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Biomedical subjects

R J Truscott

Publications and source records attributed to R J Truscott.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of indoleamine 2,3-dioxygenase, the first enzyme in UV filter biosynthesis in the human lens. Relevance for senile nuclear cataract.

3-Hydroxykynurenine (3OHKyn), the precursor of UV filters in human lens, is highly autooxidizable, generates H2O2, and binds to lens proteins, yielding a tanned/yellow product resembling senile nuclear cataractous materials. Thus, if 3OHkyn can be shown to be the causative agent in cataract, it may be possible to prevent the disease by lowering the level of 3OHKyn. To this end, indoleamine 2,3-dioxygenase, the first enzyme in UV filter synthesis, was studied using lens epithelial cell lines. The results indicated that the IDO expression is mediated by IFN-gamma. Immuno-suppressants which inhibit production of IFN-gamma may act as anti-cataract agents. Another way to lower the level of 3OHKyn is to use specific inhibitors for IDO. A recombinant human IDO was expressed to develop the inhibitors.

Aged↗
PubMed1999

Interferon-gamma-dependent/independent expression of indoleamine 2,3-dioxygenase. Studies with interferon-gamma-knockout mice.

The role of IFN-gamma in the expression of indoleamine 2,3-dioxygenase (IDO), a tryptophan oxidizing enzyme, in mouse tissues under physiological and pathological conditions was investigated using IFN-gamma-knockout mice. The results revealed that i) the expression of IDO in the large intestine or in the cecum is mediated by IFN-gamma, ii) for the systemic IDO induction under endotoxin shock, IFN-gamma is a dominant inducer but not essential, and an IFN-gamma-independent mechanism is also operative, iii) the systemic induction of IDO caused by IL-12 or Pokeweed mitogen is mediated by IFN-gamma, and iv) the constitutive IDO expression in the epididymis is IFN-gamma-independent.

Animals↗
PubMed1999

UV filter compounds in human lenses: the origin of 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside.

PURPOSE: To investigate UV filter synthesis in the human lens, in particular the biosynthetic origin of the second most abundant UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside. METHODS: Human lenses were analyzed by high-performance liquid chromatography (HPLC) after separate incubation with 3H-tryptophan (3H-Trp), beta-benzoylacrylic acid, D,L-alpha-amino-beta-benzoylpropionic acid, or D,L-3-hydroxykynurenine O-beta-D-glucoside. The effect of pH on the model compound D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside was also investigated. RESULTS: UV filters were not detected in fetal lenses, despite a 5-month postnatal lens displaying measurable levels of UV filters. In adults no radiolabel was incorporated into 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside after 3H-Trp incubations. Beta-benzoylacrylic acid was readily reduced in lenses. D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside slowly deaminated at physiological pH and were converted to beta-benzoylpropionic acid and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside, respectively, after lens incubations. CONCLUSIONS: UV filter biosynthesis appears to be activated at or near birth. Compounds containing the kynurenine side chain slowly deaminate, and in the lens, the newly formed double bond is rapidly reduced. These findings suggest that 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside is derived from L-3-hydroxykynurenine O-beta-D-glucoside through this deamination-reduction process. The slowness of the deamination presumably accounts for the absence of incorporation of radiolabel from 3H-Trp into 4(2-amino-3-hydroxyphenyl)4-oxobutanoic acid O-beta-D-glucoside.

Acrylates↗
PubMed1998

An impediment to glutathione diffusion in older normal human lenses: a possible precondition for nuclear cataract.

Human age-related nuclear cataract is associated with progressive and widespread oxidation of proteins, particularly in the centre of the lens. The reasons for the onset of cataract and why this disease should take place only in the lenses of older individuals remain unclear. However, a common feature of nuclear cataract is the low concentration of reduced glutathione (GSH) in the centre of the lens. GSH is the principal lenticular antioxidant of the lens and it is synthesized and regenerated in the lens cortex. In this study we investigated the diffusion of glutathione within the human lens as a function of age. Normal human lenses were incubated in artificial aqueous humor containing [35S]cysteine and the label was metabolically incorporated into GSH. After 48-h incubation, lenses were sectioned and phosphorimaging was used to determine the distribution of 35S label. In young lenses, label appeared to diffuse uniformly throughout the whole lens. By contrast, in lenses over the age of 30, very little 35S had penetrated to the centre of the lens. A distinct zonal pattern of label distribution was noted in the older lenses after 48 h incubation, which had dimensions of approximately 7.2 mm (diameter) by 2.8 mm (axial). In some older lenses this pattern was noticeable even after 96-h incubation. Thus a barrier to the diffusion of GSH was observed in older normal lenses which was not present in younger lenses. Furthermore, the internal zone thus delineated has dimensions that coincide with those of the coloured and sclerotic zone present in nuclear cataract lenses. Since nuclear cataract is a disease of the elderly, and maintenance of GSH is known to be vital for lens clarity, we propose that the development of a barrier to the movement of GSH from its site of synthesis and regeneration in the cortex, into the nucleus in older normal lenses, may over time allow oxidative modification of protein to take place in the nucleus, resulting ultimately in nuclear cataract.

Adolescent↗
PubMed1997

Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the Fab, F(ab')2 and deglycosylated Fc fragments obtained from the murine IgG1 B72.3 monoclonal antibody (MAb) by digestion with the sulfhydryl protease papain, in an attempt to determine the sites of cleavage and thus to clarify the mode of action of this enzyme on MAbs. ESI analysis of the Fab and F(ab')2 subunits indicated that the predominant site of papain cleavage occurred at C221 of the B72.3 MAb heavy chain. Reduction of the intra- and interchain disulfide bridges of these fragments by 1,4-dithiothreitol and subsequent electrospray analysis showed a loss of C221 from the C-terminal end of the Fd subunit. ESI analysis of the cleaved Fab fragment indicated that there was an apparent loss of amino acid residues from this fragment. Edman sequencing of the cleaved subunit revealed an intact light chain and the loss of QVQ from the N-terminal of the Fd subunit. Reduction of this subunit gave a Fd fragment approximately 32 Da greater than the predicted mass, which we have attributed to oxidation of the heavy chain methionine residues (M81 and M136). Removal of the carbohydrate portion from the Fc fragment by N-glycosidase F indicated that papain cleavage had occurred at C223 of the B72.3 MAb heavy chain. In addition, it was observed that the C-terminal lysine residue (K438) was absent from the deglycosylated Fc fragment, presumably due to carboxypeptidase B activity that occurs during the in vivo production of the B72.3 MAb in murine hosts. These data clearly illustrate the power of ESI-MS for determining small changes in mass on large proteins as well as providing a rapid and sensitive technique for assessing MAb fragments prior to use in radioimaging or radiotherapy.

Amino Acid Sequence↗
PubMed1997

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).

Amino Acid Sequence↗
PubMed1997

Oxidation products of 3-hydroxykynurenine bind to lens proteins: relevance for nuclear cataract.

3-Hydroxykynurenine (3OHKyn), present as a human lens UV filter, has also been implicated as a carcinogen and neurotoxin. It has been suggested that oxidation of 3OHKyn is involved in each of these effects. In the presence of oxygen, 3OHKyn has been found to react with bovine crystallins, to give brown-coloured products (Stutchbury and Truscott, 1993). In this study the roles of UV-light, pH, glutathione and oxygen were examined, with the objective of determining how these factors may affect the binding of 3OHKyn to crystallins under the conditions found within the lens itself. The presence of oxygen was found to be an important parameter for determining the extent to which 3OHKyn reacts with protein, and when it was totally excluded, little modification was observed. UV-light was not required for activation, but was found to augment the extent of modification and cross-linking, while an elevated pH, which is known to accelerate the rate of 3OHKyn oxidation, did not markedly increase the extent of reaction with the crystallins. 3OHKyn binding was accompanied by crystallin aggregation, pigmentation, and development of non-tryptophan fluorescence, all of which have been associated with cataract formation. The inclusion of glutathione, a ubiquitous antioxidant, in reaction mixtures resulted in a delayed onset of crystallin modification. This effect was apparent at concentrations of glutathione greater than 1 mM. When glutathione levels fell below 1 mM, crystallins became modified by 3OHKyn. Since lens glutathione concentrations decrease with age, and are known to be lower in the lens nucleus than the cortex, this region appears particularly vulnerable to modification by this UV filter. Thus, whilst the other human lens UV filters, kynurenine (Kyn) and 3-hydroxykynurenine glucoside (3HKG), appear to require activation by UV-light in order to react with proteins, 3OHKyn can modify crystallins in the absence of light, under conditions of low oxygen tension, and in the presence of glutathione concentrations found in the nucleus of an aged lens. Its reactivity is increased in the presence of both light and oxygen. The contributions of these parameters to the reactivity of 3OHKyn are discussed, with respect to the aetiology of senile nuclear cataract.

Animals↗
PubMed1997

Molecular evidence for the involvement of alpha crystallin in the colouration/crosslinking of crystallins in age-related nuclear cataract.

The proteins of the lens which become insoluble, crosslinked and coloured as a result of the onset of human nuclear cataract have been studied using a combination of enzymatic digestion and HPLC/mass spectrometry (MS). The objective was to determine if such an approach could provide information on the identities of the polypeptide components involved in the colouration and crosslinking and to discover whether any crystallins predominate in this characteristic post-translational modification process. Initially, coloured high molecular weight peptides were isolated from a tryptic/chymotryptic digest of the 6 M guanidine hydrochloride-insoluble lens protein fraction. These tryptic/chymotryptic peptides were then incubated with pronase and the small peptides released, purified by gel filtration. All but one of the peptides analysed by HPLC/MS/MS were found to contain proline. Peptides derived from alpha-crystallin were found to comprise the great majority of the peptides characterised. No gamma-crystallin peptides were observed. Both alpha A-crystallin and alpha B-crystallin were represented. Further, all but one of these peptides were derived from the N-terminal region of the alpha-crystallin subunits: a region recently implicated in the chaperone activity of alpha-crystallin. This finding suggests that the putative N-terminal domain of alpha-crystallin may be involved at the molecular level in the process of crosslinking and colouration which is known to be characteristic of age-related nuclear cataract. It is, therefore, conceivable that an early stage of these cataractous modifications may involve alpha-crystallin acting as a molecular chaperone.

Aging↗
PubMed1996

Mass spectrometry of lens crystallins: bovine beta-crystallins.

The bovine beta-crystallins have been isolated by gel permeation chromatography (GPC) and fast protein liquid chromatography (FPLC). Electrospray ionization mass spectrometric examination of the resulting fractions confirms the relative molecular masses of three bovine beta-crystallins, i.e. beta B1, beta B2 and beta A2, only one of which (beta B2) has been reported previously by mass spectrometry. The sequence of beta B3 is shown to be incorrect as the identity of this protein was confirmed by FPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass analysis of a tryptic digest of this protein. ESI analysis of the remaining beta subunits, i.e. beta A1, beta A3 and beta A4, suggests the published sequences of these proteins also contain errors. The major components observed in the beta H (i.e. octameric) and beta L (trimeric) aggregates of the beta-crystallins are shown to be consistent with subunit compositions determined by SDS-PAGE. A number of cleavage products of beta-crystallins were also identified, i.e. species with masses corresponding to beta B1(2)-203, beta B1(2)-197, beta B8(2)-204, beta B1(2)-196 were detected in the ESI mass spectra following isolation of the beta B2 fraction from one-year-old lenses by FPLC. A species with mass of beta B6(1)-244 is also observed in the ESI mass spectrum of the beta H fraction isolated by GPC. The largest and most hydrophobic of the beta-crystallins, beta B1, is detected in the beta H aggregate but not following FPLC purification of a fraction containing beta B1 and beta B2. A variety of different methods (including MALDI) were used to examine this fraction but none enabled the successful analysis of beta B1 in the deaggregated protein. Finally, this work demonstrates the advantages and some of the difficulties in mass spectrometric characterization of this class of proteins.

Amino Acid Sequence↗
PubMed1996

Age-related changes in bovine alpha-crystallin and high-molecular-weight protein.

The high-molecular-weight (HMW) protein from the lens is composed mostly of alpha-crystallin in a highly aggregated state. Bovine HMW protein was carefully separated from alpha-crystallin by size-exclusion chromatography. alpha-Crystallin has chaperone-like ability whereby it stabilizes other proteins under conditions of stress (e.g. heat). Comparison of bovine HMW protein and alpha-crystallin shows that the HMW protein has a markedly reduced chaperone ability compared to alpha-crystallin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of alpha-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the alpha-crystallin subunits with age have been quantified. Phosphorylation of alpha-crystallin occurs early in life but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of alpha-crystallin has little effect on its chaperone ability. As is found in the artificially prepared HMW complex of alpha- and gamma-crystallin, NMR spectroscopy shows that in the naturally occurring HMW protein, the short C-terminal extension of the alpha B subunit has lost its flexibility whereas the alpha A subunit extension is still flexible. Post-translational modifications therefore seem to have little effect on the chaperone action of alpha-crystallin, but alterations in the quaternary structure of alpha-crystallin via incorporation into the HMW aggregate, lead to major changes in the chaperone ability of the protein. The results are consistent with the notion that one of the contributing factors to cataract formation in the lens is the depletion of alpha-crystallin with age as it is converted into the HMW protein.

Aging↗
PubMed1996

Molecular masses of gamma-crystallins.

Bovine gamma-crystallins were isolated and analysed by electrospray mass spectrometry (ESMS). The mass of gamma II-crystallin was as predicted from the amino acid sequence and the mass of gamma IIIb-crystallin was close, but the mass of gamma IVa-crystallin was 59 Da greater than that expected. gamma IVa-Crystallin was digested with cyanogen bromide and the fragments were isolated before analysis by ESMS. The masses of the fragments did not correspond to the published sequence. The published sequence of gamma IVa-crystallin, which has been used to predict its three-dimensional structure, is incorrect.

Animals↗
PubMed1995

On the interaction of alpha-crystallin with unfolded proteins.

alpha-Crystallin, a major protein component of the lens, has chaperone-like properties whereby it prevents destabilised proteins from precipitating out of solution. It does so by forming a soluble high-molecular-weight (HMW) complex. A spectroscopic investigation of the HMW complex formed between a variety of unfolded proteins and bovine alpha-crystallin is presented in this paper. As monitored by fluorescence spectroscopy, a large amount of the hydrophobic probe, 8-anilino-1-naphthalene sulfonate (ANS) binds to the HMW complex implying that the complexed proteins (alcohol dehydrogenase (ADH), gamma-crystallin and rhodanese) are bound in an unfolded, possibly molten-globule state. The interaction between the anionic surfactant, sodium dodecyl sulfate (SDS) and ADH at high temperatures gives rise to a similar large increase in ANS fluorescence to that for the complex between alpha-crystallin and ADH. SDS, like alpha-crystallin, therefore complexes to proteins in their unfolded state leaving a large hydrophobic surface exposed to solvent. Unlike other chaperones (e.g., GroEL, DnaK and SecB), alpha-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated alpha-lactalbumin and alpha-casein). It is concluded that alpha-crystallin will only complex with proteins that are about to precipitate out of solution, i.e., ones that are severely compromised. 1H-NMR spectroscopy of the HMW complex formed between alpha-crystallin and gamma-crystallin indicates that the short C-terminal extension of alpha B-crystallin, but not that of alpha A-crystallin, has lost its flexibility in the complex implying that the former is involved in interactions with the unfolded gamma-crystallin molecule, possibly electrostatically via its two C-terminal lysine residues.

Alcohol Dehydrogenase↗
PubMed1995

Loss of the C-terminal serine residue from bovine beta B2-crystallin.

Electrospray mass spectrometric (ES-MS) examination of bovine beta-crystallins showed a significant component corresponding in mass to beta B2-crystallin less one serine residue. Tryptic digestion, followed by isolation and characterisation of the C-terminal peptide, demonstrated that this new species has arisen by the loss of the C-terminal serine residue. This phenomenon appears to be age-related since no truncation was detected in beta B2-crystallin from foetal lenses and the proportion of the truncated form, as judged by ES-MS, was lower in beta-crystallin isolated from calf lenses than that from the lenses of 3-year-old animals. This process therefore is similar to a recently reported loss of the C-terminal serine from alpha A-crystallin, which we have confirmed using ES-MS. Loss of a C-terminal serine from both crystallins may indicate the presence of carboxypeptidase-A-like activity in bovine lenses. ES-MS data provided no evidence for a significant degree of phosphorylation of beta B2-crystallin.

Aging↗
PubMed1995

Relationship between serum tryptophan and tryptophan metabolite levels after tryptophan ingestion in normal subjects and age-related cataract patients.

1. Cataract is the single major cause of blindness worldwide; however, the reasons for the development of this condition remain unknown. It has been suggested that the essential amino acid tryptophan may be implicated in the aetiology but definitive evidence has been lacking. 2. The serum levels of tryptophan and seven of its metabolites have been measured in both cataract patients and control subjects, after administration of tryptophan, in order to determine the typical response profile and to discover whether differences could be found in tryptophan metabolism in the two groups. 3. Tryptophan, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, 5-hydroxyanthranilic acid, 5-hydroxytryptophan and anthranilic acid were measured by HPLC with dual electrochemical and programmable wavelength fluorescence detection. Fasting cataract patients (n = 42) and control subjects (n = 37) were given an oral dose of L-tryptophan and sera were sampled at 0, 1, 2, 4 and 6 h. 4. Statistically significant differences in the distribution of data between the two groups were observed. The responses of kynurenine and 5-hydroxyanthranilic acid were higher in cataract patients, but those of kynurenic acid and total tryptophan were lower than in control subjects. No statistically significant differences in free tryptophan, anthranilic acid, 3-hydroxyanthranilic acid, xanthurenic acid or 5-hydroxytryptophan levels were noted. 5. We conclude that there is a major subgroup of age-related cataract patients with a dysfunction in the metabolism of tryptophan. This may be related to the onset of cataract. The mechanism remains to be established but may operate via the action of tryptophan metabolites, such as 5-hydroxyanthranilic acid, which become reactive towards protein upon oxidation.

Age Factors↗
PubMed1994

A new UV-filter compound in human lenses.

A new UV-filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, has been identified in human lenses. The structure suggests that it is derived biosynthetically from tryptophan. Quantification studies on the new compound show that it is the second-most abundant UV-filter compound in the lens with an absorption and fluorescence spectrum similar to that of 3-hydroxykynurenine glucoside.

Chromatography, High Pressure Liquid↗
PubMed1994

Alpha-crystallin: molecular chaperone and protein surfactant.

Bovine lens alpha-crystallin has recently been shown to function as a molecular chaperone by stabilizing proteins against heat denaturation (Horwitz, J. (1992) Proc. Natl. Acad. Sci. USA, 89, 10449-10453). An investigation, using a variety of physico-chemical methods, is presented into the mechanism of stabilization. alpha-Crystallin exhibits properties of a surfactant. Firstly, a plot of conductivity of alpha-crystallin versus concentration shows a distinct inflection in its profile, i.e., a critical micelle concentration (cmc), over a concentration range from 0.15 to 0.17 mM. Gel chromatographic and 1H-NMR spectroscopic studies spanning the cmc indicate no change in the aggregated state of alpha-crystallin implying that a change in conformation of the aggregate occurs at the cmc. Secondly, spectrophotometric studies of the rate of heat-induced aggregation and precipitation of alcohol dehydrogenase (ADH), beta L- and gamma-crystallin in the presence of alpha-crystallin and a variety of synthetic surfactants show that stabilization against precipitation results from hydrophobic interactions with alpha-crystallin and monomeric anionic surfactants. Per mole of subunit or monomer, alpha-crystallin is the most efficient at stabilization. alpha-Crystallin, however, does not preserve the activity of ADH after heating. After heat inactivation, gel permeation HPLC indicates that ADH and alpha-crystallin form a high molecular weight aggregate. Similar results are obtained following incubation of beta L- and gamma-crystallin with alpha-crystallin. 1H-NMR spectroscopy of mixtures of alpha- and beta L-crystallin, in their native states, reveals that the C-terminus of beta B2-crystallin is involved in interaction with alpha-crystallin. In the case of gamma- and alpha-crystallin mixtures, a specific interaction occurs between alpha-crystallin and the C-terminal region of gamma B-crystallin, an area which is known from the crystal structure to be relatively hydrophobic and to be involved in intermolecular interactions. The short, flexible C-terminal extensions of alpha-crystallin are not involved in specific interactions with these proteins. It is concluded that alpha-crystallin interacts with native proteins in a weak manner. Once a protein has become denatured, however, the soluble complex with alpha-crystallin cannot be readily dissociated. In the aging lens this finding may have relevance to the formation of high molecular weight crystallin aggregates.

Alcohol Dehydrogenase↗