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J J Harding

Publications and source records attributed to J J Harding.

At least 19 recordsLinked to original sources
PubMed2003

Decreased chaperone activity of alpha-crystallin in selenite cataract may result from selenite-induced aggregation.

PURPOSE: To investigate the role of chaperone activity of alpha-crystallin in selenite-induced cataract formation. METHODS: Selenite cataract was induced in Sprague-Dawley rats by five subcutaneous injections of sodium selenite over a 20-day period starting at 8-10 days postpartum. alpha-Crystallin was separated from the rat lenses by size-exclusion chromatography. Bovine alpha(L)-crystallin and beta(L)-crystallin were isolated for studies in vitro, and for the chaperone assays. The protective effects of both alpha(H)- and alpha(L)-crystallin were measured spectrophotometrically in four different assay procedures including the thermally induced aggregation of catalase and beta(L)-crystallin, and the fructation- and heat-induced inactivation of catalase. The bovine alpha(L)-crystallin was incubated with different concentrations of sodium selenite for 72 h and then its chaperone activity against heat-induced beta(L)-crystallin aggregation was assayed. The aggregation of selenite-treated alpha(L)-crystallin was analysed by molecular sieve high-performance liquid chromatography (HPLC). RESULTS: The protection of alpha(H)-crystallin was less than that of alpha(L)-crystallin in both normal and cataractous lenses. The chaperone activities of both alpha(H)- and alpha(L)-crystallin in selenite cataract were decreased compared with normal lenses. The protection provided by both alpha(H)-crystallin and alpha(L)-crystallin against the thermal aggregation of catalase was much greater than their protection against thermally and chemically induced inactivation. HPLC analysis demonstrated aggregation of alpha-crystallin by sodium selenite after 24 h incubation in a dose-dependent fashion. CONCLUSION: The chaperone activity of alpha-crystallin presented parallel patterns of activity with different methods, further supporting the view that the different assays measure essentially the same property. The decreased chaperone activity of alpha-crystallin in selenite cataract may result from selenite-induced aggregation.

Animals↗
PubMed2001

Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites.

A major stress protein, alpha-crystallin, functions as a chaperone. Site-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 degrees C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of alpha-crystallin. A mutant corresponding to a minor component of rodent alpha A-crystallin, alpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alpha B-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, Lys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27-->Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.

Amino Acid Sequence↗
PubMed2001

Can drugs or micronutrients prevent cataract?

Cataract is the major cause of blindness and of visual impairment worldwide, so its prevention is of the greatest importance. At present no drug therapy is licensed for use in the UK or the US, so the only treatment for cataract is by surgery, which is expensive and has adverse effects. This article reviews research on prevention of cataract by a variety of agents, including micronutrients as well as drugs. Benefits have been claimed for many compounds or mixtures and this review concentrates on those most extensively studied. Information on possible benefits of putative anticataract agents comes from a variety of approaches, from laboratory experiments, both in vitro and in vivo, to epidemiological studies in patients. Sorbitol-lowering drugs were the first to be examined systematically and progressed to clinical trials which were disappointing, and now the entire rationale for their use in prevention of cataract is questionable. Micronutrients showed little promise in animals but came to clinical trial in patients with cataract without the publication of any major benefit. Pantethine showed more promise in animal studies but the only clinical trial was abandoned early. A variety of laboratory and epidemiological evidence supports the benefits of aspirin-like drugs but there has been no trial specifically in patients with cataract. Add-on studies to trials of aspirin for other indications have not been encouraging. Research into other compounds is interesting but less advanced.

Aldehyde Reductase↗
PubMed2000

alpha-crystallin assists the renaturation of glyceraldehyde-3-phosphate dehydrogenase.

alpha-Crystallin, a major lens protein, has many of the properties of a molecular chaperone, but its ability to assist refolding of proteins has been less certain. In the present work it was shown that alpha-crystallin specifically increased the reactivation of guanidine-denatured glyceraldehyde-3-phosphate dehydrogenase with most of the activity being recovered. In the incubation mixture the recovered enzyme activity was partly free but mostly it appeared in a protective complex with alpha-crystallin. The aggregation of the denatured enzyme on dilution from the guanidine solution was prevented. Thus alpha-crystallin not only protects against aggregation and inactivation of enzymes during denaturation, but can also prevent aggregation and assist recovery of the native structure during renaturation.

Animals↗
PubMed2000

alpha-crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde.

The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether alpha-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether alpha-crystallin could renature G6PD denatured by MDA. alpha-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without alpha-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. alpha-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by alpha-crystallin, but it was stabilised and protected against further denaturation. Complex formation between alpha-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that alpha-crystallin is able to protect the enzyme against this damage.

Animals↗
PubMed2000

The lens in hereditary hyperferritinaemia cataract syndrome contains crystalline deposits of L-ferritin.

BACKGROUND/AIM: Hereditary hyperferritinaemia cataract syndrome (HHCS) is an autosomal dominant disorder characterised by elevated serum L-ferritin and bilateral cataracts. The ocular manifestations of this disorder are poorly studied. This study therefore sought to determine the origin of cataracts in HHCS. METHODS: L-ferritin ELISA, immunohistochemical and ultrastructural analysis of a lens nucleus from an HHCS individual. RESULTS: The HHCS lens L-ferritin content was 147 microg/g dry weight of lens compared with <16 microg/g for a non-HHCS control cataract lens. The cataract comprised discrete crystalline inclusions with positive staining with anti-L-ferritin but not anti-H-ferritin. CONCLUSIONS: This unusual finding of crystalline opacities in the lens may be unique to HHCS and is likely to result from disturbed metabolism of L-ferritin within the lens or an abnormal interaction between L-ferritin and lens proteins.

Animals↗
PubMed1999

Inactivation and loss of antigenicity of esterase by sugars and a steroid.

Glycation, the non-enzymic reaction of sugars with proteins, has an important role in the complications of diabetes. It has been studied mostly in structural proteins but more recently has been shown to inactivate enzymes. Previous evidence from our laboratory indicated that glycation-induced inactivation and loss of antigenicity of catalase and superoxide dismutase are simultaneous. Esterase, which decreases activity in the lens in senile cataract and diabetes, was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. Here we investigated the inactivation of carboxylesterase (EC 3.1.1.1) by sugars of different glycating power and prednisolone-21-hemisuccinate while simultaneously monitoring the loss of antigenicity. Antigenicity was assessed by immunoprecipitation and by dot-blotting the glycated and non-glycated fractions of enzymes separated by affinity chromatography. Ribose and fructose inactivated more rapidly than glucose and glucose 6-phosphate. The esterase was progressively inactivated by prednisolone-21-hemisuccinate at a lower concentration. Activity and antigenicity were lost simultaneously. The glycated enzyme had entirely lost its antigenicity. These results further support the idea that inactivation of enzyme and loss of antigenicity are simultaneous.

Aging↗
PubMed1999

Alpha-crystallin as a molecular chaperone.

The role of alpha-crystallin as a molecular chaperone may explain how the lens stays transparent for so long. Alpha-crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by "trapping" the protein in a high molecular weight complex. It also protects enzyme activities. The substrate protein may interact while in a molten globule state. Alpha-crystallin predominantly binds to proteins very early in the denaturation pathways. The amphiphilic nature of alpha-crystallin, a polar C-terminal-region and a hydrophobic N-terminal-region are all essential for chaperone function. The flexible C-terminal extension maintains solubility and can bind to opposing charged residues of unfolding proteins. Hydrophobic regions in the N-terminal region then hold the unfolded protein. Specific areas important for chaperone binding and function have been identified throughout the N-terminal-region, connecting peptide and C-terminal extension. After a substantial amount of chemical data and models, cryo-EM images of alpha-crystallin have confirmed a variable 3D surface with a hollow interior. Alpha-crystallin taken from the lens nucleus shows an age-dependent decrease in chaperone function. High molecular weight aggregates and alpha-crystallin found within the nucleus from clear and cataract lenses have reduced chaperone function. Post-translational modifications, known to occur during ageing, such as glycation, carbamylation, oxidation, phosphorylation and truncation cause a decrease in chaperone function. Alpha-crystallin is expressed outside the lens. AlphaB-crystallin can be induced by heat shock in many tissues where it is translocated from cytoplasm to nucleus. Increased expression of alphaB-crystallin has been seen in many pathological states. Conformational disorders, including cataract may have a common aetiology and potentially a common therapy.

Amino Acid Sequence↗
PubMed1999

Can cataract be prevented?

The feasibility of preventing cataract by elimination of risk factors or by anti-cataract therapy is reviewed. Elimination of the major risk factors in Western countries would be difficult, although it has potential in the developing world. Various approaches to drug therapy have been made but ran into problems. The development of aldose reductase inhibitors received by far the greatest financial support but problems with toxicity were rapidly followed by the collapse of the original hypothesis. The case of aspirin, paracetamol and ibuprofen built up more slowly with no encouragement from the pharmaceutical industry. Positive results have been achieved in vitro, and in the prevention of diabetic cataract in rats, as well as the identification of an association with a protective effect against cataract in human patients. A clinical trial of these compounds is required. Other anti-cataract agents are also being investigated.

Aldehyde Reductase↗
PubMed1999

Sugars including erythronic and threonic acids in human aqueous humour.

PURPOSE: Sugars in the aqueous humour of the eye serve both as a source of nutrients to the lens and other anterior ocular tissues, and potentially as an indicator of waste products from these tissues. In this work we intended to measure the levels of sugars in human blood and aqueous humour from cataract patients with and without diabetes. After initial results we decided to identify an unknown sugar component. METHODS: Sugars were measured by hplc. The unknown sugar peak was identified by gas chromatography/mass spectrometry RESULTS: Very little fructose and sorbitol were found. Glucose levels were higher in both blood and aqueous from diabetic patients. During these analyses we found a major component that did not correspond to any sugar reported previously in aqueous humour. This was identified as a mixture of threonic and erythronic acids. CONCLUSIONS: Glucose levels increase in human aqueous humour in diabetes without markedly raised levels of sorbitol or fructose. Erythronic and threonic acids are normal components of aqueous humour and blood. They may be derived from glycated proteins or from degradation of ascorbic acid.

Aged↗
PubMed1998

Nutrition supplements and the eye.

PURPOSE: A review of the role of vitamins, minerals, carotenoids and essential fatty acids in relation to eye health. The mode of action may be directly on the eye or by promoting bodily health on which the eye depends. RESULTS: The lens and retina suffer oxidative damage and the anti-oxidant vitamins A, C and E are implicated as protective. Studies in man give indifferent support to the role of nutrition in the development of cataract. In the elderly, vitamin intake may be inadequate, so that a vitamin supplement may be reasonable. Zinc has a role in retinal metabolism and may be beneficial in macular degeneration. Selenium has an anti-oxidant role. Other minerals including copper have a less defined role. Carotenoids are concentrated at the macula and have an anti-oxidant role. A reduced risk of macular degeneration is found in relation to a high serum level. The essential fatty acid, gamma-linolenic acid (GLA), is useful in Sjögren's syndrome and may help in other dry eye conditions. Omega-3 fatty acids are important in retinal development and have a role in preventing cardiovascular disease. CONCLUSION: All persons should be encouraged to maintain healthy nutrition. Middle-aged and elderly patients may benefit from a supplement. An intake in excess of the recommended daily intake may be beneficial, but this is not proven. Further clinical trials are indicated to define the advisability of vitamin, mineral and other supplements. Dosages for recommended intake and for supplements are given.

Carotenoids↗
PubMed1998

Cataract, Alzheimer's disease, and other conformational diseases.

Unfolding of proteins was shown to occur in human cataract more than 25 years ago. Recently, the term conformational diseases was applied to a whole group of diseases in which unfolded or misfolded proteins accumulate. In this article, common features in the biochemistry and epidemiology of cataract, Alzheimer's disease, and other conformational diseases are explored.

Aged↗
PubMed1998

Is month of birth associated with cataract in later life?

BACKGROUND: It has been suggested that season of birth might influence the susceptibility to cataract in later life. METHODS: This hypothesis was investigated using data pooled from two case-control studies carried out in Oxfordshire. RESULTS/CONCLUSION: The results showed no relation between month or season of birth and cataract in later life in an English population.

Age Factors↗
PubMed1997

Inactivation of glyceraldehyde 3-phosphate dehydrogenase by sugars, prednisolone-21-hemisuccinate, cyanate and other small molecules.

Diabetes, diarrhoea, renal failure and glucocorticoid therapy have all been identified as independent risk factors for cataract. Increased post-translational modification of proteins, leading to inactivation of enzymes and induction of conformational changes within proteins could result in lens opacification and cataract. Aspirin has been associated with many beneficial effects, including protection against cataract, in-vivo. alpha-Crystallin has been shown to act as a molecular chaperone in-vitro. This lenticular protein prevented the thermal aggregation of other lens proteins in-vitro and has sequence and functional homology with the small heat shock proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) is constitutively expressed in tissues and is susceptible to chemical modification in-vivo. In-vitro incubations of GAP-DH with sugars, cyanate and prednisolone-21-hemisuccinate, all led to significant loss of enzyme activity with time in two buffer systems. Rapid inactivation occurred when GAP-DH was incubated with fructose 6-phosphate or prednisolone-21-hemisuccinate. Slower inactivation was observed when GAP-DH was incubated with fructose, glucose 6-phosphate or potassium cyanate. Glucose did not inactivate GAP-DH under the conditions of our experiments. Aspirin and ibuprofen were shown to inactivate GAP-DH very rapidly in-vitro. Bovine lenticular alpha-crystallin conferred no protection against GAP-DH inactivation. This is the first occasion that alpha-crystallin has been demonstrated to be unable to protect against inactivation in our chemical enzyme inactivation system. This may have implications for the susceptibility of lenticular GAP-DH to post-translational inactivation.

Animals↗