Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

In vivo airway surface liquid Cl- analysis with solid-state electrodes.

The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl-] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl-] in the "thin" ASL (approximately 30 microm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl-] in vivo. In initial experiments, the electrode measured [Cl-] in physiologic salt solutions, small volume (7.6 microl) test solutions, and in in vitro cell culture models, with > or =93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl- measurements were made in the following airway regions and species. First, ASL [Cl-] was measured in normal human nasal cavity and averaged 117.3 +/- 11.2 mM (n = 6). Second, ASL [Cl-] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 +/- 1.8 mM; (n = 6) and 126.9 +/- 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 +/- 4.2 mM (n = 13); and monkey bronchus = 112.3 +/- 10.9 mM (n = 3). Third, Cl- measurements were made in small (1-2 mm) diameter airways of the rabbit (108.3 +/- 7.1 mM, n = 5) and monkey (128.5 +/- 6.8 mM, n = 3). The measured [Cl-], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology.     

Oxalate,germins, and higher-plant pathogens

Earlier surveys (1, B. G. Lane. [1991] FASEB J. 5, 2983-2901; 2, B. G. Lane. [1994] FASEB J. 8, 294-301) helped to uproot entrenched views of plant oxalate as a static substance. It is now recognized that oxalate oxidases (OXOs) found in the "true cereals" (barley, maize, oat, rice, rye, wheat), the so-called germin OXOs (G-OXOs), or simply germins, are involved in cereal defence responses to invasion by fungal pathogens and that they show promise of being valuable agents of plant defence in dicotyledons, where they are not found naturally. G-OXOs have very peculiar properties: (a) their water-soluble oligomeric structures and enzymic activity are stable during SDS-PAGE and nitrocellulose blotting, (b) their undenatured water-soluble forms are refractory to the action of broad-specificity proteases, (c) their water-insoluble forms occur abundantly (approximately 50%) in the extracellular matrix (cell walls) of wheat, and probably in varying amounts in the cell walls of other true cereals. Transfer of the wheat G-OXO coding element to dicotyledons has been found, in all cases so far examined, to result in improved resistance to fungal pathogens. The possible nature of the improved resistance is discussed in relation to (a) generation of microcidal concentrations of hydrogen peroxide when the G-OXOs act on oxalate, (b) elicitation of hypersensitive cell death at lower concentrations of hydrogen peroxide, (c) formation of effective barriers against predator penetration by the hydrogen-peroxide-mediated lignification of cell walls, and (d) destruction of oxalate, which is an inhibitor of the hypersensitive response, a strategy of particular importance in the case of ubiquitous predator organisms such as Sclerotinia sclerotiorum, which secrete high concentrations of oxalate as a toxin.     

Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle

p97 (also called VCP), a member of the AAA ATPase family, is involved in several cellular processes, including membrane fusion and extraction of proteins from the endoplasmic reticulum for cytoplasmic degradation. We have studied the conformational changes that p97 undergoes during the ATPase cycle by cryo-EM and single-particle analysis. Three-dimensional maps show that the two AAA domains, D1 and D2, as well as the N-domains, experience conformational changes during ATP binding, ATP hydrolysis, P(i) release and ADP release. The N-domain is flexible in most nucleotide states except after ATP hydrolysis. The rings formed by D1 and D2 rotate with respect to each other, and the size of their axial openings fluctuates. Taken together, our results depict the movements that this and possibly other AAA ATPases can undergo during an ATPase cycle.     

Analysis of cholera toxin-ganglioside interactions by flow cytometry

Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.     

Field parasitism of Ceratitis capitata larvae by Aganaspis daci in Chios, Greece

High parasitism rates were recorded in the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in pupae derived from field infested figs, on the Greek island of Chios in 1999 and 2000. Adult parasitoids were identified as Aganaspis daci (Weld) (Hymenoptera:Eucoilidae), previously known as Trybliographa daci (Weld). Approximately 45%of C. capitata pupae yielded adultparasitoid in both years and the totalmortality of pupae due to the parasitoid was62–65%. Development of male A. daci at25°C, reared on 3rd instar larvaeof C. capitata, was shorter than that of the female (34 and 37 days respectively). Average adultmale longevity was 4–5 days longer than female(16–17 to 11–12 days, respectively) and almostidentical in wild and F₁ parasitoids ofboth sexes. We suggest that A. daci maybe an efficient form of biological control ofC. capitata in the Mediterranean regionand probably in other areas.