Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin

Amyloid deposits of wild-type apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein, accumulate in atherosclerotic plaques where they may contribute to coronary artery disease by increasing plaque burden and instability. Using CD analysis, solid-state NMR spectroscopy, and transmission EM, we report here a surprising cooperative effect of heparin and the green tea polyphenol (-)- epigallocatechin-3-gallate (EGCG), a known inhibitor and modulator of amyloid formation, on apoA-I fibrils. We found that heparin, a proxy for glycosaminoglycan (GAG) polysaccharides that co-localize ubiquitously with amyloid in vivo, accelerates the rate of apoA-I formation from monomeric protein and associates with insoluble fibrils. Mature, insoluble apoA-I fibrils bound EGCG (KD = 30 ± 3 μM; Bmax = 40 ± 3 μM), but EGCG did not alter the kinetics of apoA-I amyloid assembly from monomer in the presence or absence of heparin. EGCG selectively increased the mobility of specific backbone and side-chain sites of apoA-I fibrils formed in the absence of heparin, but the fibrils largely retained their original morphology and remained insoluble. By contrast, fibrils formed in the presence of heparin were mobilized extensively by the addition of equimolar EGCG, and the fibrils were remodeled into soluble 20-nm-diameter oligomers with a largely α-helical structure that were nontoxic to human umbilical artery endothelial cells. These results argue for a protective effect of EGCG on apoA-I amyloid associated with atherosclerosis and suggest that EGCG-induced remodeling of amyloid may be tightly regulated by GAGs and other amyloid co-factors in vivo, depending on EGCG bioavailability

Orientation of a Diagnostic Ligand Bound to Macroscopically Aligned Amyloid-ß Fibrils Determined by Solid-state NMR

With amyloid diseases poised to become a major health burden in countries with aging populations, diagnostic molecules that aid the detection of amyloid in vitro and in vivo are of considerable clinical value. Understanding how such ligands recognize their amyloid targets would help to design diagnostics that target specific amyloid types associated with a particular disease, but methods to provide comprehensive information are underdeveloped. Here, solid-state NMR is used to determine the molecular orientation of the amyloid diagnostic 1-fluoro-2,5-bis[(E)-3-carboxy-4-hydroxystyryl]-benzene (FSB) when bound to fibrils of the Alzheimer’s amyloid-β polypeptide aligned on a planar substrate. The 19F NMR spectrum of the aligned complex reveals that FSB is oriented approximately parallel with the fibril long axis and bridges four hydrogen-bonded β-sheets. In addition to providing atomic details to aid the design of amyloid-specific diagnostics, this approach will also illuminate the molecular mechanisms of accessory molecules in amyloid disease

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

We evaluated the pharmacodynamic effects of the O6-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O6-methylguanine (O6-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O6-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O6-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O6-meG in PBMC DNA during treatment

Locomotor hyperactivity in 14-3-3Zeta KO mice is associated with dopamine transporter dysfunction

Dopamine (DA) neurotransmission requires a complex series of enzymatic reactions that are tightly linked to catecholamine exocytosis and receptor interactions on pre- and postsynaptic neurons. Regulation of dopaminergic signalling is primarily achieved through reuptake of extracellular DA by the DA transporter (DAT) on presynaptic neurons. Aberrant regulation of DA signalling, and in particular hyperactivation, has been proposed as a key insult in the presentation of schizophrenia and related neuropsychiatric disorders. We recently identified 14-3-3ζ as an essential component of neurodevelopment and a central risk factor in the schizophrenia protein interaction network. Our analysis of 14-3-3ζ-deficient mice now shows that baseline hyperactivity of knockout (KO) mice is rescued by the antipsychotic drug clozapine. 14-3-3ζ KO mice displayed enhanced locomotor hyperactivity induced by the DA releaser amphetamine. Consistent with 14-3-3ζ having a role in DA signalling, we found increased levels of DA in the striatum of 14-3-3ζ KO mice. Although 14-3-3ζ is proposed to modulate activity of the rate-limiting DA biosynthesis enzyme, tyrosine hydroxylase (TH), we were unable to identify any differences in total TH levels, TH localization or TH activation in 14-3-3ζ KO mice. Rather, our analysis identified significantly reduced levels of DAT in the absence of notable differences in RNA or protein levels of DA receptors D1–D5. Providing insight into the mechanisms by which 14-3-3ζ controls DAT stability, we found a physical association between 14-3-3ζ and DAT by co-immunoprecipitation. Taken together, our results identify a novel role for 14-3-3ζ in DA neurotransmission and provide support to the hyperdopaminergic basis of pathologies associated with schizophrenia and related disorders.H Ramshaw, X Xu, EJ Jaehne, P McCarthy, Z Greenberg, E Saleh, B McClure, J Woodcock, S Kabbara, S Wiszniak, Ting-Yi Wang, C Parish, M van den Buuse, BT Baune, A Lopez and Q Schwar

A phase I study of the safety and tolerability of olaparib (AZD2281, KU0059436) and dacarbazine in patients with advanced solid tumours

BACKGROUND: Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) is essential in cellular processing of DNA damage via the base excision repair pathway (BER). The PARP inhibition can be directly cytotoxic to tumour cells and augments the anti-tumour effects of DNA-damaging agents. This study evaluated the optimally tolerated dose of olaparib (4-(3--4-fluorophenyl) methyl-1(2H)-one; AZD2281, KU0059436), a potent PARP inhibitor, with dacarbazine and assessed safety, toxicity, clinical pharmacokinetics and efficacy of combination treatment. PATIENTS AND METHODS: Patients with advanced cancer received olaparib (20-200 mg PO) on days 1-7 with dacarbazine (600-800 mg m(-2) IV) on day 1 (cycle 2, day 2) of a 21-day cycle. An expansion cohort of chemonaive melanoma patients was treated at an optimally tolerated dose. The BER enzyme, methylpurine-DNA glycosylase and its substrate 7-methylguanine were quantified in peripheral blood mononuclear cells. RESULTS: The optimal combination to proceed to phase II was defined as 100 mg bd olaparib with 600 mg m(-2) dacarbazine. Dose-limiting toxicities were neutropaenia and thrombocytopaenia. There were two partial responses, both in patients with melanoma. CONCLUSION: This study defined a tolerable dose of olaparib in combination with dacarbazine, but there were no responses in chemonaive melanoma patients, demonstrating no clinical advantage over single-agent dacarbazine at these doses

Ecosystem development after mangrove wetland creation : plant–soil change across a 20-year chronosequence

This paper is not subject to U.S. copyright. The definitive version was published in Ecosystems 15 (2012): 848-866, doi:10.1007/s10021-012-9551-1.Mangrove wetland restoration and creation efforts are increasingly proposed as mechanisms to compensate for mangrove wetland losses. However, ecosystem development and functional equivalence in restored and created mangrove wetlands are poorly understood. We compared a 20-year chronosequence of created tidal wetland sites in Tampa Bay, Florida (USA) to natural reference mangrove wetlands. Across the chronosequence, our sites represent the succession from salt marsh to mangrove forest communities. Our results identify important soil and plant structural differences between the created and natural reference wetland sites; however, they also depict a positive developmental trajectory for the created wetland sites that reflects tightly coupled plant-soil development. Because upland soils and/or dredge spoils were used to create the new mangrove habitats, the soils at younger created sites and at lower depths (10–30 cm) had higher bulk densities, higher sand content, lower soil organic matter (SOM), lower total carbon (TC), and lower total nitrogen (TN) than did natural reference wetland soils. However, in the upper soil layer (0–10 cm), SOM, TC, and TN increased with created wetland site age simultaneously with mangrove forest growth. The rate of created wetland soil C accumulation was comparable to literature values for natural mangrove wetlands. Notably, the time to equivalence for the upper soil layer of created mangrove wetlands appears to be faster than for many other wetland ecosystem types. Collectively, our findings characterize the rate and trajectory of above- and below-ground changes associated with ecosystem development in created mangrove wetlands; this is valuable information for environmental managers planning to sustain existing mangrove wetlands or mitigate for mangrove wetland losses

Biophysical Factors Affecting the Distribution of Demersal Fish around the Head of a Submarine Canyon Off the Bonney Coast, South Australia

We sampled the demersal fish community of the Bonney Canyon, South Australia at depths (100–1,500 m) and locations that are poorly known. Seventy-eight species of demersal fish were obtained from 12 depth-stratified trawls along, and to either side, of the central canyon axis. Distributional patterns in species richness and biomass were highly correlated. Three fish assemblage groupings, characterised by small suites of species with narrow depth distributions, were identified on the shelf, upper slope and mid slope. The assemblage groupings were largely explained by depth (ρw = 0.78). Compared to the depth gradient, canyon-related effects are weak or occur at spatial or temporal scales not sampled in this study. A conceptual physical model displayed features consistent with the depth zonational patterns in fish, and also indicated that canyon upwelling can occur. The depth zonation of the fish assemblage was associated with the depth distribution of water masses in the area. Notably, the mid-slope community (1,000 m) coincided with a layer of Antarctic Intermediate Water, the upper slope community (500 m) resided within the core of the Flinders Current, and the shelf community was located in a well-mixed layer of surface water (<450 m depth)

X-ray Absorption and Reflection in Active Galactic Nuclei

X-ray spectroscopy offers an opportunity to study the complex mixture of emitting and absorbing components in the circumnuclear regions of active galactic nuclei, and to learn about the accretion process that fuels AGN and the feedback of material to their host galaxies. We describe the spectral signatures that may be studied and review the X-ray spectra and spectral variability of active galaxies, concentrating on progress from recent Chandra, XMM-Newton and Suzaku data for local type 1 AGN. We describe the evidence for absorption covering a wide range of column densities, ionization and dynamics, and discuss the growing evidence for partial-covering absorption from data at energies > 10 keV. Such absorption can also explain the observed X-ray spectral curvature and variability in AGN at lower energies and is likely an important factor in shaping the observed properties of this class of source. Consideration of self-consistent models for local AGN indicates that X-ray spectra likely comprise a combination of absorption and reflection effects from material originating within a few light days of the black hole as well as on larger scales. It is likely that AGN X-ray spectra may be strongly affected by the presence of disk-wind outflows that are expected in systems with high accretion rates, and we describe models that attempt to predict the effects of radiative transfer through such winds, and discuss the prospects for new data to test and address these ideas.Comment: Accepted for publication in the Astronomy and Astrophysics Review. 58 pages, 9 figures. V2 has fixed an error in footnote

The fitness for the Ageing Brain Study II (FABS II): protocol for a randomized controlled clinical trial evaluating the effect of physical activity on cognitive function in patients with Alzheimer's disease

Background: Observational studies have documented a potential protective effect of physical exercise in older adults who are at risk for developing Alzheimer's disease. The Fitness for the Ageing Brain II (FABS II) study is a multicentre randomized controlled clinical trial (RCT) aiming to determine whether physical activity reduces the rate of cognitive decline among individuals with Alzheimer's disease. This paper describes the background, objectives of the study, and an overview of the protocol including design, organization and data collection methods

Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the μ1 ϕ Domain

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein μ1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of μ1 indicates that the μ1 ϕ domain is sufficient to elicit a cell death response. To evaluate the contribution of the μ1 ϕ domain to the induction of apoptosis following reovirus infection, ϕ mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in ϕ diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in ϕ affect the apoptotic potential of reovirus, suggesting that ϕ initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective ϕ mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the ϕ domain of μ1 plays an important regulatory role in reovirus-induced apoptosis and disease