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Biomedical subjects

E C Abraham

Publications and source records attributed to E C Abraham.

At least 19 recordsLinked to original sources
PubMed1995

Role of glycine 1 and lysine 2 in the glycation of bovine gamma B-crystallin. Site-directed mutagenesis of lysine to threonine.

To determine the role of Gly-1 and Lys-2 of bovine gamma B-crystallin in glycation and cross-linking, Lys-2 was changed to Thr by site-directed mutagenesis. A polymerase chain reaction was used to perform site-directed mutagenesis on the third codon (AAG-->ACG) of bovine gamma B-crystallin cDNA. The wild type and mutant cDNAs were cloned into pMON5743 and expressed in JM101 Escherichia coli cells, and the identity of gamma B-crystallin was confirmed by Western blotting after purification by cation exchange high performance liquid chromatography. Glycation of gamma B-crystallin by [14C]glucose was reduced significantly due to the mutation of Lys-2, supporting the view that Lys-2 is a major glycation site. Peptide mapping showed the presence of two major labeled peptides containing N-terminal sequences, and in the mutant these peptides had longer retention times and reduced radioactivity. Amino acid analysis, after glycation with [14C]glucose, revealed N-terminal glycine as the most predominant glycation site. Lys-2 was glycated slower than Gly-1 but faster than Lys-163. Glycation with DL-glyceraldehyde showed an important role for both Gly-1 and Lys-2 in the glycation-mediated gamma B-crystallin cross-linking.

Amino Acid Sequence↗
PubMed1995

Diabetes affects alpha-crystallin chaperone function.

In vitro glycation was previously shown to influence alpha-crystallin chaperone function. In the present study we show that this function is compromised in diabetes. The alpha H, alpha L and the total alpha fractions were isolated by gel permeation chromatography from the water-soluble protein of streptozotocin-diabetic as well as age-matched normal rats. Based on the beta L-crystallin thermal denaturation assay the chaperone function was significantly decreased in the diabetic rats.

Animals↗
PubMed1995

Decreased molecular chaperone property of alpha-crystallins due to posttranslational modifications.

We studied the effect of oxidation, mixed disulfide formation and glycation of alpha-crystallins on their molecular chaperone property. The ability of alpha-crystallins to protect heat-induced denaturation and aggregation of beta L-crystallin was significantly diminished by these modifications. alpha-Crystallin from senile human lenses also showed significant loss of chaperone-like property. Age-dependent increase in posttranslationally modified alpha-crystallins is the likely cause for this change.

Adolescent↗
PubMed1995

Effect of germanium-132 on galactose cataracts and glycation in rats.

Germanium compounds have been shown to be effective in preventing the formation of advanced glycation end-products and for reversible solubilization of glycated proteins. As protein glycation has been proposed to play a role in lens opacification, we initiated studies to evaluate the effects of 2-carboxyethyl germanium sesquioxide (germanium compound 132 or Ge-132) on galactose-induced cataractogenesis. For this study young Sprague-Dawley rats were fed a 50% galactose diet. One group of rats received topical saline and another group was administered Ge-132 in saline four times a day. The lenses were periodically examined with an ophthalmoscope and at desired intervals processed for light and scanning electron microscopy. Our observations, beginning at 3 days and continuing to 21 days of galactose feeding, exhibited the characteristic galactose-induced morphological alterations, which include the formation of vacuoles, cysts, membrane disruption and swelling of fibers and epithelial cells as well as disorganization of the bow in lenses of rats in both groups. However, in the majority of rats administered Ge-132 these alterations were delayed as compared to the lenses of rats administered saline. Our findings show that, although the initiation, progression and pattern of lens opacification in rats receiving saline and Ge-132 were similar, in the majority of lenses the progression and establishment of mature cataracts in the Ge-132 group of rats were delayed. Analysis of the water-soluble and water-insoluble lens-protein fractions for glycated proteins showed increased levels of the Amadori products and advanced glycation related fluorescent products in galactosemic rats treated with saline eye drops. In rats receiving the topical Ge-132 treatment the levels of these glycation products were substantially reduced to levels lower than control values. Prevention of glycation seems to be a mechanism by which cataract progression is delayed.

Animals↗
PubMed1994

Site selectivity in the glycation of alpha A- and alpha B-crystallins by glucose.

We determined the site selectivity of glycation by glucose (glucosylation) in alpha A- and alpha B-crystallins using two independent approaches. HPLC purified 14C-glucose labeled chymotryptic peptides and affinity chromatography/HPLC purified fully glycated peptides were identified by FAB-MS. Lys 11 and 78 of alpha A-crystallin and Lys 90 and/or 92 of alpha B-crystallin were the fast reacting sites of glucosylation.

Animals↗
PubMed1993

Glycation mediated lens crystallin aggregation and cross-linking by various sugars and sugar phosphates in vitro.

Glycation of lens crystallins results in protein conformational changes, oxidation, browning and aggregation. Though glucose is the major sugar, other sugars and sugar phosphates generated as intermediates of metabolic pathways are present in the lens, albeit at low concentrations. In this study we incubated bovine lens soluble fraction with various sugars and sugar phosphates (5mM for 10 days). The reactivity was in the order trioses > tetroses > pentoses > hexoses. High molecular weight (HMW) aggregates were also formed at a comparable rate. Increased levels of fluorescence were associated with the HMW aggregates with fast reacting sugars. The phosphorylated derivatives were only slightly more reactive than their respective sugars. Interestingly, fructose-1,6-diphosphate was more reactive and cross-linked more readily than fructose-6-phosphate. Gel electrophoresis under reducing and nonreducing conditions showed formation of disulfide linked protein aggregates with slow reacting sugars such as glucose and non-disulfide covalent linked protein aggregates with fast reacting sugars such as erythrose. In contrast, if 0.1 m DTT was present in erythrose incubations (a fast reacting sugar), the HMW aggregate formation was significantly reduced. In order to show the reactivity among the slow reacting hexoses, we incubated lens proteins with 1 M hexoses for 30 days and the results showed that galactose was more reactive and showed higher cross-linking than fructose and glucose. These results thus indicate that relatively low levels of some sugars and sugar phosphates in the lens could be compensated by enhanced lens protein cross-linking and the combined effect could be rather significant with respect to cataractogenesis.

Animals↗
PubMed1993

In vitro glycation and acetylation (by aspirin) of rat crystallins.

In vitro studies with rat lens crystallins were conducted to explore the mechanism by which aspirin (ASA-acetylsalicylic acid) could inhibit cataractogenesis. The purpose of the present study is to show whether gamma-crystallin is the primary target for glycation by glucose and acetylation by ASA. Lens soluble fractions from one and seven month old Sprague-Dawley rats were incubated with 5 mM [14C]glucose with and without 10 mM ASA. alpha, beta, and gamma-crystallins were separated by molecular sieve HPLC and specific activities of each crystallin determined. In vitro acetylation was also studied by measuring protein bound [14C]acetyl groups after incubation with [14C]acetyl ASA. There was 2 to 4-fold faster glycation of gamma-crystallin than all other crystallins from 1-month-old rats and ASA inhibited glycation of gamma-crystallin four times more than that of alpha and beta-crystallins, thus showing preferential glycation of gamma-crystallin and its selective inhibition by ASA. [14C]acetyl incorporation showed increased acetylation of gamma-crystallin in one month old rats, whereas in older lenses acetylation of other crystallins predominated. Treatment with 10 mM ASA showed 35% decrease in free -NH2 groups but protein thiols remained unchanged.

Acetylation↗
PubMed1993

Glycation of human lens crystallins: effect of age and aspirin treatment.

Human lenses of three different ages were used to study the effect of age and aspirin treatment on glycation of alpha-, beta- and gamma-crystallins. Soluble lens proteins were subjected to in vitro glycation with 5 mM [14C]glucose in the presence and absence of 10 mM aspirin. With crystallins from a 27-year-old lens alpha-crystallin was the most readily glycated protein. Glycation of all crystallins decreased substantially (37-77%) in 46- and 67-year-old lenses indicating an age-dependent decline in glycation sites. On the basis of a sensitive chemical assay for protein-bound glycogroups in lenses of 2-82 years of age this decline is apparently due to a 60% increase in in vivo glycation. Aspirin did not show any selectivity with regard to its ability to inhibit glycation of various crystallins. Irrespective of the age glycation of all crystallins was inhibited to a varying extent.

Acetylation↗
PubMed1992

Glycation of lens MIP26 affects the permeability in reconstituted liposomes.

We studied the role of glycation of lens putative gap junctional protein, MIP26, on the permeability as well as on calmodulin mediated gating activity in reconstituted liposomes. Calf lens membranes were incubated with 0-100 mM glucose for 3 days and MIP26 was isolated. There was a glucose concentration dependent increase in the glycation of MIP26 which reached to 2.48 moles/mole of protein with 100 mM glucose. Gel electrophoresis showed that there was no degradation of MIP26 to MIP22 during incubation. Channel permeability was determined by reconstituting MIP26 into asolectin liposomes. There was a MIP26 glycation dependent decrease in the permeability to sucrose. Furthermore, proteoliposomes containing nonglycated MIP26 showed complete uncoupling of the channels with calmodulin whereas the channels containing glycated MIP26 were only partially uncoupled. These results suggest that glycation of MIP26 does interfere with the gating activity in reconstituted liposomes.

Animals↗
PubMed1992

Glycation of human lens proteins: preferential glycation of alpha A subunits.

Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16-85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [3H]NaBH4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg-1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (Ex: 370 nm; Em: 440 nm), expressed as relative fluorescence U mg-1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of alpha-crystallin, while the increases in beta and gamma were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10-50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Glycation of lens membrane intrinsic proteins.

Changes occurring at the membrane are believed to be the decisive factors in the initiation of diabetic cataract. During diabetic hyperglycemia lens crystallins were shown to undergo glycation. Several studies indicated that glycation brings about protein conformational changes thus implicated in cataractogenesis. Since the membrane proteins are the first targets for glycation, in this study we measured the glycation of alkali washed urea-insoluble membrane proteins from control and diabetic rats by two different methods, phenyl-boronate affinity chromatography and [3H]NaBH4 reduction, and confirmed by amino acid analysis. There was a significant increase in the glycation of membrane proteins in diabetic cataract lenses when compared to controls. It appears that lysine is the major site of glycation. Concomitant to early glycation, there was an increase in non-tryptophan fluorescence (Ex: 350 nm/Em: 440 nm) in the diabetic lens membrane proteins suggesting the presence of advanced glycation mediated protein cross-links. In order to identify whether the major membrane intrinsic protein, MIP26, undergoes glycation, we isolated MIP26 along with its degradatory product MIP22 as one peak on molecular sieve HPLC. HPLC isolated MIP26/MIP22 was further separated on SDS-PAGE followed by slicing and counting. This analysis revealed that MIP26 and MIP22 were more or less equally glycated in controls, however, in diabetic rats glycation of MIP22 was glycated slightly higher than MIP26. Moreover, the proportion of MIP22 increased by about 2-fold in diabetic lenses compared to controls. Thus it appears that major glycation sites are still retained in MIP22 in diabetic rat lenses. In vitro glycation studies with bovine lens membranes were also done using 14C glucose, followed by SDS-PAGE and autoradiography. The major protein glycated in vitro also seems to be MIP26. Interestingly, MIP22 was less glycated than MIP26 in vitro.

Animals↗
PubMed1991

Differential glycation of rat alpha-, beta- and gamma-crystallins.

Crystallin glycation seems to play an important role in the development of diabetic cataract. In order to understand the role of glycation in cataractogenesis, levels of glycation of different crystallins were determined by in vitro glycation of rat lens soluble fraction with 50 mM glucose or glucose-6-phosphate (G6P) for up to 5 days and in streptozotocin-diabetic rats during various stages of cataract development. All samples were reduced with [3H]NaBH4 and the tritium incorporation was taken as a measure of glycation. Proteins were routinely separated by molecular sieve HPLC. In vitro studies with glucose showed that gamma-crystallin was readily glycated and reached a plateau by 3 days, while alpha- and beta-crystallins were glycated slowly initially up to 3 days followed by a steep increase as seen on the fifth day. Incubation with 50 mM G6P resulted in an approximately two fold increase in glycation compared to glucose of all crystallins. In the diabetic animals also gamma-crystallin glycation increased approximately twofold within 15 days after the onset of diabetes and an additional threefold within the next 45 days followed by a slight decrease during the following 90-120 days. Increase in glycation, on the contrary, was very slow up to 30 days for alpha-crystallin and up to 60 days for beta-crystallin, followed by a steep increase during the remainder of the experimental period. The high molecular weight (HMW) aggregates had higher levels of glycation than other proteins; the insoluble HMW aggregates contained higher levels of glycation than the soluble HMW aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Reverse-phase HPLC analysis of human alpha crystallin.

A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.

Adolescent↗
PubMed1989

The fate of gamma L crystallins in rat lens during diabetic cataractogenesis as determined by a monoclonal antibody.

We developed a monoclonal antibody against HPLC purified rat lens gamma L crystallins. This antibody was specific to both the polypeptides (19,000 and 21,000 daltons) which constituted the HPLC gamma L peak. Least reactivity was shown against gamma H (24,000 daltons). This antibody was used as a probe to detect the presence of and quantitate gamma L crystallins in lens soluble, insoluble and urea-insoluble fractions during diabetes. Utilizing a direct binding immunoassay (ELISA) we calculated the absolute quantities of gamma L crystallins present in these fractions. Our results show, in normal animals there was a minimal change in total quantities of gamma L crystallins in soluble fraction from 1 month to 5 months of age, but a slow accumulation of these crystallins in insoluble and urea-insoluble fractions was seen during the same period. Diabetes resulted in a depletion of gamma L crystallins from the soluble fraction, both in terms of relative proportion and absolute quantities. In insoluble and urea-insoluble fractions the relative proportions of these crystallins were increased dramatically up until 60 days followed by a decrease during 90-120 days of diabetes, whereas, the absolute quantities remained more or less steady after reaching the maximum on 60 days. Although the relative proportions of gamma L crystallins in the insoluble fraction seem to be less when compared to urea-insoluble fraction, the total quantity of these crystallins was much higher due to abundance of this fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Inhibition of lens crystallin glycation and high molecular weight aggregate formation by aspirin in vitro and in vivo.

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)

Animals↗