Differential glycation of rat α-, β- and γ-crystallins
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We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.
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Previous studies have shown that glycation of lens proteins could be a contributory factor in the development of diabetic and senile cataracts. Acetylation by aspirin (acetylsalicylic acid or ASA) has been used as an inhibitor of glycation which blocks the potential glycation sites (epsilon-NH2 groups). If glycation is a contributory factor, inhibition of glycation by acetylation should bring about a corresponding decrease in cataractogenic changes. We relied on in vitro glycation system and streptozotocin-diabetic rats to study the effects of ASA on lens crystallin glycation, thiol oxidation and aggregation. For in vitro studies, sterile lens soluble crystallin preparations from 1-month-old rats were incubated, under nitrogen, with 50 mM glucose and 20 mM ASA up to 15 days at 37 degrees C. To study the in vivo effect in diabetic rats, ASA feeding (200 mg/kg body wt/day) was initiated 1 week prior to streptozotocin administration, and sacrificed on 15, 30, 60 and 90 days after injection. The in vitro data show the inhibitory effect on glycation of ASA with all concentrations that were tested (5, 10, 20 mM ASA); the percentage inhibition increased with increasing ASA concentration and time. For example, with 50 mM glucose and 20 mM ASA incubated for 15 days, there was a significant decrease in glycation (P less than 0.05), thiol oxidation (P less than 0.05) and aggregation (P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)
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The effect of various concentrations of N,N″ -bis[ p -( N′ -methylamidino)phenyl]terephthalamidine, tetrahydrochloride (NSC 57155)[4][1] for various incubation periods on P815Y cells in tissue culture and on sensitive P815 and resistant P815/NSC 57155 cells in mice was studied. A 50% growth inhibition in tissue culture after 72 hr was produced by incubation with 300 µg/ml for 15–30 min, 100 µg/ml for 1–2 hr, or 1 µg/ml for 72 hr.
The incorporation of NSC 57155-14C into cells and its distribution in nuclei and cytoplasm were studied with a 2-hr incubation at a concentration of 100 µg/ml. After that period most of the activity was found in the cytoplasm and only very little in the cell nuclei. More than 50% of this activity could be extracted with nonpolar solvents.
Incorporation of NSC 57155-14C occurred at a slower rate in resistant P815/NSC 57155 cells than in sensitive P815 cells.
When P815 and P815/NSC 57155 cells were bioassayed after in vitro incubation with 100 µg/ml of NSC 57155, the cytostatic effect was found to be time dependent. P815/NSC 57155 cells were somewhat sensitive to these high concentrations of the drug, but a longer incubation period was required than with the sensitive cells.
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Because of minimal or no turnover, lens proteins are subjected to substantial post-translational modifications which in turn disrupt lens architecture and change the optical properties leading to senile cataract formation. Progressive glycation is believed to have the potential to initiate the changes that are conducive to lens opacification. Fisher 344 rats were systematically followed from juvenile to older and aged phases of their life to study the relationship between lens glycation and high molecular weight (HMW) aggregate formation as well as quantitative and qualitative changes in lens crystallins. Levels of glycated proteins were quantified by affinity chromatography. Changes in lens crystallin composition and HMW aggregate formation were monitored by molecular sieve HPLC, further confirmed by SDS-PAGE and IEF techniques. As the age advances HMW and insoluble proteins increase with a concomitant disappearance of gamma-crystallins from soluble fraction. This disappearance of gamma-crystallins coincided with increased glycation (approximately 2-fold higher in insoluble fraction) and decreased sulfhydryl groups from soluble fraction. It appears that lens protein glycation, disappearance of gamma-crystallins and sulfhydryls from soluble fraction and increase of insoluble fraction and HMW aggregate are interrelated.
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