Analysis Of Secreted Phosphoprotein (2ar; Osteopontin) Gene Expression And Association With Carcinogenesis

The murine cDNA encoding secreted phosphoprotein 1 (SPP) was cloned in this lab on the basis of induction of the mRNA (originally designated 2ar) by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in JB6 epidermal cells. The polypeptide predicted from the cDNA sequence is 84% identical to rat osteopontin (44 kDa bone phosphoprotein; sialoprotein I). Many primary structural features are conserved, including an ArgGlyAsp (RGD) cell adhesion site and an unusual series of 9-10 consecutive aspartate residues. SPP mRNA is most abundant in vivo in bone and kidney, and was localized to the medulla region of adult mouse kidney by in situ hybridization. Portions of the predicted SPP polypeptide were expressed as cro-{dollar}\beta{dollar}-galactosidase fusion proteins, and polyclonal antisera were obtained. The SPP antisera recognized the major ({dollar}\sp{lcub}32{rcub}{dollar}P) orthophosphate-labeled protein secreted by all mouse and rat cell lines studied. This observation led to the discovery that SPP is closely related or identical to a transformation-related phosphoprotein, also known as pp69. SPP mRNA was transiently induced by treatment of mouse epidermis in vivo with 12-O-tetradecanoylphorbol-13-acetate, and was constitutively expressed in 2 of 3 epidermal papillomas and 7 of 7 squamous cell carcinomas. SPP expression was elevated in T24 H-ras-transformed mouse fibroblasts, and the level of expression correlated with the metastatic ability of these cells. It is proposed that SPP may act as an autocrine adhesion factor for tumor cells. SPP is encoded by a single copy mouse gene which spans approximately 9 kilobasepairs (kb). A genomic clone containing the first 6 exons and 16 kb of 5{dollar}\sp\prime{dollar} flanking DNA was isolated. The SPP promoter region contains a TATA-like box (TTTAAA), a CCAAT box (reverse complement), and a number of potential regulatory elements. A small fragment ({dollar}-{dollar}235 to +79) was able to direct high level tumor promoter-inducible expression of a fused marker gene

The Stability of Carotenogenic Food Colorants and Strategies to Prolong the Shelf Life in Process Cheese Spread

Food quality is often times measured by the way one perceives the food, particularly with respect to color and texture. Color quality often pre-determines expectation making it an important parameter to understand. The same can be said for textural properties of food. Maintaining color and texture are just two ways in which the shelf-life of food can be measured. One particular product that has experienced problems in this area is processed cheese spread in which the US military uses as part of the Meal, Ready-to-Eat (MRE) rations. The cheese spread is one of the most highly accepted products in the MRE’s; therefore, research was necessary to determine formulary changes that could be made in order to improve product quality and increase the parameters of its shelf life. Studies were done to determine the cheese-age effect and ingredient effects for the addition of vitamins, colorants, emulsifiers, and stabilizers. The greatest improvement for the problems of hardening and darkening over time was observed when vitamins were removed from the product. Colorants were studied in the cheese spread, as well as in model systems. Carotenoid pigments were selected to determine stability against the effects of light and oxygen, and to measure antioxidant capacity after exposure to ozone. These compounds are responsible for the yellow, orange, and reds observed in fruits, vegetables, and some algal species. Extraction from the natural source has made carotenoid pigments commercially available to the food industry. Environmental influences such as atmosphere and lighting do affect the stability of carotenogenic compounds by causing structural degradation which in turn causes changes in antioxidant abilities

Inhibitory Synapses Get Madd for Neuroligin

The mechanisms mediating the appropriate clustering of neurotransmitter receptors opposite release sites are poorly understood. Two studies in this issue of Neuron, Maro et al. (2015) and Tu et al. (2015), identify a new extracellular effector for neuroligin in GABAergic postsynaptic differentiation

Comparison of Raw Dairy Manure Slurry and Anaerobically Digested Slurry as N Sources for Grass Forage Production

We conducted a 3-year field study to determine how raw dairy slurry and anaerobically digested slurry (dairy slurry and food waste) applied via broadcast and subsurface deposition to reed canarygrass (Phalaris arundinacea) affected forage biomass, N uptake, apparent nitrogen recovery (ANR), and soil nitrate concentrations relative to urea. Annual N applications ranged from 600 kg N ha−1 in 2009 to 300 g N ha−1 in 2011. Forage yield and N uptake were similar across slurry treatments. Soil nitrate concentrations were greatest at the beginning of the fall leaching season, and did not differ among slurry treatments or application methods. Urea-fertilized plots had the highest soil nitrate concentrations but did not consistently have greatest forage biomass. ANR for the slurry treatments ranged from 35 to 70% when calculations were based on ammonium-N concentration, compared with 31 to 65% for urea. Slurry ANR calculated on a total N basis was lower (15 to 40%) due to lower availability of the organic N in the slurries. No consistent differences in soil microbial biomass or other biological indicators were observed. Anaerobically digested slurry supported equal forage production and similar N use efficiency when compared to raw dairy slurry

Interaction of the Postsynaptic Density-95/Guanylate Kinase Domain-Associated Protein Complex with a Light Chain of Myosin-V and Dynein

NMDA receptors interact directly with postsynaptic density-95 (PSD-95), a scaffold protein that organizes a cytoskeletal- signaling complex at the postsynaptic membrane. The molecular mechanism by which the PSD-95-based protein complex is trafficked to the postsynaptic site is unknown but presumably involves specific motor proteins. Here we demonstrate a direct interaction between the PSD-95-associated protein guanylate kinase domain-associated protein (GKAP) and dynein light chain (DLC), a light chain subunit shared by myosin-V (an actin-based motor) and cytoplasmic dynein (a microtubule-based motor). A yeast two-hybrid screen with GKAP isolated DLC2, a novel protein 93% identical to the previously cloned 8 kDa dynein light chain (DLC1). A complex containing PSD-95, GKAP, DLC, and myosin-V can be immunoprecipitated from rat brain extracts. DLC colocalizes with PSD-95 and F-actin in dendritic spines of cultured neurons and is enriched in biochemical purifications of PSD. Immunogold electron microscopy reveals a concentration of DLC in the postsynaptic compartment of asymmetric synapses of brain in which it is associated with the PSD and the spine apparatus. We discuss the possibility that the GKAP/DLC interaction may be involved in trafficking of the PSD-95 complex by motor proteins

CRIPT, a Novel Postsynaptic Protein that Binds to the Third PDZ Domain of PSD-95/SAP90

AbstractThe synaptic protein PSD-95/SAP90 binds to and clusters a variety of membrane proteins via its two N-terminal PDZ domains. We report a novel protein, CRIPT, which is highly conserved from mammals to plants and binds selectively to the third PDZ domain (PDZ3) of PSD-95 via its C terminus. While conforming to the consensus PDZ-binding C-terminal sequence (X-S/T-X-V-COOH), residues at the -1 position and upstream of the last four amino acids of CRIPT determine its specificity for PDZ3. In heterologous cells, CRIPT causes a redistribution of PSD-95 to microtubules. In brain, CRIPT colocalizes with PSD-95 in the postsynaptic density and can be coimmunoprecipitated with PSD-95 and tubulin. These findings suggest that CRIPT may regulate PSD-95 interaction with a tubulin-based cytoskeleton in excitatory synapses

Clinical use of amyloid-positron emission tomography neuroimaging: Practical and bioethical considerations

Until recently, estimation of β-amyloid plaque density as a key element for identifying Alzheimer's disease (AD) pathology as the cause of cognitive impairment was only possible at autopsy. Now with amyloid-positron emission tomography (amyloid-PET) neuroimaging, this AD hallmark can be detected antemortem. Practitioners and patients need to better understand potential diagnostic benefits and limitations of amyloid-PET and the complex practical, ethical, and social implications surrounding this new technology. To complement the practical considerations, Eli Lilly and Company sponsored a Bioethics Advisory Board to discuss ethical issues that might arise from clinical use of amyloid-PET neuroimaging with patients being evaluated for causes of cognitive decline. To best address the multifaceted issues associated with amyloid-PET neuroimaging, we recommend this technology be used only by experienced imaging and treating physicians in appropriately selected patients and only in the context of a comprehensive clinical evaluation with adequate explanations before and after the scan

A Fully Integrated Real-Time Detection, Diagnosis, and Control of Community Diarrheal Disease Clusters and Outbreaks (the INTEGRATE Project):Protocol for an Enhanced Surveillance System

BACKGROUND:Diarrheal disease, which affects 1 in 4 people in the United Kingdom annually, is the most common cause of outbreaks in community and health care settings. Traditional surveillance methods tend to detect point-source outbreaks of diarrhea and vomiting; they are less effective at identifying low-level and intermittent food supply contamination. Furthermore, it can take up to 9 weeks for infections to be confirmed, reducing slow-burn outbreak recognition, potentially impacting hundreds or thousands of people over wide geographical areas. There is a need to address fundamental problems in traditional diarrheal disease surveillance because of underreporting and subsequent unconfirmed infection by patients and general practitioners (GPs); varying submission practices and selective testing of samples in laboratories; limitations in traditional microbiological diagnostics, meaning that the timeliness of sample testing and etiology of most cases remains unknown; and poorly integrated human and animal surveillance systems, meaning that identification of zoonoses is delayed or missed. OBJECTIVE:This study aims to detect anomalous patterns in the incidence of gastrointestinal disease in the (human) community; to target sampling; to test traditional diagnostic methods against rapid, modern, and sensitive molecular and genomic microbiology methods that identify and characterize responsible pathogens rapidly and more completely; and to determine the cost-effectiveness of rapid, modern, sensitive molecular and genomic microbiology methods. METHODS:Syndromic surveillance will be used to aid identification of anomalous patterns in microbiological events based on temporal associations, demographic similarities among patients and animals, and changes in trends in acute gastroenteritis cases using a point process statistical model. Stool samples will be obtained from patients' consulting GPs, to improve the timeliness of cluster detection and characterize the pathogens responsible, allowing health protection professionals to investigate and control outbreaks quickly, limiting their size and impact. The cost-effectiveness of the proposed system will be examined using formal cost-utility analysis to inform decisions on national implementation. RESULTS:The project commenced on April 1, 2013. Favorable approval was obtained from the Research Ethics Committee on June 15, 2015, and the first patient was recruited on October 13, 2015, with 1407 patients recruited and samples processed using traditional laboratory techniques as of March 2017. CONCLUSIONS:The overall aim of this study is to create a new One Health paradigm for detecting and investigating diarrhea and vomiting in the community in near-real time, shifting from passive human surveillance and management of laboratory-confirmed infection toward an integrated, interdisciplinary enhanced surveillance system including management of people with symptoms. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):DERR1-10.2196/13941