Effect of cessation of phospholipid synthesis on the synthesis of a specific membrane-associated bacteriophage protein in Escherichia coli.

The major coat protein of the bacteriophage f1 is synthesized during infection of Escherichia coli and becomes tightly associated with the host membrane. This synthesis was studied in conjunction with the strain BB26-36, a mutant defective in phospholipid synthesis, to investigate basic questions concerning membrane protein and phospholipid synthesis. Coat protein synthesis is decreased in the absence of net phospholipid synthesis. The coat protein produced under these conditions is still found tightly associated with the membrane. Resumption of phospholipid synthesis leads to an increase in the synthesis and accumulation of the coat protein. Therefore, a correlation between coat protein and phospholipid synthesis seems to exist. However, the packaging of phage deoxyribonucleic acid into phage particles proceeds in the absence of phospholipid synthesis, and the number of phage particles produced appears to depend only on the amount of coat protein in the membrane.     

The effect of conjugated linoleic acid on transepithelial calcium transport and mediators of paracellular permeability in human intestinal-like Caco-2 cells

Conjugated linoleic acid (CLA) increases paracellular permeability across human intestinal-like Caco-2 cell monolayers, which transport Ca predominantly by the transcellular route. In vivo, however, paracellular Ca transport is the predominant route of Ca transport. Therefore, the objective of this study was to investigate the effect of CLA on transepithelial Ca transport in Caco-2 cells transporting Ca predominantly by the paracellular route. Cells were seeded onto permeable transport membranes and allowed to differentiate, over 14 d, into intestinal-like cell monolayers. Monolayers (n=9/treatment) were exposed to 0 (control) or 80 microM- 18:2, -cis-9, trans-11 CLA or -trans-10, cis-12 CLA for 14 d prior to Ca transport studies. Overall transepithelial Ca transport as well as transcellular and parcellular Ca transport was significantly increased (P<0.001) by exposure of Caco-2 cells to both isomers of CLA, an effect which appeared to be related to altered localization of zona occludens 1 (a tight junction protein).     

Synthesis and in vitro biological evaluation of a carbon glycoside analogue of morphine-6-glucuronide

Attachment of a glucose moiety to 6-beta-aminomorphine afforded compound 3, where the glucose moiety was linked to the C-6 nitrogen atom by a two-carbon bridge. The synthesis of 3 was accomplished in eight steps from 3-triisopropylsilyl-6-beta-aminomorphine and 2,3,4,6-tetra-O-benzyl-D-glucose. The C-glycoside 3 was prepared with the objective of examining a metabolically stable analogue of morphine-6-glucuronide and determining the potency and selectivity of opioid receptor binding. Competition binding assays showed that 3 bound to the mu opioid receptor with a Ki value of 3.5 nM. The C-glycoside 3 exhibited delta/mu and kappa/mu selectivity ratios of 76 and 165, respectively. The synthetic intermediate (i.e., benzyl precursor, compound 11) bound to the mu opioid receptor with a Ki value of 0.5 nM, was less selective for the mu opioid receptor. The [35S]GTPgammaS assay was used to evaluate the functional properties of compounds 3 and 11. Compound 3 was determined to be a full agonist at the mu opioid receptor, whereas compound 11 was found to be a partial agonist. Compound 3 was determined to be very stable in the presence of human liver S9, and rat and monkey liver microsomes: no detectable loss of 3 was observed up to 90 min. Compound 3 was also very stable at pH 2 and pH 7.4, suggesting that 3 possessed properties for sustained duration of action.     

Disease Mechanisms in ALS: Misfolded SOD1 Transferred Through Exosome-Dependent and Exosome-Independent Pathways

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20 % of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-β and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naïve cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1.     

Planning for psychiatric emergencies.

Hospital emergency services have been used increasingly in recent years. This has resulted in questions as to the true nature of patients'' complaints and the appropriateness of this type of care. Since the increase in the number of psychiatric emergency patients has paralleled that for patients at other types of emergency clinics a study was conducted at the Clarke Institute of Psychiatry, Toronto, to examine the situation prior to the inception in 1977 of a crisis intervention unit. It was found that most patients had both psychiatric and social difficulties, and it was considered that planning should concentrate on strategies for efficient management of the clinical problems.     

Nanopore analysis reveals differences in structural stability of ovine PrPC proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.     

Nanopore Analysis of Wild-Type and Mutant Prion Protein (PrPC): Single Molecule Discrimination and PrPC Kinetics

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP^C) in the central nervous system into the infectious isoform (PrP^Sc). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP^Sc. A number of pathogenic PrP^C mutants exist that are characterized by an increased propensity for conversion into PrP^Sc and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP^C conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP^C, (a PrP^C mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP^C capture event. Moreover, we present a four-state model to describe wild-type PrP^C kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP^C. These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.