Role of the non-neuronal cholinergic system in the eye: a review

Pharmacologically active preparations directed towards modulating muscarinic receptor activity in the eye have been used for over 2000 years when extracts from Atropa belladonna were first applied to enhance eye appearance through pupillary dilation. The first clinically active drugs targeting a specific eye disease were anticholinesterases (e.g. ecothiophate) applied as eye drops to treat glaucoma in the 1960's. However, cataract was soon detected as a relatively frequent side effect and such drugs are now only used to treat glaucoma as a last resort. As muscarinic agonists have been found to reduce intraocular pressure both by decreasing aqueous humour production (through Na,K-ATPase pump inhibition) and increasing outflow (by muscle contraction), it is likely that treatments will be developed that target specific muscarinic subtypes. Recently, it has been shown that the M1 receptor subtype predominates in the lens. It is therefore important that this subtype is not targeted in future ocular therapies so that the side-effect of cataract is avoided. Form-deprived myopia resulting from an increased axial length in the affected eye can be reduced by the application of atropine. This effect has been achieved both in a chick model system and in human clinical trials, and in the former system atropine has been shown to reduce the production of scleral extracellular proteins. Carbachol stimulates tear fluid production through the activation of muscarinic receptors. Interestingly, at least part of the stimulation occurs via epidermal growth factor (EGF) receptors and although the precise signalling mechanisms are not completely understood, it has been shown that calcium mobilisation plays a critical role in both muscarinic and EGF receptor activity. It should be noted that in the four examples described above, the cell types responsible for producing the physiological output are non-neuronal in origin. Therefore cholinergic receptor activation plays diverse roles in the eye and pharmacological intervention based on specific receptor sub-types has potential benefit in a number of ocular problems. However, potential side effects have also recently been identified.     

Studies on the active site of pig plasma amine oxidase.

Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity.     

Development and Application of an Assay for Uranyl Complexation by Fungal Metabolites, Including Siderophores

An assay to detect UO₂²⁺ complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe³⁺ dye and the other containing a CAS-UO₂²⁺ dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO₂²⁺ agar to the discoloration on the CAS-Fe³⁺ agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO₂²⁺ chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO₂²⁺ chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.     

Can Inferred Provenance and Its Visualisation Be Used to Detect Erroneous Annotation? A Case Study Using UniProtKB

A constant influx of new data poses a challenge in keeping the annotation in biological databases current. Most biological databases contain significant quantities of textual annotation, which often contains the richest source of knowledge. Many databases reuse existing knowledge; during the curation process annotations are often propagated between entries. However, this is often not made explicit. Therefore, it can be hard, potentially impossible, for a reader to identify where an annotation originated from. Within this work we attempt to identify annotation provenance and track its subsequent propagation. Specifically, we exploit annotation reuse within the UniProt Knowledgebase (UniProtKB), at the level of individual sentences. We describe a visualisation approach for the provenance and propagation of sentences in UniProtKB which enables a large-scale statistical analysis. Initially levels of sentence reuse within UniProtKB were analysed, showing that reuse is heavily prevalent, which enables the tracking of provenance and propagation. By analysing sentences throughout UniProtKB, a number of interesting propagation patterns were identified, covering over sentences. Over sentences remain in the database after they have been removed from the entries where they originally occurred. Analysing a subset of these sentences suggest that approximately are erroneous, whilst appear to be inconsistent. These results suggest that being able to visualise sentence propagation and provenance can aid in the determination of the accuracy and quality of textual annotation.Source code and supplementary data are available from the authors website at http://homepages.cs.ncl.ac.uk/m.j.bell1/sentence_analysis/.     

A comparison of protein S-thiolation (protein mixed-disulfide formation) in heart cells treated with t-butyl hydroperoxide or diamide

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide, and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30-45 min. Cells stopped beating 25 min after the addition of t-butyl hydroperoxide, and beating did not resume. t-Butyl hydroperoxide caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42 and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment. S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by t-butyl hydroperoxide or diamide, and that the specificity of the response depends on the agent used.     

Protein mixed-disulfides in cardiac cells. S-thiolation of soluble proteins in response to diamide

Protein mixed-disulfides in cultured rat heart cells were analyzed by gel electrophoresis under conditions that eliminated artifactual formation of these protein derivatives. Protein S-thiolation (protein mixed-disulfide formation) was detectable under normal culture conditions. Diamide oxidized intracellular glutathione in these cells and produced extensive protein S-thiolation. The specificity of this protein modification indicates a role in the regulation of cardiac metabolism.     

Investigations of Amavadin

The development of the understanding of the co-ordination chemistry and the properties of Amavadin, the chemical form in which vanadium is accumulated by the Amanita genus of mushrooms, is reviewed.