Association of DJ-1 and parkin mediated by pathogenic DJ-1 mutations and oxidative stress

The identification of rare monogenic forms of Parkinson's disease (PD) has provided tremendous insight into the molecular pathogenesis of this disorder. Heritable mutations in alpha-synuclein, parkin, DJ-1 and PINK1 cause familial forms of PD. In the more common sporadic form of PD, oxidative stress and derangements in mitochondrial complex-I function are considered to play a prominent role in disease pathogenesis. However, the relationship of DJ-1 with other PD-linked genes and oxidative stress has not been explored. Here, we show that pathogenic mutant forms of DJ-1 specifically but differentially associate with parkin, an E3 ubiquitin ligase. Chemical cross-linking shows that pathogenic DJ-1 mutants exhibit impairments in homo-dimer formation, suggesting that parkin may bind to monomeric DJ-1. Parkin fails to specifically ubiquitinate and enhance the degradation of L166P and M26I mutant DJ-1, but instead promotes their stability in cultured cells. The interaction of parkin with L166P DJ-1 may involve a larger protein complex that contains CHIP and Hsp70, perhaps accounting for the lack of parkin-mediated ubiquitination. Oxidative stress also promotes an interaction between DJ-1 and parkin, but this does not result in the ubiquitination or degradation of DJ-1. Parkin-mediated alterations in DJ-1 protein stability may be pathogenically relevant as DJ-1 levels are dramatically increased in the detergent-insoluble fraction from sporadic PD/DLB brains, but are reduced in the insoluble fraction from parkin-linked autosomal recessive juvenile-onset PD brains. These data potentially link DJ-1 and parkin in a common molecular pathway at multiple levels that may have important implications for understanding the pathogenesis of inherited and sporadic PD.     

Brief intense exercise followed by passive recovery modifies the pattern of fuel use in humans during subsequent sustained intermittent exercise

The role of work period duration as the principal factor influencing carbohydrate metabolism during intermittent exercise has been investigated. Fuel oxidation rates and muscle glycogen and free carnitine content were compared between two protocols of sustained intermittent intense exercise with identical treadmill speed and total work duration. In the first experiment subjects (n=6) completed 40 min of intermittent treadmill running involving a work : recovery cycle of 6 : 9 s or 24 : 36 s on separate days. With 24 : 36 s exercise a higher rate of carbohydrate oxidation approached significance (P=0.057), whilst fat oxidation rate was lower (P ≤ 0.01) and plasma lactate concentration higher (P ≤ 0.01). Muscle glycogen was lower post‐exercise with 24 : 36 s (P ≤ 0.05). Muscle free carnitine decreased (P ≤ 0.05), but there was no difference between protocols. In the second experiment a separate group of subjects (n=5) repeated the intermittent exercise protocols with the addition of a 10‐min bout of intense exercise, followed by 43 ± 5 min passive recovery, prior to sustained (40 min) intermittent exercise. For this experiment the difference in fuel use observed previously between 6 : 9 s and 24 : 36 s was abolished. Carbohydrate and fat oxidation, plasma lactate and muscle glycogen levels were similar in 6 : 9 s and 24 : 36 s. When compared with the first experiment, this result was because of reduced carbohydrate oxidation in 24 : 36 s (P ≤ 0.05). There was no difference, and no change, in muscle free carnitine between protocols. A 10‐min bout of intense exercise, followed by 43 ± 5 min of passive recovery, substantially modifies fuel use during subsequent intermittent intense exercise.     

Sequence relations and coding properties of a subgenomic RNA isolated from barley stripe mosaic virus

A subgenomic (SG) RNA ( approximately 800 nucleotides) is a minor component of barley stripe mosaic virus RNAs. The SG-RNAs isolated from the Type and North Dakota 18 (ND18) strains of BSMV have sequence homology with RNA 2 of the ("pseudo-two component") Type strain, which has two electrophoretic components, but only limited homology is evident with RNA 2 of the ND18 and Norwich strains, which have three electrophoretic components ("three component" strain). Instead, eDNAs from SG-RNA hybridize most efficiently with RNA 3 of the ND18 and Norwich strains. In wheat germ extracts the SG-RNAs direct the synthesis of two polypeptides with apparent molecular weights of 20 to 21 x 10(3). However, these two polypeptides were difficult to detect by polyacrylamide gel electrophoresis of extracts from Type- or ND18-infected barley and so appear not to accumulate during infection.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Effect of a Polymeric Additive on the Pore‐Size Distribution and Shrinking Process of a Hydrogel Network

A systematic comparison of the effect of architectural modifications to the network structure on the internal microstructure of N‐isopropylacrylamide (NIPA) based hydrogels showed that the addition of a second component to the network significantly increased the proportion of macropores in the network. The second components considered were short poly(N‐isopropylacrylamide) (PNIPAM) chains grafted to the network backbone, high‐molecular‐weight polyacrylamide (PAM) chains, or microsphere particles of PNIPAM. Structures are proposed for each of the modified gel networks taking into account the new structural information. Through a combination of the pore size and network structure data reported here, and with the shrinking data obtained previously, shrinking mechanisms are proposed for each of the modified network structures. In all cases, the enhanced shrinking rates were directly caused by the presence of the second component, which acted as nuclei for shrinking (graft‐PNIPAM and PNIPAM microspheres) or as water‐release channels (PAM gel), and indirectly caused by the second components via their affect on the network microstructure.

Proposed structures for the architecturally modified gels based on the pore‐size information. Graft‐PNIPAM gel. The freely mobile graft chains prevent chains from meeting resulting in larger pores.     

The effect of age on the fibrinolytic enzyme system

Components of the fibrinolytic enzyme system, FR-antigen and fibrinogen, were measured in 20 healthy volunteers aged 20-40 years and in 61 elderly subjects aged 66-96 years. Plasminogen activator levels did not significantly differ between the 20-40 and 66-75 age groups, but were higher in those over 75. Plasminogen showed no change with age except for a fall in those over 75. Fibrinogen, FR-antigen, alpha(1)-antitrypsin, and alpha(2)-macroglobulin all rose with age, but the mean fibrinogen concentration fell in the very elderly.