Genetic evidence strongly supports the view that Aβ amyloid production is central to the cause of Alzheimer's disease. The kinetics, compartmentation, and form of Aβ and its temporal relation to the neurodegenerative process remain uncertain. The levels of soluble and insoluble Aβ were determined by using western blot techniques, and the findings were assessed in relation to indices of severity of disease. The mean level of soluble Aβ is increased threefold in Alzheimer's disease and correlates highly with markers of disease severity. In contrast, the level of insoluble Aβ (also a measure of total amyloid load) is found only to discriminate Alzheimer's disease from controls, and does not correlate with disease severity or numbers of amyloid plaques. These findings support the concept of several interacting pools of Aβ, that is, a large relatively static insoluble pool that is derived from a constantly turning over smaller soluble pool. The latter may exist in both intracellular and extracellular compartments, and contain the basic forms of Aβ that cause neurodegeneration. Reducing the levels of these soluble Aβ species by threefold to levels found in normal controls might prove to be a goal of future therapeutic intervention.

Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.

SummaryPlasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in Plasmodium berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro, and reduced capacity to cause severe disease in a cerebral malaria model. Thus, while not essential for parasite survival, TRX2 may help to optimize PTEX activity. Importantly, the generation of TRX2 knockout parasites that display altered phenotypes provides a much-needed tool to dissect PTEX function.

Background: Skeletal muscle fiber formation requires myoblast cell-cell membrane contact and fusion. Results: A versican-rich pericellular matrix surrounding myoblasts is proteolytically cleared by ADAMTS versicanases facilitating myoblast contact and fusion. Conclusion: Versican processing by ADAMTS versicanases contribute to muscle fiber formation. Significance: Targeting versican remodeling could enhance the regenerative capacity of muscle by improving muscle fiber fusion during regeneration.

Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta peptide (Abeta), the major biomarker of AD, in the presence of copper ions reduces Cu(2+) to Cu(+) and catalyses the formation of H(2)O(2) that subsequently induces radicals through Fenton chemistry. Abeta is also subject to attack by free radicals, where the presence of Cu(2+) in conjunction with H(2)O(2) catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of Abeta, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of Abeta (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of Abeta and may relate to changes in properties of the peptide following MCO.

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenes ulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (rDPH and rTMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, rDPH was decreased, whereas there was no change in rTMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.

The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.