataset of thousands of pedestrian images generated from a synthetic model, we can perform accurate inference of the 3D locations of 19 joints on the body based on observed silhouette contours from real images.

Learning World Models in Environments with Manifest Causal Structure

This thesis examines the problem of an autonomous agent learning a causal world model of its environment. Previous approaches to learning causal world models have concentrated on environments that are too "easy" (deterministic finite state machines) or too "hard" (containing much hidden state). We describe a new domain --- environments with manifest causal structure --- for learning. In such environments the agent has an abundance of perceptions of its environment. Specifically, it perceives almost all the relevant information it needs to understand the environment. Many environments of interest have manifest causal structure and we show that an agent can learn the manifest aspects of these environments quickly using straightforward learning techniques. We present a new algorithm to learn a rule-based causal world model from observations in the environment. The learning algorithm includes (1) a low level rule-learning algorithm that converges on a good set of specific rules, (2) a concept learning algorithm that learns concepts by finding completely correlated perceptions, and (3) an algorithm that learns general rules. In addition this thesis examines the problem of finding a good expert from a sequence of experts. Each expert has an "error rate"; we wish to find an expert with a low error rate. However, each expert's error rate and the distribution of error rates are unknown. A new expert-finding algorithm is presented and an upper bound on the expected error rate of the expert is derived.

A dichotomic search algorithm for mining and learning in domain-specific logics

Ferr?, S. and King, R. D. (2004) A dichotomic search algorithm for mining and learning in domain-specific logics. Fundamenta Informaticae. IOS Press. To appear

Phase estimation receiver for full-field detection: a novel receiver structure for electronic dispersion compensation of metropolitan area networks

The development of ultra high speed (~20 Gsamples/s) analogue to digital converters (ADCs), and the delayed deployment of 40 Gbit/s transmission due to the economic downturn, has stimulated the investigation of digital signal processing (DSP) techniques for compensation of optical transmission impairments. In the future, DSP will offer an entire suite of tools to compensate for optical impairments and facilitate the use of advanced modulation formats. Chromatic dispersion is a very significant impairment for high speed optical transmission. This thesis investigates a novel electronic method of dispersion compensation which allows for cost-effective accurate detection of the amplitude and phase of the optical field into the radio frequency domain. The first electronic dispersion compensation (EDC) schemes accessed only the amplitude information using square law detection and achieved an increase in transmission distances. This thesis presents a method by using a frequency sensitive filter to estimate the phase of the received optical field and, in conjunction with the amplitude information, the entire field can be digitised using ADCs. This allows DSP technologies to take the next step in optical communications without requiring complex coherent detection. This is of particular of interest in metropolitan area networks. The full-field receiver investigated requires only an additional asymmetrical Mach-Zehnder interferometer and balanced photodiode to achieve a 50% increase in EDC reach compared to amplitude only detection.

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.

Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Measuring morphological features using light-scattering spectroscopy and Fourier-domain low-coherence interferometry.

We present measurements of morphological features in a thick turbid sample using light-scattering spectroscopy (LSS) and Fourier-domain low-coherence interferometry (fLCI) by processing with the dual-window (DW) method. A parallel frequency domain optical coherence tomography (OCT) system with a white-light source is used to image a two-layer phantom containing polystyrene beads of diameters 4.00 and 6.98 mum on the top and bottom layers, respectively. The DW method decomposes each OCT A-scan into a time-frequency distribution with simultaneously high spectral and spatial resolution. The spectral information from localized regions in the sample is used to determine scatterer structure. The results show that the two scatterer populations can be differentiated using LSS and fLCI.

Intraoperative spectral domain optical coherence tomography for vitreoretinal surgery.

We demonstrate in vivo human retinal imaging using an intraoperative microscope-mounted optical coherence tomography system (MMOCT). Our optomechanical design adapts an Oculus Binocular Indirect Ophthalmo Microscope (BIOM3), suspended from a Leica ophthalmic surgical microscope, with spectral domain optical coherence tomography (SD-OCT) scanning and relay optics. The MMOCT enables wide-field noncontact real-time cross-sectional imaging of retinal structure, allowing for SD-OCT augmented intrasurgical microscopy for intraocular visualization. We experimentally quantify the axial and lateral resolution of the MMOCT and demonstrate fundus imaging at a 20Hz frame rate.

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex.

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.

The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0 Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATPBinding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse Pgp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.

15N NMR relaxation data reveal significant chemical exchange broadening in the alpha-domain of human α-lactalbumin

Human alpha-lactalbumin (alpha-LA), a 123-residue calcium-binding protein, has been studied using (15)N NMR relaxation methods in order to characterize backbone dynamics of the native state at the level of individual residues. Relaxation data were collected at three magnetic field strengths and analyzed using the model-free formalism of Lipari and Szabo. The order parameters derived from this analysis are generally high, indicating a rigid backbone. A total of 46 residues required an exchange contribution to T(2); 43 of these residues are located in the alpha-domain of the protein. The largest exchange contributions are observed in the A-, B-, D-, and C-terminal 3(10)-helices of the alpha-domain; these residues have been shown previously to form a highly stable core in the alpha-LA molten globule. The observed exchange broadening, along with previous hydrogen/deuterium amide exchange data, suggests that this part of the alpha-domain may undergo a local structural transition between the well-ordered native structure and a less-ordered molten-globule-like structure.