UV filters in the lens of the thirteen lined ground squirrel (Spermophilus tridecemlineatus).

Major UV filters have been identified in the lens of the 13 lined ground squirrel (Spermophilus tridecemlineatus). These were found to be N-acetyl-3-hydroxykynurenine and N-acetyl-kynurenine, in addition to a small quantity of 3-hydroxykynurenine. The level of N-acetyl-3-hydroxykynurenine measured in the ground squirrel lens, 8.2mM, is approximately 11 times the concentration of 3-hyroxykynurenine glucoside reported previously for the human lens. Two additional UV filters of related structure were also present; however, their structures are still under investigation. HPLC elution profiles indicated that the ground squirrel lens cortex and nucleus contained comparable amounts of alpha-, beta(H)-, beta(L)-, and gamma-crystallins. Levels of GSH in the cortex and nucleus were 12.4 and 7.4mM, respectively. Such high concentrations of GSH may act to inhibit oxidation of the 3-hydroxykynurenine and N-acetyl-3-hydroxykynurenine. N-Acetylated kynurenines are less labile than those with free alpha-amino groups since N-acetyl-alpha-amino groups do not undergo spontaneous deamination. This modification thus stabilises the squirrel UV filters. In addition, because deamination is prevented, the decomposition products will not be involved in binding to lens proteins. Because of the similarity of the UV filters present in the ground squirrel to those in man, this species may be a suitable animal model for investigating the effects of UV radiation on cataract, and other ocular diseases, thought to involve exposure to light.

Sequencing and two-dimensional structure prediction of a phospholipase A(2) inhibitor from the serum of the common tiger snake (Notechis scutatus).

A phospholipase A(2) inhibitor has been identified in the serum of the common tiger snake (Notechis scutatus). The inhibitor is composed of two chains, an alpha-chain and a beta-chain, that form a non-covalently associated complex capable of inhibiting the enzymatic activity of all phospholipase A(2) enzymes it was tested against. The alpha and beta-chains have been purified to homogeneity, digested and sequenced. From the peptide sequence generated, degenerate PCR primers were designed and used to elucidate the complete cDNA sequence of the chains using 5' and 3' RACE PCR. A total of three alpha-chain isoforms were identified, only one isoform of the beta-chain was detected. The two-dimensional structure of the three alpha-chains and one beta-chain were predicted using five prediction programs (discrimination of secondary structure class; nearest neighbour secondary structure, profile network from Heidelberg; self-optimised prediction method from multiple alignment, SSPAL). For each protein chain a consensus prediction was generated. Results are discussed in relation to the function of the protein, and how they may influence the three-dimensional structure of the inhibitor. Additionally, the sequences of several snake phospholipase A(2) inhibitors were used as the input for a motif prediction algorithm (MEME). The results are discussed in relation to the activity of these proteins.

Low molecular weight analysis of tears using matrix assisted laser desorption ionization-time of flight mass spectrometry.

Many low molecular weight substances in human tears, including protein and lipid species, have yet to be characterized. Some of these uncharacterized substances may well be important in the pathogenesis of ocular surface disease or in ocular discomfort. The aim of this study was to build a biochemical profile of low molecular weight species in tears, and to determine its repeatability. A total of 80 tear samples were collected from 11 subjects. Tear samples were dialysed to remove salts, added to a matrix of alpha-cyano-4-hydroxycinnamic acid, and analysed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Species were separated based on their mass to charge ratio (m:z). The repeatability of the appearance of the different species was analysed using logistic regression and diurnal and day-to-day repeatability were ascertained. Peptides were identified in the range of 848 3897 Da. Of these, 39 peptides were found to be present in more than 10/80 samples. There was no diurnal variation in the peptides. All species were found to occur repeatably, with the exception of peptide 1653 Da.This study has demonstrated that the majority of low molecular weight species in tears are repeatably present and do not exhibit diurnal variation. Further study aims to characterize these species and to identify changes in tear profiles between subject groups.

Characterisation of the biochemical and biological variations from the venom of the death adder species (Acanthophis antarcticus, A. praelongus and A. pyrrhus).

We report on species variation in the venoms of the three species of death adder; the Common death adder (Acanthophis antarcticus), the Northern death adder (Acanthophis praelongus) and the Desert death adder (Acanthophis pyrrhus). The venoms were found to vary in their biochemical (chromatography) and biological (PLA(2) activity, anticoagulant activity and reactivity with commercial death adder antivenom) properties. Each species produced significant differences in the profile and distribution of PLA(2) activity, when whole venom was applied to a cation-exchange Mono-S column. PLA(2) enzymes were purified from each venom and termed acanthoxin B (from A. praelongus), acanthoxin C (from A. pyrrhus) and the previously characterised acanthoxin A (from A. antarcticus). Acanthoxin B and C showed lower enzymatic activities than acanthoxin A (4.0, 13.7 and 23.9 micromol of phospholipid hydrolyzed/min/mg protein, respectively). N-terminal sequencing revealed acanthoxin B to share highest homology with the numerous PLA(2) isozymes (Pa-12C, Pa-1G, Pa-12A) from the King brown snake (Pseudechis australis) and Acanthin I from the Common death adder. Similar to acanthoxin A, acanthoxin C showed highest homology with Acanthin I/II, and pseudexin A-chain from the Red-bellied black snake (Pseudechis porphyriacus). Whole venom from A. antarcticus, A. praelongus and A. pyrrhus each showed weak anticoagulant activity (being able to prolong coagulation of the plasma for 107, 220 and 195 s, respectively). By immunodiffusion, each venom produced precipitation bands against commercial death adder antivenom.

Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera.

Although the resistance of snakes to their own venom is well known, until now no investigators have examined the serum of Australian snakes. Here we describe the identification and purification of a range of phospholipase A(2) (PLA(2)) inhibitors from the serum of Australian elapids. All PLA(2) inhibitors were composed of two protein chains, an alpha-chain and a beta-chain. The alpha-chains were approx. 22.5 kDa in size and variably glycosylated, whereas the beta-chains were approx. 19.8 kDa in size and not glycosylated. Identification of isoforms of the two subunit chains was significant because three of the six sera examined were from single snake specimens. In addition, the glycosylation patterns of the alpha-chains were thoroughly investigated in these unpooled sera. The functional and structural properties of the purified inhibitors were studied. Uniquely, a snake PLA(2) inhibitor was found to inhibit human type II PLA(2) enzyme, which has implications for the treatment of the many diseases in which PLA(2) enzymes have been implicated. Further, we demonstrate that the inhibitor forms a non-covalent association with a purified PLA(2) enzyme. Finally, the purified PLA(2) inhibitor was shown to protect in vivo against the lethal affects of a homologous PLA(2) enzyme, suggesting a role for PLA(2) inhibitors in the treatment of snake bite victims.

Functional characteristics of a phospholipase A(2) inhibitor from Notechis ater serum.

A phospholipase A(2) inhibitor has been purified p6om the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (alpha and beta) that form the intact complex of approximately 110 kDa. The alpha-chain is a 30-kDa glycoprotein and the beta-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A(2) inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A(2) enzymes and inhibited the enzymatic activity of all phospholipase A(2) enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A(2) than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor.

Cross-matching marsupial proteins with eutherian mammal databases: proteome analysis of cells from UV-induced skin tumours of an opossum (Monodelphis domestica).

The identification and characterisation of Monodelphis proteins has required cross-species analysis. Protein expression was investigated in normal, nonirradiated adult fibroblasts and also in fibroblastic cells from a benign cutaneous tumour after chronic ultraviolet (UVB) exposure and a metastatic cutaneous tumour after intermittent exposure. Proteins were separated and visualised by two-dimensional gel electrophoresis (2-D PAGE) and a peptide mass fingerprint (PMF) was obtained for protein spots using matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDITOF-MS). Cross-species PMF database analysis facilitated the identification of 120 proteins, constituting 46.5% of the proteins analysed. The identification of two proteins was confirmed by internal amino acid sequencing using tandem MS. Differential protein expression was observed between normal fibroblasts and those in tumours chronically or intermittently exposed. A number of tropomyosin and vimentin isoforms were expressed only in cells from the metastatic tumour induced by intermittent exposure to UV radiation. These results highlight the value of cross-species PMF analysis for the rapid characterisation of proteins from a poorly defined species and also show how proteomics can be used to detect changes in protein expression in differentially treated cells.

The microbial proteome database--an automated laboratory catalogue for monitoring protein expression in bacteria.

Laboratories devoted to high-throughput characterisation of purified proteins arrayed via two-dimensional (2-D) gel electrophoresis face an arduous task in maintaining a centralised and constantly evolving record of information relating to the characterisation of proteins and their responses following biological challenges. The Microbial Proteome Database (MPD) has been conceived as an in-house resource for complementing the plethora of genomic databases available for such organisms. The database utilises commercially available software to provide an electronic 'lab book' of information obtained daily from 2-D electrophoresis gels, image analysis packages, protein characterisation methodologies, and biological experimentation. The MPD begins from a single 2-D gel image (a 2-D 'reference map') with clickable spots that link to a 'protein catalogue' (ProtCat) with spot information including protein identity, changes in expression determined under experimental conditions, cellular location, mass, and pI. The entry for each protein then contains further links to gel images corresponding to the presence of the particular protein within different subproteomes (as defined by the pH of narrow- and wide-range immobilised pH gradients or from differential extraction methods used to determine the location of the protein within a functional cell). The database currently contains information from strains of three microbial species (Escherichia coil, Pseudomonas aeruginosa and Staphylococcus aureus) and 32 master gel images. The rapid accessibility of information obtained from microbial proteomes is an essential step towards the integrated analysis of these organisms at the gene, transcript, protein and functional levels and will aid in reducing turnaround times between sample preparation and the discovery of molecules of biological significance.

Modeling of acanthoxin A1, a PLA2 enzyme from the venom of the common death adder (Acanthophis antarcticus).

The phospholipase A2 enzyme, acanthoxin, found in the venom of the common death adder (Acanthophis antarcticus) as with other snake PLA2 enzymes displays neurotoxic activity. It is unclear whether this neurotoxic activity particular to some snake PLA2 enzymes is a result of structural differences solely within the catalytic sites or at a distant location upon the molecules. We have predicted the three-dimensional structure of one of the two predominant isoforms of acanthoxin (A1) using comparative protein modeling techniques. Given the high degree of homology and the availability of a high quality crystallographic structure, notexin was used as a molecular template to construct an all atom model of acanthoxin. The model was made using the program MODELLER3 and then refined with X-PLOR. Comparison between the predicted structure of acanthoxin and several X-ray structures of toxic and nontoxic PLA2 enzymes has led to a testable two-step proposal of neurotoxic PLA2 activity; involving the favorable binding to acceptor molecules followed by enzymatic intrusion upon the target membrane. The electrostatic potentials across the molecular surfaces of toxic and nontoxic PLA2 enzymes were calculated (GRASP) and it was found that the toxic PLA2 enzymes possessed a charge distribution on the noncatalytic surface not identified in the nontoxic PLA2 enzymes. Thus we have identified residues potentially involved in the interaction of the PLA2 enzymes with their acceptor molecules. Furthermore, the proposed acceptor molecule recognition site is distant from the catalytic site which upon binding of the PLA2 to the acceptor molecule may enhance the enzymatic ability of the toxic PLA2 enzymes on particular cell types.