Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages

In previous publications, we found that the membrane of macrophage phagolysosomes could be selectively radioiodinated in living cells. The technique required phagocytosis of lactoperoxidase covalently coupled to latex spheres (LPO-latex), followed by iodination on ice with Na125I and hydrogen peroxide. In this paper, we use the LPO-latex system to further analyze the composition and recycling of phagocytic vacuole membrane. Three approaches were employed to examine the polypeptide composition of the phagolysosome (PL) and plasma membranes (PM). The efficiency of intracellular iodination was increased by increasing lysosomal pH with chloroquine. By one-dimensional SDS PAGE, the heavily labeled chloroquine-treated PL exhibited the same labeled polypeptides as PM iodinated extracellularly with LPO-latex. Iodinated PL and PM were compared by two-dimensional gel electrophoresis. No differences in the isoelectric point and molecular weight of the major iodinated species were detected. Quantitative immune precipitation was performed with five specific antibodies directed against cell surface antigens. Four antibodies precipitated similar relative amounts of labeled antigen on the cell surface and endocytic vacuole. One antibody, secreted by hybridoma 2.6, detected a 21-kdalton polypeptide that was enriched sevenfold in PL membrane. This enrichment was cell surface-derived, since the amount of labeled 2.6 was increased sevenfold when iodinated PM was driven into the cell during latex uptake. Therefore, intracellular iodination primarily detects PL proteins that are identical to their PM counterparts. Additional studies employed electron microscope autoradiography to monitor the centrifugal flow of radiolabeled polypeptides from PL to PM. Cells were iodinated intralysosomally and returned to culture for only 5-10 min at 37°C. Most of the cell-associated label then redistributed to the cell surface or its adjacent area. Significant movement out of the lysosome compartment occurred even at 2°C and 22°C. Extensive and rapid membrane flow through the secondary lysosome presumably contributes to the great similarity between PM and PL membrane polypeptides

Diagnostic Classification Modeling of Rubric-Scored Constructed-Response Items

The need for formative assessments has led to the development of a psychometric framework known as diagnostic classification models (DCMs), which are mathematical measurement models designed to estimate the possession or mastery of a designated set of skills or attributes within a chosen construct. Furthermore, much research has gone into the practice of “retrofitting” diagnostic measurement models to existing assessments in order to improve their diagnostic capability. Although retrofitting DCMs to existing assessments can theoretically improve diagnostic potential, it is also prone to challenges including identifying multidimensional traits from largely unidimensional assessments, a lack of assessments that are suitable for the DCM framework, and statistical quality, specifically highly correlated attributes and poor model fit. Another recent trend in assessment has been a move towards creating more authentic constructed-response assessments. For such assessments, rubric-based scoring is often seen as method of providing reliable scoring and interpretive formative feedback. However, rubric-scored tests are limited in their diagnostic potential in that they are usually used to assign unidimensional numeric scores. It is the purpose of this thesis to propose general methods for retrofitting DCMs to rubric-scored assessments. Two methods will be proposed and compared: (1) automatic construction of an attribute hierarchy to represent all possible numeric score levels from a rubric-scored assessment and (2) using rubric criterion score level descriptions to imply an attribute hierarchy. This dissertation will describe these methods, discuss the technical and mathematical issues that arise in using them, and apply and compare both methods to a prominent rubric-scored test of critical thinking skills, the Collegiate Learning Assessment+ (CLA+). Finally, the utility of the proposed methods will be compared to a reasonable alternative methodology: the use of polytomous IRT models, including the Graded Response Model (GRM), the Partial Credit Model (PCM), and the Generalized-Partial Credit Model (G-PCM), for this type of test score data

The Membrane Polypeptides of the Vacuolar System: Composition and Recycling

A method has been developed to deliver an iodinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable enzymatically active form. The addition of LPO-Iatex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membranederived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrate both acid phosphatase and lactoperoxidase activities. The exposure of LPO-Iatex containing cells to 125-- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4°C leads to incorporation of label into TCA-precipitable material. The incorporated cell-associated label was present as monoiodotyrosine; negligible amounts were found in lipids. Cell viability remained\u3e 99%. Autoradiography at both the light and EM level revealed that \u3e 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents

An extended hybrid density functional (X3LYP) with improved descriptions of nonbond interactions and thermodynamic properties of molecular systems

We derive here the form for the exact exchange energy density for a density that decays with Gaussian-type behavior at long range. This functional is intermediate between the B88 and the PW91 exchange functionals. Using this modified functional to match the form expected for Gaussian densities, we propose the X3LYP extended functional. We find that X3LYP significantly outperforms Becke three parameter Lee–Yang–Parr (B3LYP) for describing van der Waals and hydrogen bond interactions, while performing slightly better than B3LYP for predicting heats of formation, ionization potentials, electron affinities, proton affinities, and total atomic energies as validated with the extended G2 set of atoms and molecules. Thus X3LYP greatly enlarges the field of applications for density functional theory. In particular the success of X3LYP in describing the water dimer (with Re and De within the error bars of the most accurate determinations) makes it an excellent candidate for predicting accurate ligand–protein and ligand–DNA interactions

Brain delivery of vasoactive intestinal peptide (VIP) following nasal administration to rats

The aim of this work was to study in rats the nasal route for the brain delivery of the vasoactive intestinal peptide (VIP) neuropeptide. After evaluating VIP stability in solutions obtained from nasal washes, the effect of formulation parameters (pH 4-9, 0-1% (w/v) lauroylcarnitine (LC), hypo- or isoosmolality) on the brain uptake of intranasally administered VIP (10(-8)M)/125I-VIP (300,000 cpm/ml) was studied, using an in situ perfusion technique. Brain radioactivity distribution was assessed by quantitative autoradiographic analysis. Results were compared to intravenously administered VIP. With a hypotonic formulation at pH 4 containing 0.1% LC and 1% bovine serum albumin, VIP stability was satisfactory and loss by adsorption was minimal. Using this formulation, around 0.11% of initial radioactivity was found in the brain after 30 min perfusion and was located in the olfactory bulbs, the midbrain and the cerebellum. HPLC analysis of brain and blood extracts demonstrated the presence of intact VIP in brain and its complete degradation in the blood compartment. By intravenous administration, no intact VIP was found either in brain or in blood. In conclusion, intact VIP could be delivered successfully to the brain using the intranasal route for administration

Resistance to the influences of others: Limits to the formation of a collective memory through conversational remembering

People often form collective memories by sharing their memories with others. Warnings about the reliability of one conversational participant can limit the extent to which conversations or other forms of postevent information can influence subsequent memory. Although this attenuation is consistently found for prewarnings, there are substantial reasons to suspect that, by carefully manipulating both individual characteristics of the listener in a conversation and the dynamics of the postevent conversation, one can restrict the effect even prewarnings have on the influence a speaker might have on the memory of a listener. Indeed, in situations in which a speaker contributes substantially to a conversation and the quality of memory of a listener is poor, prewarnings have the paradoxical effect of increasing the influence of the speaker on a listener's memory. Warnings may not always limit the formation of a collective memory.Fil: Muller, Felipe Juan. Universidad de Belgrano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hirst, William. New School for Social Research; Estados Unido

Anticancer drug delivery with transferrin targeted polymeric chitosan vesicles

The study reports the initial biological evaluation of targeted polymeric glycol chitosan vesicles as carrier systems for doxorubicin (Dox). Transferrin (Tf) was covalently bound to the Dox-loaded palmitoylated glycol chitosan (GCP) vesicles using dimethylsuberimidate (DMSI). For comparison, glucose targeted niosomes were prepared using N-palmitoyl glucosamine. Biological properties were studied using confocal microscopy, flow cytometry, and cytotoxicity assays as well as a mouse xenograft model. Tf vesicles were taken up rapidly with a plateau after 1-2 h and Dox reached the nucleus after 60-90 min. Uptake was not increased with the use of glucose ligands, but higher uptake and increased cytotoxicity were observed for Tf targeted as compared to GCP Dox alone. In the drug-resistant A2780AD cells and in A431 cells, the relative increase in activity was significantly higher for the Tf-GCP vesicles than would have been expected from the uptake studies. All vesicle formulations had a superior in vivo safety profile compared to the free drug. The in vitro advantage of targeted Tf vesicles did not translate into a therapeutic advantage in vivo. All vesicles reduced tumor size on day 2 but were overall less active than the free drug