Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-2M complexes

By combining two previously generated null mutations, Ii° and M°, we produced mice lacking the invariant chain and H-2M complexes, both required for normal cell-surface expression of major histocompatibility complex class II molecules loaded with the usual diverse array of peptides. As expected, the maturation and transport of class II molecules, their expression at the cell surface, and their capacity to present antigens were quite similar for cells from Ii°M° double-mutant mice and from animals carrying just the Ii° mutation. More surprising were certain features of the CD4+ T cell repertoire selected in Ii°M° mice: many fewer cells were selected than in Ii+M° animals, and these had been purged of self-reactive specificities, unlike their counterparts in Ii+M° animals. These findings suggest (i) that the peptides carried by class II molecules on stromal cells lacking H-2M complexes may almost all derive from invariant chain and (ii) that H-2M complexes edit the peptide array displayed on thymic stromal cells in the absence of invariant chain, showing that it can edit, in vivo, peptides other than CLIP.

Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins

An emerging theme in transforming growth factor-β (TGF-β) signalling is the association of the Smad proteins with diverse groups of transcriptional regulatory proteins. Several Smad cofactors have been identified to date but the diversity of TGF-β effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addressed the possible interaction of Smad proteins with the myocyte enhancer-binding factor 2 (MEF2) transcriptional regulators. Our studies indicate that Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins in a GAL4-based one-hybrid reporter gene assay. We have also observed in vivo interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S-transferase pull-down analysis. Immunofluorescence studies in C2C12 myotubes show that Smad2 and MEF2A co-localise in the nucleus of multinuclear myotubes during differentiation. Interestingly, phospho-acceptor site mutations of MEF2 that render it unresponsive to p38 MAP kinase signalling abrogate the cooperativity with the Smads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad–MEF2 interaction. Thus, the association between Smad2 and MEF2A may subserve a physical link between TGF-β signalling and a diverse array of genes controlled by the MEF2 cis element.

A framework for interpreting the leucine-rich repeats of the Listeria internalins

The surface protein InlB of the bacterial pathogen Listeria monocytogenes is required for inducing phagocytosis in various nonphagocytic mammalian cell types in vitro. InlB causes tyrosine phosphorylation of host cell adaptor proteins, activation of phosphoinositide 3-kinase, and rearrangements of the actin cytoskeleton. These events lead to phagocytic uptake of the bacterium by the host cell. InlB belongs to the internalin family of Listeria proteins, which also includes InlA, another surface protein involved in host cell invasion. The internalins are the largest class of bacterial proteins containing leucine-rich repeats (LRR), a motif associated with protein–protein interactions. The LRR motif is found in a functionally diverse array of proteins, including those involved in the plant immune system and in the mammalian innate immune response. Structural and functional interpretations of the sequences of internalin family members are presented in light of the recently determined x-ray crystal structure of the InlB LRR domain.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.

Preparation and properties of nido-carborane-specific monoclonal antibodies for potential use in boron neutron capture therapy for cancer.

As the first step of a research program aimed at developing a bispecific monoclonal antibody system for the delivery of boron-rich molecules to tumor cells for boron neutron capture therapy, monoclonal antibodies (mAbs) were produced against an anionic nido-carborane derivative, 4-[7,8-dicarbadodecahydroundecaborat(-1)-7-yl]butanoic acid. Two IgG subclass mAbs, designated HAW101 and HAW102, were identified that specifically bound the anionic nido-carborane hapten, as well as a variety of other anionic nido-carborane cage derivatives. By using surface plasmon resonance technology, the affinity constants of HAW101 and HAW102 were determined to be 1.9 x 10(9) and 6.8 x 10(8) M-1, respectively. A diverse array of 7-substituted and 7,8-disubstituted anionic nido-carborane derivatives reacted with the mAb HAW101 in competition ELISA, whereas anionic closo-polyhedral boranes showed negligible binding, suggesting a role for the open nido-carborane cage structure. These results suggest that mAbs such as HAW101, which bind anionic nido-carboranes, are useful in the development of bispecific mAbs for specific targeting and enhanced boron delivery to tumor sites.

Photogenerated o-Azaxylylenes: Mechanistic Studies and Synthetic Applications

This research sets out to build upon excited state o-azaxylylene cycloaddition. The mechanism behind the excitation and cycloaddition process of photogenerated o-azaxylylenes was determined experimentally. Time-correlated single-photon counting, steady-state spectroscopy, triplet quenching experiments, and quantum yield studies provided evidence suggesting that excited state intramolecular proton transfer is followed by intersystem crossing and stepwise addition to the tethered unsaturated pendant. In keeping with the principles of diversity oriented synthesis, a modular approach was taken to gain access to a diverse array of N,O,S-Polyheterocycles which were modified postphotochemically via Suzuki coupling to yield fused biaryls. Cycloaddition products, outfitted with halogens in the aromatic ring of the o-azaxylylene, proved to be reactive with a variety of boronic acids resulting in a rapid growth in structural complexity. A novel procedure was developed that utilized multiple o-azaxylylene cores in a photochemical cascade transformation yielding complex scaffolds of unprecedented topology. The photoprecursors were produced in a one-pot two-step sequence from commercially available starting materials, and upon irradiation yield structures containing up to five fused hetrocyclic rings, and showed complete diastereoselectivity.

Flotillins and the PHB domain protein family: Rafts, worms and anaesthetics

While our understanding of lipid microdomains has advanced in recent years, many aspects of their formation and dynamics are still unclear. In particular, the molecular determinants that facilitate the partitioning of integral membrane proteins into lipid raft domains are yet to be clarified. This review focuses on a family of raft-associated integral membrane proteins, termed flotillins, which belongs to a larger class of integral membrane proteins that carry an evolutionarily conserved domain called the prohibitin homology (PHB) domain. A number of studies now suggest that eucaryotic proteins carrying this domain have affinity for lipid raft domains. The PHB domain is carried by a diverse array of proteins including stomatin, podocin, the archetypal PHB protein, prohibitin, lower eucaryotic proteins such as the Dictyostelium discoideum proteins vacuolin A and vacuolin B and the Caenorhabditis elegans proteins unc-1, unc-24 and mec-2. The presence of this domain in some procaryotic proteins suggests that the PHB domain may constitute a primordial lipid recognition motif. Recent work has provided new insights into the trafficking and targeting of flotillin and other PHB domain proteins. While the function of this large family of proteins remains unclear, studies of the C. elegans PHB proteins suggest possible links to a class of volatile anaesthetics raising the possibility that these lipophilic agents could influence lipid raft domains. This review will discuss recent insights into the cell biology of flotillins and the large family of evolutionarily conserved PHB domain proteins.

Molecular control of cell type diversity in the developing spinal cord

1, During embryonic development, a diverse array of neurons and glia are generated at specific positions along the dorsoventral and rostro-caudal axes of the spinal cord from a common pool of precursor cells. 2. This cell type diversity can be distinguished by the spatially and temporally coordinated expression of several transcription factors that are also linked to cell type specification at a very early stage of spinal cord development. 3, Recent studies have started to uncover that the generation of cell type diversity in the developing spinal cord. Moreover, distinct cell types in the spinal cord appear to be determined by the spatially and temporally coordinated expression of transcription factors. 4. The expression of these factors also appears to be controlled by gradients of factors expressed by ventral and dorsal midline cells, namely Sonic hedgehog and members of the transforming growth factor-beta family. 5, Changes in the competence of precursor cells and local cell interactions may also play important roles in cell type specification within the developing spinal cord.

Changes in information processing in children and their implications for marketing strategy

This thesis examines children's consumer choice behaviour using an information processing perspective, with the fundamental goal of applying academic research to practical marketing and commercial problems. Proceeding a preface, which describes the academic and commercial terms of reference within which this interdisciplinary study is couched, the thesis comprises four discernible parts. Initially, the rationale inherent in adopting an information processing perspective is justified and the diverse array of topics which have bearing on children's consumer processing and behaviour are aggregated. The second part uses this perspective as a springboard to appraise the little explored role of memory, and especially memory structure, as a central cognitive component in children's consumer choice processing. The main research theme explores the ease with which 10 and 11 year olds retrieve contemporary consumer information from subjectively defined memory organisations. Adopting a sort-recall paradigm, hierarchical retrieval processing is stimulated and it is contended that when two items, known to be stored proximally in the memory organisation are not recalled adjacently, this discrepancy is indicative of retrieval processing ease. Results illustrate the marked influence of task conditions and orientation of memory structure on retrieval; these conclusions are accounted for in terms of input and integration failure. The third section develops the foregoing interpellations in the marketing context. A straightforward methodology for structuring marketing situations is postulated, a basis for segmenting children's markets using processing characteristics is adopted, and criteria for communicating brand support information to children are discussed. A taxonomy of market-induced processing conditions is developed. Finally, a case study with topical commercial significance is described. The development, launch and marketing of a new product in the confectionery market is outlined, the aetiology of its subsequent demise identified and expounded, and prescriptive guidelines are put forward to help avert future repetition of marketing misjudgements.

Psychological traits influence autonomic nervous system recovery following esophageal intubation in health and functional chest pain

Background: Esophageal intubation is a widely utilized technique for a diverse array of physiological studies, activating a complex physiological response mediated, in part, by the autonomic nervous system (ANS). In order to determine the optimal time period after intubation when physiological observations should be recorded, it is important to know the duration of, and factors that influence, this ANS response, in both health and disease. Methods: Fifty healthy subjects (27 males, median age 31.9 years, range 20-53 years) and 20 patients with Rome III defined functional chest pain (nine male, median age of 38.7 years, range 28-59 years) had personality traits and anxiety measured. Subjects had heart rate (HR), blood pressure (BP), sympathetic (cardiac sympathetic index, CSI), and parasympathetic nervous system (cardiac vagal tone, CVT) parameters measured at baseline and in response to per nasum intubation with an esophageal catheter. CSI/CVT recovery was measured following esophageal intubation. Key Results: In all subjects, esophageal intubation caused an elevation in HR, BP, CSI, and skin conductance response (SCR; all p < 0.0001) but concomitant CVT and cardiac sensitivity to the baroreflex (CSB) withdrawal (all p < 0.04). Multiple linear regression analysis demonstrated that longer CVT recovery times were independently associated with higher neuroticism (p < 0.001). Patients had prolonged CSI and CVT recovery times in comparison to healthy subjects (112.5 s vs 46.5 s, p = 0.0001 and 549 s vs 223.5 s, p = 0.0001, respectively). Conclusions & Inferences: Esophageal intubation activates a flight/flight ANS response. Future studies should allow for at least 10 min of recovery time. Consideration should be given to psychological traits and disease status as these can influence recovery. The psychological trait of neuroticism retards autonomic recovery following esophageal intubation in health and functional chest pain. © 2013 John Wiley & Sons Ltd.

Site-selective direct arylation of unprotected adenine nucleosides mediated by palladium and copper:insights into the reaction mechanism

Reaction conditions facilitating the site-selective direct aryl functionalisation at the C-8 position of adenine nucleosides have been identified. Many different aromatic components may be effectively cross-coupled to provide a diverse array of arylated adenine nucleoside products without the need for ribose or adenine protecting groups. The optimal palladium catalyst loading lies between 0.5 and 5 mol %. Addition of excess mercury to the reaction had a negligible affect on catalysis, suggesting the involvement of a homogeneous catalytic species. A study by transmission electron microscopy (TEM) shows that metal containing nanoparticles, ca. 3 nm with good uniformity, are formed during the latter stages of the reaction. Stabilised PVP palladium colloids (PVP=N-polyvinylpyrrolidone) are catalytically active in the direct arylation process, releasing homogenous palladium into solution. The effect of various substituted 2-pyridine ligand additives has been investigated. A mechanism for the site-selective arylation of adenosine is proposed. © 2008 Elsevier Ltd. All rights reserved.

Mosquito larvicides from cyanobacteria

Cyanobacteria (blue-green algae) produce a diverse array of toxic or otherwise bioactive metabolites. These allelochemicals may also play a role in defense against potential predators and grazers, particularly aquatic invertebrates and their larvae, including mosquitoes. Compounds derived from cyanobacteria collected from the Florida Everglades and other Florida waterways were investigated as insecticides against the mosquito Aedes aegypti, a vector of dengue and yellow fever. Screening of cyanobacterial biomass revealed several strains that exhibited mosquito larvicidal activity. Guided via bioassay guided fractionation, a non-polar compound from Leptolyngbya sp. 21-9-3 was found to be the most active component. Characterization revealed the prospective compound to be a monounsaturated fatty acid with the molecular formula C16H30O2. This is the first evidence of mosquito larvicidal activity for this particular fatty acid. With larvicidal becoming more prevalent, fatty acids should be explored for future mosquito control strategies.^

Sources, Fate and Transformation of Organic Matter in Wetlands and Estuaries

Dissolved organic matter (DOM) is a complex mixture of organic compounds and represents the largest reservoirs of carbon (C) on earth. Particulate organic matter (POM) is another important carbon component in C cycling and controls a variety of biogeochemical processes. Estuaries, as important interfaces between land and ocean, play important roles in retaining and transforming such organic matter (OM) and serve as both sources and sinks of DOM and POM. There is a diverse array of both autochthonous and allochthonous OM sources in wetland/estuarine ecosystems. A comprehensive study on the sources, transformation and fate of OM in such ecosystems is essential in advancing our understanding of C cycling and better constraining the global C budget. In this work, DOM characteristics were investigated in different estuaries. Dissolved organic matter source strengths and dynamics were assessed in a seagrass-dominated subtropical estuarine lagoon. DOM dynamics controlled by hydrology and seagrass primary productivity were confirmed, and the primary source of DOM was quantified using the combination of excitation emission matrix fluorescence with parallel factor analysis (EEM-PARAFAC) and stable C isotope analysis. Seagrass can contribute up to 72% of the DOM in the study area. The spatial and temporal variation of DOM dynamics was also studied in a freshwated dominated estuary fringed with extensive salt marshes. The data showed that DOM was primarily derived from freshwater marshes and controlled by hydrology while salt marsh plants play a significant role in structuring the distribution patterns of DOM quality and quantity. The OM dynamics was also investigated in a mangrove-dominate estuary and a comparative study was conducted between the DOM and POM pools. The results revealed both similarity and dissimilarity in DOM and POM composition. The dynamics of both OM pools are largely uncoupled as a result of source differences. Fringe mangrove swamps are suggested to export similar amounts of DOM and POM and should be considered as an important source in coastal C budgets. Lastly, chemical characterizations were conducted on the featured fluorescence component in OM in an attempt to better understand the composition and origins of the specific PARAFAC component. The traditionally defined ‘protein-like’ fluorescence was found to contain both proteinaceous and phenolic compounds, suggesting that the application of this parameter as a proxy for amino acid content and bioavailability may be limited.

Prevalence of Aspergillus section Fumigati in Portuguese slaughterhouses: a fungal and mycotoxin concern

Introduction - Within the Aspergillus genus, Aspergillus fumigatus species is one of the most ubiquitous saprophytic fungi and is considered the species with higher clinical relevance. The fungi belonging to the Fumigati section are the most common cause of invasive aspergillosis and a major source of infection related mortality in immunocompromised patients. One of the most abundant metabolites produced by Aspergillus fumigatus is the metabolite gliotoxin, which exhibits a diverse array of biologic effects on the immune system. Further, environments contaminated with A. fumigatus may be the cause or enhance respiratory problems in the workers of those specific settings. These species produce specific allergens and mycotoxins that could cause respiratory disorders. Aim of the study - The aim of the present work was to determine the prevalence of Aspergillus section Fumigati by cultural and molecular methods in poultry; swine and bovine; and large animal (bovine and horses) slaughterhouses.

Bioexploration of wild Alaskan berries: From field screening to functional food

Wild berries are fundamental components of traditional diet and medicine for Native American and Alaska Native tribes and contain a diverse array of phytochemicals, including anthocyanins and proanthocyanidins, with known efficacy against metabolic disorders. Bioexploration represents a new paradigm under which bioactive preparations are screened in coordination with indigenous communities, to prepare for subsequent in-depth chemical and biological analysis. The inclusive, participatory philosophical approach utilized in bioexploration has additional benefits that could be realized in seemingly disparate areas, such as education and economics. Five species of wild Alaskan berries (Vaccinium uliginosum, V. ovalifolium, Empetrum nigrum, Rubus chamaemorus, and R. spectabilis) were tested using “Screens-to-Nature” (STN), a community-participatory approach to screen for potential bioactivity, in partnership with tribal members from three geographically distinct Alaskan villages: Akutan, Seldovia, and Point Hope. Berries were subsequently evaluated via HPLC and LC-MS2, yielding significant species and location-based variation in anthocyanins (0.9-438.6 mg eq /100g fw) and proanthocyanins (73.7-625.2 mg eq /100g fw). A-type proanthocyanidin dimers through tetramers were identified in all species tested. Berries were analyzed for in vitro and in vivo activity related to diabetes and obesity. R. spectabilis samples increased preadipocyte-factor-1 levels by 82% over control, and proanthocyanidin-rich fractions from multiple species reduced lipid accumulation in 3T3-L1 adipocytes. Furthermore, extracts of V. uliginosum and E. nigrum (Point Hope) reduced serum glucose levels in C57bl/6j mice up to 45%. The same precepts of bioexploration, especially the inclusion of indigenous community perspectives and knowledge, have relevance in other areas of study, such as education and economics. Studies have established the apathetic, low-motivational environment characteristic of many introductory science laboratory classes is detrimental to student interest, learning, and continuation in scientific education. A primary means of arresting this decline and stimulating the students’ attention and excitement is via engagement in hands-on experimentation and research. Using field workshops, the STN system is investigated as to its potential as a novel participatory educational tool, using assays centered around bioexploration and bioactive plant compounds that hold the potential to offset human health conditions. This evaluation of the STN system provided ample evidence as to its ability to augment and improve science education. Furthermore, Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis was employed as a theoretical framework to review the potential benefits and hurdles associated with developing a wild Alaskan berry commodity. Synthesizing various sources of information – including logistics and harvest costs, sources of initial capital, opportunities in the current superfruit industry, and socioeconomic factors – the development of a berry commodity proves to be a complex amalgam of competing factors which would require a delicate balance before proceeding.