Structural Differences between Aβ(1-40) Intermediate Oligomers and Fibrils Elucidated by Proteolytic Fragmentation and Hydrogen/Deuterium Exchange

The aggregation of amyloid-β protein (Aβ) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Aβ(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Aβ peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.     

CHARACTER DISCRIMINATORY POWER,CHARACTER-SET CONGRUENCE,AND THE CLASSIFICATION OF INDIVIDUALS FROM HYBRID ZONES: AN EXAMPLE USING STONE CRABS (MENIPPE)

Many investigators categorize individuals from hybrid zones to facilitate comparisons among genotypic classes (e.g., parental, F₁, backcross) for comparative studies in which components of fitness or geographic variation are being analyzed. Frequently, multiple character sets representing genetically independent traits are used to classify these individuals and various methodologies are employed to combine the classifications obtained from the different character sets. We adapted the principles of total evidence and taxonomic congruence (two formalized approaches used by systematists in formulating phylogenetic hypotheses) to address the problem of discriminating hybridizing species and classifying individuals from hybrid zones. As our model, we used two morphological (coloration and morphometric) and two molecular (allozyme and mitochondrial DNA restriction-fragment-length polymorphism) character sets that differentiate two stone crab species (Menippe adina and M. mercenaria). Using principal-components analysis, we determined that combining character sets and eliminating characters or character sets that did not have large eigenvector coefficients for the principal component that best separated the two species yielded the highest level of discrimination between species and allowed us to classify a broad range of morpho-genotypes as hybrids. For the stone crabs, three diagnostic allozyme loci and five diagnostic coloration characters best separated the species. The two character sets were not completely congruent, but they agreed in their classification of 50% of the individuals from the hybrid zone and rarely strongly disagreed in their classifications. Classification discrepancies between the two character sets probably represent variation between traits in interspecific gene flow rather than intraspecific, ecologically mediated variation. Our results support the assertions of previous investigators who espoused the benefits associated with using multiple character sets to classify individuals from hybrid zones and demonstrate that, if character sets are reasonably congruent and numerically balanced, combining diagnostic characters from multiple character sets (a total-evidence approach) can enhance discriminatory power between species and facilitate the assignment of hybrid-zone individuals to genotypic classes. On the contrary, classifying hybrid-zone individuals using character sets separately (a taxonomic-congruence approach) provides the opportunity to compare levels of introgression between species and to assess reasons for discordance among the data sets.     

Responses of pyriform cortex neurons to excitatory amino acids: Voltage dependence,conductance changes,and effects of divalent cations

The actions of ionophoretically applied N-methyl aspartate (NMA), quisqualate, and kainate, thought to activate three different types of excitatory amino acid receptors, were studied on pyramidal neurons of the rat pyriform cortex, maintained in an isolated, submerged, and perfused brain slice. Intracellular recordings were made with either K acetate or CsCl electrodes. In most neurons all three agonists elicited monophasic responses which could be evoked at 20-sec intervals. Some neurons showed biphasic responses, most commonly to kainate but, on occasion, also for quisqualate. The slower component appeared to be correlated with excitotoxicity and, consequently, was difficult to study. As a result the kainate responses studied were from neurons selected for having a single component. In neurons selected for having a linear current-voltage relationship or neurons loaded with Cs to suppress K conductance and linearize the current-voltage relationship, the average changes in resistance recorded during ionophoretic responses at resting potential were as follows: NMA, 131.2 +/- 6.7% of control; kainate, 104.7 +/- 5.8% of control; and quisqualate, 92.8 +/- 2.8% of control. The magnitude and direction of the conductance change were very reproducible in any one neuron, but especially for kainate some cells showed clear conductance increases, while others showed clear conductance decreases. Using CsCl electrodes it was possible to reduce K+ conductance and depolarize the neurons over a wider range. By passing depolarizing current it was possible to reverse the responses. The response to all three agonists reversed at the same depolarized potential. This observation indicates that while there are differences in the ionic channels associated with the three agonists at resting potential, the channels have similar properties at more depolarized potentials. Responses to all three agonists were influenced by the concentrations of divalent cations in the perfusion medium. The NMA responses were most sensitive to Mg, increasing in amplitude in the absence of Mg and being depressed by Mg elevation. All responses were sensitive to Ca, with discharges being greatly increased by low Ca and depressed by high Ca. The kainate response was most sensitive to Ca concentration changes. Unlike reports from other preparations the apparent conductance decreases to NMA were not altered by the perfusion of solutions with either no added Mg or no added Ca. The NMA response was very much reduced in either Co (1-2 mM) or Zn (100-200 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 22 human primary and metastatic liver tumors, and expanded in vitro in the presence of either interleukin-2 (IL-2, 100 U/ml) plus tumor necrosis factor (TNF, 1000 U/ml), IL-2 (1000 U/ml) plus IL-4 (1000 U/ml) or IL-2 (1000 U/ml) alone. TIL proliferated in culture in 20/22 cases. Among different cytokine combinations, TNF and IL-2 were most effective in promoting the outgrowth of CD3⁺ CD8⁺ T lymphocytes (mean ± SEM: 90%±5) in the cultures of TIL from primary liver tumors. Cytotoxicity against autologous tumor cells was demonstrated in all early cultures of TIL from primary liver cancers in the presence of IL-2 plus TNF. In contrast, cultures of TIL derived from colon cancer metastatic to liver had significantly lower levels of autotumor cytotoxicity and proportions of CD3⁺ CD8⁺ cells (40%±13) than those of TIL from primary liver tumors. The addition on day 0 of interferons ( or ) to TIL cultured in the presence of TNF and IL-2, significantly augmented cytotoxicity against autologous tumor. In contrast, incubation of TIL in the presence of IL-4 and IL-2 did not result in increased autotumor responses in the cultures of TIL from primary liver tumors. The expansion (-fold) of TIL (day 30) cultured in the presence of IL-2 alone compared to that in the presence of TNF and IL-2 was significantly greater for hepatocellular carcinoma (median, 280 vs 260) than for autologous peripheral blood lymphocytes (36 vs 27), cholangiocarcinoma (42 vs 51) or TIL from metastatic colon cancer (39 vs 30). Outgrowth of TIL in IL-2 plus TNF offers an opportunity for in vitro enrichment in cells with autotumor cytotoxicity in primary liver tumors. However, this cytokine combination was unable to promote and sustain growth of autotumor effectors from TIL in metastatic liver cancer.Supported by ACS grants IM27 077 and IM588 A (TLW) and Organ Transplant Program Project 1P01-CA-4744501 AZ     

The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K⁺ which was accompanied by an equimolar net influx of H⁺. These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K⁺ during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K⁺ and H⁺ fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K⁺ concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K⁺ efflux and H⁺ influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K⁺/H⁺ exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.     

Gas mixing in the airways of dog lungs during high-frequency ventilation

Washout of insoluble inert test gases of different diffusivity (He and SF6 or He and Ar) from dog lungs was studied during high-frequency ventilation (HFV). Test gas equilibrium and subsequent washout were performed with HFV, succeeding measurements being performed at different stroke volumes (1.5-2.5 ml/kg body wt), oscillation frequencies (10-30 Hz), and with different lung volumes (32-74 ml X kg-1). Test gas concentrations were continuously measured by a mass spectrometer. The time course of washout could be described as the sum of two exponentials. There were no consistent differences in the time courses of washout between He and SF6 or between He and Ar. It is concluded that gas mixing in the airways during HFV is not significantly limited by diffusion, and this is suggested to apply during HFV to steady-state transport of respiratory gases (e.g., O2 and CO2) as well as to the transient state of inert gas washout.