Neutron Reflectometry Studies Define Prion Protein N-terminal Peptide Membrane Binding

The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and α-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions.     

Specificity of catecholamine-induced growth in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica

The present study demonstrates that catecholamine responsiveness in Yersinia enterocolitica, a bacterial pathogen whose infectious spectrum is principally limited to the gut, is limited to norepinephrine and dopamine, and not epinephrine; this behavior contrasts with observations for two pathogens with a wider extra-gastrointestinal spectrum, Escherichia coli O157:H7 and Salmonella enterica, which respond to all three catecholamines. Epinephrine showed lower potency than norepinephrine and dopamine in inducing growth of E. coli and S. enterica, and was a potent antagonist of norepinephrine and dopamine growth responsiveness in Y. enterocolitica. Given that only norepinephrine and dopamine and not epinephrine-containing neurons are found with the enteric nervous system, the results suggest that certain of the more exclusive enteric pathogens may have developed response systems preferentially for those neuroendocrine hormones that are produced by the enteric nervous system as host-derived signals by which to sense the environment and initiate pathogenic processes.     

Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.     

Neural mechanisms of the cognitive model of depression

In the 40 years since Aaron Beck first proposed his cognitive model of depression, the elements of this model--biased attention, biased processing, biased thoughts and rumination, biased memory, and dysfunctional attitudes and schemas--have been consistently linked with the onset and maintenance of depression. Although numerous studies have examined the neural mechanisms that underlie the cognitive aspects of depression, their findings have not been integrated with Beck's cognitive model. In this Review, we identify the functional and structural neurobiological architecture of Beck's cognitive model of depression. Although the mechanisms underlying each element of the model differ, in general the negative cognitive biases in depression are facilitated by increased influence from subcortical emotion processing regions combined with attenuated top-down cognitive control.     

Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain

Although the N terminus of the prion protein (PrP^C) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23–110) and N2 (23–89) fragments derived from constitutive processing of PrP^C and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.     

Pollination of greenhouse tomatoes by the Australian bluebanded bee Amegilla (Zonamegilla) holmesi (Hymenoptera: Apidae)

The pollination effectiveness of bluebanded bees of the species Amegilla (Zonamegilla) holmesi Rayment (Hymenoptera: Apidae) was evaluated in tomato plants, Lycopersicon esculentum Miller (Solanaceae), cultivated in two greenhouse chambers. Bluebanded bee pollination was compared with mechanical pollination and no supplementary pollination. Pollination effectiveness was compared between treatments by using the percentage of fruit set, fruit weight, fruit diameter, fruit roundness, and the number of seeds per fruit. Both the bluebanded bee pollination and the mechanical pollination treatments significantly increased fruit set, individual fruit weight, and diameter compared with the control treatment. Fruit were also significantly rounder and contained significantly more seeds. Positive correlations were found for fruit weight versus seed number, maximum diameter versus seed number and minimum diameter versus seed number. We conclude that the use of A. holmesi for pollinating greenhouse tomatoes in Australia may be an effective alternative to the use of mechanical pollination.     

Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH

Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the ‘rhombic’ fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the ‘axial’ form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.     

A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus)

The Tsaatan (or Dhuka) peoples of northern-western Mongolia are one of the few remaining reindeer-herding cultural groups in the world. Recently a disease condition that involves sudden death of reindeer and cases involving fever, lethargy, and pale mucous membranes has been reported. Examination of blood smears collected in the 2005 field season resulted in the identification of intra-erythrocytic inclusions resembling Anaplasma spp. in smears from clinically sick animals. Using universal polymerase chain reaction (PCR) primers for the amplification of the 60 kDa chaperonin gene (cpn60, also known as hsp60 or groEL), we detected sequences corresponding to Anaplasma ovis in reindeer blood samples. Species-specific PCR primers for A. ovis were designed and validated and used to screen blood samples from Mongolian reindeer. Screening of 66 blood samples collected in the 2006 field season resulted in the detection of A. ovis in 80% of the samples. Our results indicate a high prevalence of A. ovis in the Tsaatan reindeer herds and an association with clinical disease that is likely to be anaplasmosis. To our knowledge this is the first report of natural A. ovis infection in reindeer.     

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.