The senescence accelerated mouse (SAMP8) as a model for oxidative stress and Alzheimer's disease

The senescence accelerated mouse (SAMP8) is a spontaneous animal model of overproduction of amyloid precursor protein (APP) and oxidative damage. It develops early memory disturbances and changes in the blood-brain barrier resulting in decreased efflux of amyloid-β protein from the brain. It has a marked increase in oxidative stress in the brain. Pharmacological treatments that reduce oxidative stress improve memory. Treatments that reduce amyloid-β (antisense to APP and antibodies to amyloid-β) not only improve memory but reduce oxidative stress. Early changes in lipid peroxidative damage favor mitochondrial dysfunction as being a trigger for amyloid-β overproduction in this genetically susceptible mouse strain. This sets in motion a cycle where the increased amyloid-beta further damages mitochondria. We suggest that this should be termed the Inflammatory-Amyloid Cycle and may well be similar to the mechanisms responsible for the pathophysiology of Alzheimer's disease. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP⁺ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP⁺. A comparison of several crystal forms of OTEMO bound to FAD and NADP⁺ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k_cat/K_m) favors 2-n-hexyl cyclopentanone (4.3 × 10⁵ M⁻¹ s⁻¹) as a substrate, although its affinity (K_m = 32 μM) was lower than that of the CoA-activated substrate (K_m = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.     

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase

Tissue transglutaminase undergoes thermal inactivation with first-order kinetics at moderate temperatures, in a process which is affected in opposite way by the regulatory ligands calcium and GTP, which stabilize different conformations. We have explored the processes of inactivation and of unfolding of transglutaminase and the effects of ligands thereon, combining approaches of differential scanning calorimetry (DSC) and of thermal analysis coupled to fluorescence spectroscopy and small angle scattering. At low temperature (38-45°C), calcium promotes and GTP protects from inactivation, which occurs without detectable disruption of the protein structure but only local perturbations at the active site. Only at higher temperatures (52-56°C), the protein structure undergoes major rearrangements with alterations in the interactions between the N- and C-terminal domain pairs. Experiments by DSC and fluorescence spectroscopy clearly indicate reinforced and weakened interactions of the domains in the presence of GTP and of calcium, and different patterns of unfolding. Small angle scattering experiments confirm different pathways of unfolding, with attainment of limiting values of gyration radius of 52, 60 and 90?? in the absence of ligands and in the presence of GTP and calcium. Data by X-rays scattering indicate that ligands influence retention of a relatively compact structure in the protein even after denaturation at 70°C. These results suggest that the complex regulation of the enzyme by ligands involves both short- and long-range effects which might be relevant for understanding the turnover of the protein in vivo.     

Lead optimisation of pyrazoles as novel FPR1 antagonists

Optimisation of a series of pyrazole inhibitors of the human FPR1 receptor has been achieved. The use of an in vitro media loss assay was utilised to identify sub-series with more robust DMPK profiles. These were subsequently improved to generate analogues with attractive overall profiles.     

The dynamics of sexual contact networks: effects on disease spread and control

Sexually transmitted pathogens persist in populations despite the availability of biomedical interventions and knowledge of behavioural changes that would reduce individual-level risk. While behavioural risk factors are shared between many sexually transmitted infections, the prevalence of these diseases across different risk groups varies. Understanding this heterogeneity and identifying better control strategies depends on an improved understanding of the complex social contact networks over which pathogens spread. To date, most efforts to study the impact of sexual network structure on disease dynamics have focused on static networks. However, the interaction between the dynamics of partnership formation and dissolution and the dynamics of transmission plays a role, both in restricting the effective network accessible to the pathogen, and in modulating the transmission dynamics. We present a simple method to simulate dynamical networks of sexual partnerships. We inform the model using survey data on sexual attitudes and lifestyles, and investigate how the duration of infectiousness changes the effective contact network over which disease may spread. We then simulate several control strategies: screening, vaccination and behavioural interventions. Previous theory and research has advanced the importance of core groups for spread and control of STD. Our work is consistent with the importance of core groups, but extends this idea to consider how the duration of infectiousness associated with a particular pathogen interacts with host behaviours to define these high risk subpopulations. Characteristics of the parts of the network accessible to the pathogen, which represent the network structure of sexual contacts from the “point of view” of the pathogen, are substantially different from those of the network as a whole. The pathogen itself plays an important role in determining this effective network structure; specifically, we find that if the pathogen’s duration of infectiousness is short, infection is more concentrated in high-activity, high-concurrency individuals even when all other factors are held constant. Widespread screening programmes would be enhanced by follow-up interventions targeting higher-risk individuals, because screening shortens the expected duration of infectiousness and causes a greater relative decrease in prevalence among lower-activity than in higher-activity individuals. Even for pathogens with longer durations of infectiousness, our findings suggest that targeting vaccination and behavioural interventions towards high-activity individuals provides comparable benefits to population-wide interventions.     

Tracking the expression of excitatory and inhibitory neurotransmission-related proteins and neuroplasticity markers after noise induced hearing loss

Excessive exposure to loud noise can damage the cochlea and create a hearing loss. These pathologies coincide with a range of CNS changes including reorganisation of frequency representation, alterations in the pattern of spontaneous activity and changed expression of excitatory and inhibitory neurotransmitters. Moreover, damage to the cochlea is often accompanied by acoustic disorders such as hyperacusis and tinnitus, suggesting that one or more of these neuronal changes may be involved in these disorders, although the mechanisms remain unknown. We tested the hypothesis that excessive noise exposure increases expression of markers of excitation and plasticity, and decreases expression of inhibitory markers over a 32-day recovery period. Adult rats (n = 25) were monaurally exposed to a loud noise (16 kHz, 1/10(th) octave band pass (115 dB SPL)) for 1-hour, or left as non-exposed controls (n = 5). Animals were euthanased at either 0, 4, 8, 16 or 32 days following acoustic trauma. We used Western Blots to quantify protein levels of GABA(A) receptor subunit α1 (GABA(A)α1), Glutamic-Acid Decarboxylase-67 (GAD-67), N-Methyl-D-Aspartate receptor subunit 2A (NR2A), Calbindin (Calb1) and Growth Associated Protein 43 (GAP-43) in the Auditory Cortex (AC), Inferior Colliculus (IC) and Dorsal Cochlear Nucleus (DCN). Compared to sham-exposed controls, noise-exposed animals had significantly (p<0.05): lower levels of GABA(A)α1 in the contralateral AC at day-16 and day-32, lower levels of GAD-67 in the ipsilateral DCN at day-4, lower levels of Calb1 in the ipsilateral DCN at day-0, lower levels of GABA(A)α1 in the ipsilateral AC at day-4 and day-32. GAP-43 was reduced in the ipsilateral AC for the duration of the experiment. These complex fluctuations in protein expression suggests that for at least a month following acoustic trauma the auditory system is adapting to a new pattern of sensory input.     

AVPR1A and SLC6A4 polymorphisms in choral singers and non-musicians: a gene association study

Amateur choral singing is a common pastime and worthy of study, possibly conferring benefits to health and social behaviour. Participants might be expected to possess musical ability and share some behavioural characteristics. Polymorphisms in genes concerned with serotonergic neurotransmission are associated with both behaviour and musical aptitude. Those investigated previously include the variable number tandem repeats RS1, RS3 and AVR in the AVPR1A (arginine vasopressin receptor 1a) gene and STin2 in the SLC6A4 (solute carrier family 6 [neurotransmitter transporter, serotonin], member 4) gene, as well as the SLC6A4 promoter region polymorphism, 5-HTTLPR. We conducted a genetic association study on 523 participants to establish whether alleles at these polymorphisms occur more commonly in choral singers than in those not regularly participating in organised musical activity (non-musicians). We also analysed tagging single nucleotide polymorphisms (SNPs) for AVPR1A and SLC6A4 to determine whether other variants in these genes were associated with singer/non-musician status. At the STin2 polymorphism, overall association with singer/non-musician status was evident at P = 0.006. The 9-repeat (P = 0.04) and 12-repeat (P = 0.04) alleles were more common in singers and the 10-repeat allele less so (P = 0.009). Odds ratios were 0.73 (95% CI 0.57-0.94) for the 10-repeat allele and 2.47 (95% CI 0.88-6.94) for the rarer 9-repeat allele. No overall association was detected at P<0.05 between any other polymorphism and singer/non-musician status. Our null findings with respect to RS3, RS1 and AVR, polymorphisms associated with musical ability by other authors, suggest that choir membership may depend partly on factors other than musical ability. In a related musical project involving one participating choir, a new 40-part unaccompanied choral work, "Allele", was composed and broadcast on national radio. In the piece, each singer's part incorporated their personal RS3 genotype.     

Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O(2)) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.