Implementation of Organ Culture storage of donor corneas: a 3 year study of its impact on the corneal transplant wait list at the Lions New South Wales Eye Bank

Organ Culture corneal storage offers an extended storage time and increased donor pool and tissue assessment opportunities. In September 2011, the Lions New South Wales Eye Bank (LNSWEB) moved from hypothermic storage to Organ Culture corneal storage. This study evaluates the impact of implementation of Organ Culture on donor eye retrieval and the corneal transplant waiting list over a 3 year period in NSW, Australia. Retrospective review of the LNSWEB data from September 2011 to August 2014. Tissue collection, waiting list and tissue utilization data were recorded. The data from September 2008 to August 2011 for Optisol-GS storage was used for comparison. The annual donor and cornea collection rate increased 35 % and 44 % respectively with Organ Culture compared to Optisol-GS storage. The utilization rate of corneal tissue increased from 73.4 % with hypothermic storage to 77.2 % with Organ Culture storage. The transplant wait list decreased by 77.3 % from September 2011 to August 2014 and correlated with the increased rate of corneal transplantation (r = ?0.9381, p < 0.0001). No other factors impacting the wait list changed over this period. Corneas not used from either storage method were due to unacceptable endothelial cell density/viability. The contamination rate of corneas stored in Organ Culture medium was low at 1.74 %. The Organ Culture storage method increases the corneal donor pool available to Eye banks. The practical benefits of the extended storage time and increased donor assessment opportunities have directly led to an increase in corneal utilization rate and a significant decrease in recipient wait list time.     

The reactivity of heme in biological systems: autocatalytic formation of both tyrosine-heme and tryptophan-heme covalent links in a single protein architecture

We have previously shown that introduction of an engineered Met160 residue in ascorbate peroxidase (S160M variant) leads to the formation of a covalent link between Met160 and the heme vinyl group [Metcalfe, C. L., et al. (2004) J. Am. Chem. Soc. 126, 16242-16248]. In this work, we have used electronic spectroscopy, HPLC, and mass spectrometry to show that the introduction of a tyrosine residue at the same position (S160Y variant) leads, similarly, to the formation of a heme-tyrosine covalent link in an autocatalytic reaction that also leads to formation of a second covalent link from the heme to Trp41 [Pipirou, Z., et al. (2007) Biochemistry 46, 2174-2180]. Stopped-flow and EPR data implicate the involvement of a tyrosyl radical in the reaction mechanism. The results indicate that the heme can support the formation of different types of covalent links under appropriate conditions. The generality of this idea is discussed in the context of other heme enzymes.     

Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms.

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two-step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore-induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nüssler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem. 272, 20 884-20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm's immune system destroy non-self components.     

The electron paramagnetic resonance characterisation of a copper-containing extracellular peroxidase from Thermomonospora fusca BD25.

The actinomycete Thermomonospora fusca BD25 contains a peroxidase with a high activity over a broad range of temperature and pH and a high stability against denaturing agents. Unusually this peroxidase (PO) is a non-haem enzyme. As prepared PO is characterised by two electron paramagnetic resonance (EPR) signals, detected at liquid helium temperature, a free radical signal (g=2.0045) and a broad signal at g=2.056. The peroxidase activity of the purified enzyme was assayed using H(2)O(2) and 2,4-dichlorophenol (DCP). The intensity of the free radical EPR signal correlated with the peroxidase activity in a variety of enzyme preparations. Furthermore, when DCP and H(2)O(2) were added to PO a significant increase of both the free radical signal and the broad signal at g=2.056 was observed. We associate the increase of the broad signal with the oxidation of the preparation since a similar increase can be achieved by the addition of ferricyanide. The high intensity of the broad signal in the ferricyanide treated PO allowed us to deconvolute the signal into several components using the difference in their relaxation characteristics: two distinct copper signals were detected, one of which was similar to a type 2 centre. Furthermore a symmetrical singlet was detected at g=2.059, consistent with the presence of an iron complex with a high degree of symmetry and weakly coordinated ligands.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.     

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.     

Altered expression of the xenobiotic transporter P-glycoprotein in liver and liver tumours of mummichog Fundulus heteroclitus from a creosote-contaminated environment

P-glycoproteins (Pgps) are involved in efflux of xenobiotics from drug-resistant cell lines and tumours, and in excretion of toxicants from normal tissues. Recently, investigators have proposed that Pgp activity contributes to resistance or tolerance of certain aquatic species to pollutants. In the present study using immunoblot and immunohistochemical techniques, we found elevation of Pgp in liver and liver tumours of creosote-resistant mummichog from a contaminated site in the Elizabeth River, Virginia. Immunoblots of mummichog liver extracts showed an immunoreactive band at 170 kDa and indicated two- to three-fold elevation of Pgp in livers of resistant fish relative to those from a reference site. Laboratory exposures of reference site fish to a model PAH (3-methylcholanthrene), however, produced no increase in liver Pgp levels as measured by immunoblot. Normal mummichog liver sections showed specific immunohistochemical staining for Pgp on the canalicular surface of hepatocytes. In the majority of hepatic neoplasms we observed a high level of over-expression and altered patterns of Pgp expression. However we did not observe Pgp over-expression in early proliferative lesions. Elevation of Pgp in livers and liver tumoursof these resistant mummichog may contribute to their survival in a heavily contaminated environment.