Reduction of a-tocopherylquinone Model Compound With Various Reductant

In order to study the possibility of tranformation of a-tocopherylquinone (TQ) into a more oxidiseable compound and also to find out the recycling effect in the cells, an experiment was conducted by reducing the model compound 2-(3- hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ) with various reductants. In the experiment it was shown that glutathione did not reduce PQ,nor NADH by itself, so the effective reductant in the NADH/FAD combination must have been FADH2. Thus there is a probability that in a biological system, the most probable reductant for TQ would be a flavin enzyme rather that ascorbic acid or glutathione. The non-physiological dithiothreitol was as effective as NADH/ FAD which is interesting because of its similarity to the physiologically important reduced lipoic acid. The reactivity of the various reductants used in this experiment decrease in the order of dithiothreitol ~ NADH/FAD (8/10) > sodium dithionite > NADH/FAD (2:10) > sodium ascorbate > ascorbic acid (Fig.8)

Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time

Substitutions in the redox-sensing PAS domain of the NifL regulatory protein define an inter-subunit pathway for redox signal transmission

The Per-ARNT-Sim (PAS) domain is a conserved a/ß fold present within a plethora of signalling proteins from all kingdoms of life. PAS domains are often dimeric and act as versatile sensory and interaction modules to propagate environmental signals to effector domains. The NifL regulatory protein from Azotobacter vinelandii senses the oxygen status of the cell via an FAD cofactor accommodated within the first of two amino-terminal tandem PAS domains, termed PAS1 and PAS2. The redox signal perceived at PAS1 is relayed to PAS2 resulting in conformational reorganization of NifL and consequent inhibition of NifA activity. We have identified mutations in the cofactor-binding cavity of PAS1 that prevent 'release' of the inhibitory signal upon oxidation of FAD. Substitutions of conserved ß-sheet residues on the distal surface of the FAD-binding cavity trap PAS1 in the inhibitory signalling state, irrespective of the redox state of the FAD group. In contrast, substitutions within the flanking A'a-helix that comprises part of the dimerization interface of PAS1 prevent transmission of the inhibitory signal. Taken together, these results suggest an inter-subunit pathway for redox signal transmission from PAS1 that propagates from core to the surface in a conformation-dependent manner requiring a flexible dimer interface

Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of β-sheets into α-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Characterisation of FAD-family folds using a machine learning approach

Flavin adenine dinucleotide (FAD) and its derivatives play a crucial role in biological processes. They are major organic cofactors and electron carriers in both enzymatic activities and biochemical pathways. We have analysed the relationships between sequence and structure of FAD-containing proteins using a machine learning approach. Decision trees were generated using the C4.5 algorithm as a means of automatically generating rules from biological databases (TOPS, CATH and PDB). These rules were then used as background knowledge for an ILP system to characterise the four different classes of FAD-family folds classified in Dym and Eisenberg (2001). These FAD-family folds are: glutathione reductase (GR), ferredoxin reductase (FR), p-cresol methylhydroxylase (PCMH) and pyruvate oxidase (PO). Each FADfamily was characterised by a set of rules. The “knowledge patterns” generated from this approach are a set of rules containing conserved sequence motifs, secondary structure sequence elements and folding information. Every rule was then verified using statistical evaluation on the measured significance of each rule. We show that this machine learning approach is capable of learning and discovering interesting patterns from large biological databases and can generate “knowledge patterns” that characterise the FADcontaining proteins, and at the same time classify these proteins into four different families

Final report on the farmer's aid in plant disease diagnoses

This report is the final report on the FAD project. The FAD project was initiated in september 1985 to test the expert system shell Babylon by developing a prototype crop disease diagnosis system in it. A short overview of the history of the project and the main problems encountered is given in chapter 1. Chapter 2 describes the result of an attempt to integrate JSD with modelling techniques like generalisation and aggregation and chapter 3 concentrates on the method we used to elicit phytopathological knowledge from specialists. Chapter 4 gives the result of knowledge acquisition for the 10 wheat diseases most commonly occurring in the Netherlands. The user interface is described briefly in chapter 5 and chapter 6 gives an overview of the additions to the implementation we made to the version of FAD reported in our second report. Chapter 7, finally, summarises the conclusions of the project and gives recommendations for follow-up projects

Pharmacological And Genetic Reversal Of Age-Dependent Cognitive Deficits Attributable To Decreased Presenilin Function

Alzheimer\u27s disease (AD) is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are attributable to mutations in a single copy of the Presenilin (PS) and amyloid precursor protein genes. The dominant inheritance pattern of FAD indicates that it may be attributable to gain or change of function mutations. Studies of FAD-linked forms of presenilin (psn) in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of Drosophila metabotropic glutamate receptor (DmGluRA), the inositol trisphosphate receptor (InsP(3)R), or inositol polyphosphate 1-phosphatase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age-onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP(3)R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced

Reinforcement-Driven Spread of Innovations and Fads

We propose kinetic models for the spread of permanent innovations and transient fads by the mechanism of social reinforcement. Each individual can be in one of M+1 states of awareness 0,1,2,...,M, with state M corresponding to adopting an innovation. An individual with awareness k<M increases to k+1 by interacting with an adopter. Starting with a single adopter, the time for an initially unaware population of size N to adopt a permanent innovation grows as ln(N) for M=1, and as N^{1-1/M} for M>1. The fraction of the population that remains clueless about a transient fad after it has come and gone changes discontinuously as a function of the fad abandonment rate lambda for M>1. The fad dies out completely in a time that varies non-monotonically with lambda.Comment: 4 pages, 2 columns, 5 figures, revtex 4-1 format; revised version has been expanded and put into iop format, with one figure adde

A Novel Model of Mixed Vascular Dementia Incorporating Hypertension in a Rat Model of Alzheimer's Disease.

Alzheimer's disease (AD) and mixed dementia (MxD) comprise the majority of dementia cases in the growing global aging population. MxD describes the coexistence of AD pathology with vascular pathology, including cerebral small vessel disease (SVD). Cardiovascular disease increases risk for AD and MxD, but mechanistic synergisms between the coexisting pathologies affecting dementia risk, progression and the ultimate clinical manifestations remain elusive. To explore the additive or synergistic interactions between AD and chronic hypertension, we developed a rat model of MxD, produced by breeding APPswe/PS1ΔE9 transgenes into the stroke-prone spontaneously hypertensive rat (SHRSP) background, resulting in the SHRSP/FAD model and three control groups (FAD, SHRSP and non-hypertensive WKY rats, n = 8-11, both sexes, 16-18 months of age). After behavioral testing, rats were euthanized, and tissue assessed for vascular, neuroinflammatory and AD pathology. Hypertension was preserved in the SHRSP/FAD cross. Results showed that SHRSP increased FAD-dependent neuroinflammation (microglia and astrocytes) and tau pathology, but plaque pathology changes were subtle, including fewer plaques with compact cores and slightly reduced plaque burden. Evidence for vascular pathology included a change in the distribution of astrocytic end-foot protein aquaporin-4, normally distributed in microvessels, but in SHRSP/FAD rats largely dissociated from vessels, appearing disorganized or redistributed into neuropil. Other evidence of SVD-like pathology included increased collagen IV staining in cerebral vessels and PECAM1 levels. We identified a plasma biomarker in SHRSP/FAD rats that was the only group to show increased Aqp-4 in plasma exosomes. Evidence of neuron damage in SHRSP/FAD rats included increased caspase-cleaved actin, loss of myelin and reduced calbindin staining in neurons. Further, there were mitochondrial deficits specific to SHRSP/FAD, notably the loss of complex II, accompanying FAD-dependent loss of mitochondrial complex I. Cognitive deficits exhibited by FAD rats were not exacerbated by the introduction of the SHRSP phenotype, nor was the hyperactivity phenotype associated with SHRSP altered by the FAD transgene. This novel rat model of MxD, encompassing an amyloidogenic transgene with a hypertensive phenotype, exhibits several features associated with human vascular or "mixed" dementia and may be a useful tool in delineating the pathophysiology of MxD and development of therapeutics