Spatial and temporal expression patterns of the epithelial cell adhesion molecule (EpCAM/EGP-2) in developing and adult kidneys.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is expressed by most epithelia and is involved in processes fundamental for morphogenesis, including cell-cell adhesion, proliferation, differentiation, and migration. Previously, a role for EpCAM in pancreatic morphogenesis was confirmed in vitro. Furthermore, changes in the EpCAM expression pattern were found in developing lung and thymus and in the regenerating liver. Therefore, EpCAM was proposed to be a morphoregulatory molecule. METHODS: Using immunohistochemistry, the expression pattern of human and murine homologues of EpCAM was characterized in adult and embryonic kidneys from humans and human-EpCAM (hEpCAM)-transgenic mice. RESULTS: EpCAM expression was found in the ureteric bud throughout nephrogenesis. EpCAM was not expressed in the metanephric mesenchyme. In comma- and S-shaped bodies, both metanephric mesenchyme derived structures, EpCAM expression appeared by E13.5. In adult kidneys, most epithelia expressed varying levels of EpCAM, as confirmed by double staining for human EpCAM and segment-specific nephron markers. Podocytes were EpCAM negative. At the cellular level, the EpCAM expression shifted from apical in embryonic to basolateral in adult kidneys. CONCLUSIONS: The spatiotemporal expression pattern of EpCAM changes during nephrogenesis. In the adult kidney, the expression varies markedly along the nephron. These data provide a basis for further studies on EpCAM in developing and adult kidneys.

Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.

The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of approximately 40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis.

Redox control of K+ channel remodeling in rat ventricle.

Electrical remodeling of the diseased heart contributes to contractile dysfunction and arrhythmias, and is characterized by down-regulation of K(+) channels that control action potential morphology. We have recently shown that remodeling of K(+) channels underlying the transient outward current (I(to)) involves a shift in cell redox balance that is reflected by a depletion of the endogenous redox buffer, glutathione (GSH). This study used a pharmacological model to further examine the role of redox-mediated mechanisms in regulating cardiac K(+) currents. Inhibition of major redox pathways was elicited in normal rats by daily injections of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of thioredoxin and glutathione reductases, and buthionine sulfoximine (BSO), a blocker of GSH synthesis. Fluorescence microscopy studies showed that [GSH] in isolated ventricular myocytes was decreased ~50% from control after 3 days of BCNU/BSO treatment (P<0.05), consistent with a shift in cell redox state. In voltage-clamp experiments, maximum I(to) density was decreased 33% from control in left ventricular myocytes from BCNU/BSO-treated rats (P<0.05), while the inward rectifier and steady state outward currents were not significantly altered. Decreased I(to) density correlated with significant decreases in Kv4.2 mRNA and proteins levels of Kv4.2 and Kv1.4. Down-regulation of I(to) in myocytes from BCNU/BSO rats was reversed in vitro by exogenous GSH or N-acetylcysteine, a GSH precursor and antioxidant. I(to) density and [GSH] were also up-regulated by receptor tyrosine kinase activation with insulin or a tyrosine phosphatase inhibitor. The effect of these activators on I(to) was blocked by inhibitors of PI 3-kinase, MEK and p38 MAP kinases. These data suggest that expression of cardiac I(to) channels is regulated by endogenous oxidoreductase systems and that receptor tyrosine kinase signaling functionally impacts K(+) channel remodeling through its control of cell redox state.

The presence of a transsulfuration pathway in the lens: a new oxidative stress defense system.

The finding that a lens under oxidative stress accumulated free and protein-bound cysteine (protein-S-S-cysteine) in the fiber cells prompted us to examine if there is an alternative source for cysteine pools besides the active cysteine transport system in the lens, namely, the transsulfuration pathway of homocysteine-cystathionine-cysteine, which utilises methionine through transmethylation. We examined the presence of the gene for cystathionine-beta-synthase (CBS), the rate limiting enzyme that converts homocysteine to cystathionine in the transsulfuration pathway, in human lens epithelial (HLE) B3 cells using PCR with primers designed based on the sequence of human liver CBS (Forward 5'-CCA CAC TGC CCC GGC AAA AT-3'; Reverse 5'-CTG GCA ATG CCC GTG ATG GT-3'). The purified DNA fragment (586 bp) from PCR analysis was sequenced and confirmed the homology with CBS gene from other human tissues. The CBS protein band (67 kDa) was present in the HLE cells, which reacted positively with the human liver anti-CBS antibody. The enzyme protein was detected in the pig and human lenses with the highest intensity in the epithelial layer, lower but equal quantities of CBS was present in the cortical and nuclear regions. Human nuclear CBS increased while epithelial CBS decreased with aging. Oxidative stress transiently upregulated the gene expression of CBS both in HLE cells (0.1 mMH2O2) and in pig lens cultured in TC 199 medium (0.5 mMH2O2). The catalytic activity for CBS, which was assayed by measuring the production of C14-cystathionine from C14-serine in the presence of homocysteine, S-adenosyl-methionine and pyridoxal phosphate, was detectable in the HLE cells and transiently activated with H2O2. Free cystathionine accumulated when HLE B3 cells were treated with propargylglycine (PGG), an inhibitor of cystathionase, the downstream enzyme that converts cystathionine to cysteine. More cystathionine accumulation occurred when the cells were simultaneously exposed to PGG and 0.1 mMH2O2. We have shown that oxidative stress of H2O2 could increase the flux of this transsulfuration pathway by committing more homocysteine to cysteine and glutathione production as H2O2 (0.1 mM) inhibited the remethylation enzyme of methionine synthase while concurrently activating the CBS enzyme. This is the first evidence that a transsulfuration pathway is present in the lens, and that it can be upregulated under oxidative stress to provide additional redox potential for the cells.

Effect of H(2)O(2)on human lens epithelial cells and the possible mechanism for oxidative damage repair by thioltransferase.

Human lens epithelial (HLE) B3 cells were used to study the oxidative damage and cellular repair with respect to the redox homeostasis, the oxidative defense enzymes and the glucose metabolic pathway. The effect of oxidative stress on cell growth was initially analyzed by culturing the cells with a bolus amount (0.02--0.1m M) of hydrogen peroxide (H(2)O(2)) in minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) for 1 week. Concentration of H(2)O(2)greater than 0.03m M showed progressive inhibition of cell growth. However, the cells were also shown to tolerate H(2)O(2)concentrations up to 0.5m M by detoxifying the exogenous oxidant within 3hr without any detectable DNA damage. Therefore, this short-term H(2)O(2)exposure model was chosen to study the effect of oxidative stress on the cellular redox homeostasis. HLE B3 cells were first grown to confluence in MEM with 20% FBS. Approximately 1.6 million cells were gradually weaned off serum by subculturing in 2% FBS overnight, followed by serum-free medium for 30 min before subjecting to a bolus of 0.1m M H(2)O(2)for up to 180 min. These cells were used for biochemical analysis, which included H(2)O(2)detoxification (H(2)O(2)in the medium), glutathione (GSH) level and lactate production. Activity measurements were conducted on the oxidation defense enzymes: glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx); the dethiolating enzyme, thioltransferase (TTase); and a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (G-3PD). While the B3 cells were shown to tolerate and detoxify 0.1m M H(2)O(2)within 60 min, the GSH pool was transiently depleted in the first 60 min before fully recovered. GPx suffered more than 80% loss in activity and was unable to recover fully. GST showed slight inactivation but neither GR nor TTase was affected. G-3PD was inactivated to < 50% within 15 min of oxidative stress and was reactivated gradually to 80% of normal at the end of 180 min, concurrent with the transient loss of lactate production in the same cells. The reactivation of G-3PD was both temperature- and GSH-dependent, occurring only at physiological temperature and failing to reactivate when the intracellular GSH pool was depleted by BCNU (GR inhibitor) pretreatment. The inactivated cellular G-3PD in the cell extract could be partially reactivated by DTT (6m M) or by recombinant human lens thioltransferase (RHLT) but not by GSH (1m M), GR or GST. HLE cells cultured in the presence of L-(35)S-cystine and cycloheximide displayed an extra radiolabelled protein band on the autoradiograph in the H(2)O(2)treated cells. The labelled band was positively reacted with G-3PD antibody and could be removed by RHLT, indicating that S-thiolation of G-3PD occurred. The H(2)O(2)pre-exposed cells also transiently accumulated proteins modified by thiolation, including protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). It can be concluded that HLE could endure up to 0.1m M of H(2)O(2)oxidative stress since the cell could be protected by its effective repair systems, including dethiolating the inactivated key SH-sensitive enzymes. TTase may play a role in this. One of the mechanisms may be through preserving glucose metabolism and supplying ATP needed for maintaining cell viability.

Localization of a recessive juvenile cataract mutation to proximal chromosome 7 in mice.

OBJECTIVE: To localize the chromosomal position of a novel cataract mutation (juvenile recessive cataract; jrc) in mice. METHODS: A mapping population was developed by crossing cataract males (albino MH) to wild-type females (black C57BL/6J). F1 females were backcrossed to albino MH males with cataracts. RESULTS: The results were consistent with a model of a single autosomal recessive gene [153 cataract, 169 wild-type; chi2 = 0.8, 1 degree of freedom (d.f.), p > 0.35]. Linkage with the albino (tyrosinase; Tyr) locus was evident (chi2 = 61.5, 1 d.f., p < 0.0001), implicating chromosome 7 as the location of jrc. Recombination percentages (+/- SE) between jrc and D7Mit340 (1.2 cM location), D7Mit227 (16.0 cM) and D7Mit270 (18.0 cM) were 17.1 +/- 2.1, 3.7 +/- 1.1 and 6.2 +/- 1.3%, respectively. Multi-point mapping determined that the most likely order of these loci is D7Mit340 - jrc - D7Mit227 - D7Mit270 - Tyr. Although animals with the mutant phenotype appeared to have little or no sense of sight, their growth was not different (p >0.20) from that of normal mice. CONCLUSION: The jrc mutation model may be useful in the study of the genetics of cataracts in other animal species, including humans.

Identification and characterization of a new mammalian glutaredoxin (thioltransferase), Grx2.

A thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a variety of cellular redox pathways. A single cytosolic Grx (Grx1) was previously described in mammals. We now report identification and characterization of a second mammalian Grx, designated Grx2. Grx2 exhibited 36% identity with Grx1 and had a disulfide active center containing the Cys-Ser-Tyr-Cys motif. Grx2 was encoded in the genomes of mammals and birds and expressed in a variety of cell types. The gene for human Grx2 consisted of four exons and three introns, spanned 10 kilobase pairs, and localized to chromosome 1q31.2-31.3. The coding sequence was present in all exons, with the first exon encoding a mitochondrial signal peptide. The mitochondrial leader sequence was also present in mouse and rat Grx2 sequences and was shown to direct either Grx2 or green fluorescent protein to mitochondria. Alternative splicing forms of mammalian Grx2 mRNAs were identified that differed in sequences upstream of exon 2. To functionally characterize the new protein, human and mouse Grx2 proteins were expressed in Escherichia coli, and the purified proteins were shown to reduce mixed disulfides formed between GSH and S-sulfocysteine, hydroxyethyldisulfide, or cystine. Grx1 and Grx2 were sensitive to inactivation by iodoacetamide and H(2)O(2) and exhibited similar pH dependence of catalytic activity. However, H(2)O(2)-inactivated Grx2 could only be reactivated with 5 mm GSH, whereas Grx1 could also be reactivated with dithiothreitol or thioredoxin/thioredoxin reductase. The Grx2 structural model suggested a common reaction mechanism for this class of proteins. The data provide the first example of a mitochondrial Grx and also indicate the occurrence of a second functional Grx in mammals.

Regulation of thioltransferase expression in human lens epithelial cells.

PURPOSE: To study how the expression of thioltransferase (TTase), a critical thiol repair and dethiolating enzyme, is regulated in human lens epithelial cells under oxidative stress. Also to examine whether depleting the primary cellular antioxidant glutathione (GSH) in these cells has any influence on TTase expression under the same conditions. METHODS: Human lens epithelial cells (B3) were grown to confluence (1.6 million) and gradually weaned from serum in the medium before exposing to 0.1 mM H2O2 for 2 hours. Cells were removed at the time intervals of 0, 5, 10, 15, 30, 60, and 120 minutes for protein measurements of GSH and TTase activity and for reverse transcription-polymerase chain reaction (RT-PCR) or Northern hybridization analysis to quantify TTase mRNA. The effect of GSH depletion on TTase mRNA expression was examined by treating the cells with buthionine S,R-sulfoximine (BSO); 1-chloro, 2,4-dinitrobenzene (CDNB); or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Lens epithelial cells, depleted of cellular GSH by treatment with BCNU, were subjected to oxidative stress to examine the effect on TTase activity and mRNA level. RESULTS: A transient increase was detected in TTase mRNA after 5 minutes of H2O2 treatment. The upregulation reached a maximum of 80% above the normal level by 10 minutes and gradually decreased as the oxidant was detoxified by the cells. Manipulation of cellular GSH level by treatment with BSO, CDNB, and BCNU resulted in a minimum change in TTase expression. It is noteworthy that when cells depleted of GSH were subjected to oxidative stress, TTase expression was also found to be strongly upregulated. CONCLUSIONS: These observations suggest that the upregulation of TTase expression in the lens epithelial cells could be an adaptive response of the cells to combat oxidative stress to restore the vital functions of the lens proteins and enzymes. Such regulation is independent of cellular GSH concentration.

Human lens thioltransferase: cloning, purification, and function.

PURPOSE: To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT). METHODS: The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2. RESULTS: The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells. CONCLUSIONS: The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

The use of music to decrease agitated behaviour of the demented elderly: the state of the science.

This paper reviews the state of the science of interventions using music to decrease the agitated behaviour of the demented elderly person. Seven research articles were located through computerized databases. The review of the literature suggested that music therapy is a useful intervention to help patients deal with a range of behaviour problems. However, overall weakness and limitations of studies are considerable. More rigorous research designs are required to evaluate the immediate and sustained physiological, psychological and sociological effects of music therapy on agitation behaviours of demented elderly. Some recommendations for future research are provided.

Risk factors and incidence of postoperative delirium in elderly Chinese patients.

OBJECTIVES: To investigate the incidence of postoperative delirium among elderly patients and to examine the interrelationship between basic vulnerability and precipitating factors for delirium. DESIGN AND METHODS: This was a prospective cohort study. Data were collected in a tertiary medical center in Taipei, Taiwan. From the 1st to the 5th postoperative day, nurses assessed patients using a confusion-screening tool. Patients with signs of delirium were closely examined for changes in behavior and cognitive status and vital signs, and laboratory data were collected to further validate the organic etiology of delirium. Patients were finally diagnosed according to the DSM-IV criteria in consensus meetings. SUBJECTS: Seven hundred and one elderly patients, that were admitted consecutively for elective orthopedic or urologic surgery, were enrolled in this study. All subjects met the following criteria: (1) 65 years of age or older; (2) able to communicate orally in Chinese, and (3) not unconscious, delirious, deaf, or aphasic upon admission. RESULTS: The overall incidence of delirium among these subjects was 5.1%. Logistic regression analysis identified that older age and preexisting cognitive impairment were vulnerability factors, and that the use of psychoactive drugs was a precipitating factor for delirium. Patients with both basic vulnerability and the precipitating factor had a 56-fold increased probability of delirium (0.28 vs. 0.005 in comparison with those who did not exhibit these factors). CONCLUSION: Few risk factors of postoperative delirium in the older Chinese sample were identified. The only modifiable risk factor appears to be the use of psychoactive drugs.

Modulation of lens glycolytic pathway by thioltransferase.

The observation that the level of S-thiolated proteins (protein-thiol mixed disulfides) was transiently increased in the lens epithelial cells correlation with the transient inactivation of glyceraldehyde-3-phosphate dehydrogenase (G-3PD), a key glycolytic enzyme, when the cells were treated with a bolus of hydrogen peroxide, prompted our speculation that G-3PD may have been transiently thiolated at the SH sensitive active center. In the meantime, thioltransferase (TTase), a thiol regulating enzyme, whose activity remained constant under the same condition, may be regulating G-3PD and other sulfhydryl-sensitive glycolytic enzymes through thiol-disulfide exchange reactions ( Lou et al., 1998 ). To prove this hypothesis, several purified glycolytic enzymes from a commercial source, including hexokinase (HK), G-3PD, pyruvate kinase (PK) and fructose 1,6-bisphosphatase (FBPase), an enzyme in gluconeogenesis, were made into protein-thiol mixed disulfide and used for this study. Glycolytic enzymes in cultured rabbit lens epithelial cells pre-exposed to H(2)O(2)(0.5 m M for 15 min) were also studied for this purpose. Recombinant human lens thioltransferase (RHLT), which was isolated and purified previously in this laboratory, reactivated these pure glycolytic enzymes inactivated by forming protein-S-S-gluthathione (PSSG), protein-S-S-cysteine (PSSC) or, protein-S-S-cysteamine after thiolating with oxidized glutathione, cystine or cystamine respectively. RHLT also reactivated these enzymes in the cell extract of cultured rabbit lens epithelial cells after being briefly exposed to 0.5 m M H(2)O(2). The S-thiolation and dethiolation of FBPase however, showed an opposite effect to that of glycolytic enzymes. These results suggest that TTase may participate in the repair process of glycolytic enzymes during oxidative stress and restore their activities in situ.

Thiol regulation in the lens.

The high content of glutathione (GSH) in the lens is believed to protect the thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size (4-6 mM). However, we have observed that, in aging lenses or lenses under oxidative stress, the size of GSH pool is diminished; and some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC). We have shown in an H2O2-induced cataract model that PSSG formation precedes a cascade of events starting with protein disulfide crosslinks, protein solubility loss, and eventual lens opacification. Recently, we discovered that this early oxidative damage in protein thiols could be spontaneously reversed in H2O2 pretreated lenses if the oxidant was removed in time. This dethiolation process is likely mediated through a redox regulating enzyme, thioltransferase (TTase), which has been discovered recently in the lens. To understand if the role of oxidative defense and repair is the physiological function of TTase in the lens, we cloned the TTase gene and purified the recombinant human lens TTase. Although TTase required GSH for its activity, TTase was far more efficient in dethiolating lens proteins than GSH alone. It favored PSSG over PSSC and dethiolated gamma-crystallin-S-S-G better than the alpha-crystallin counterparts. Furthermore, TTase showed a remarkable resistance to oxidation (H2O2) in cultured rabbit lens epithelial cells when GSH peroxidase, GSH reductase, and glyceraldehyde-3-phosphate dehydrogenase were severely inactivated. We further showed that activity loss in those SH sensitive enzymes could be attributed to S-thiolation, but reactivation via dethiolation could be attributed to TTase. We conclude that TTase can regulate and repair the thiols in lens proteins and enzymes through its dethiolase activity, thus contributing to the maintenance of the function of the lens.

Relative importance of aldose reductase versus nonenzymatic glycosylation on sugar cataract formation in diabetic rats.

The relative importance of sorbitol formation versus nonenzymatic glycosylation and advanced glycosylation end products (AGEs) on sugar cataract formation was examined in diabetic rats. Diabetes was experimentally induced in young, 50 g rats with streptozotocin, and aldose reductase inhibitors were administered in the diet for up to 8 weeks at concentrations of 0.06% for tolrestat or ponalrestat and 0.0125% for AL-1576. Cataract formation was monitored by hand-held slit lamp for up to 11 weeks. Lens polyol levels were monitored by GLC, glycosylated protein levels were spectrophotometrically determined, and AGE products were estimated by fluorescence measurements and ELISA. Sugar cataract formation was observed in all untreated diabetic rats while cataract formation was inhibited in all diabetic rats treated with the AR inhibitors. Lens sorbitol levels were reduced in all ARI-treated rats. Glycosylated lens protein levels were elevated in the diabetic rats, and these levels were not significantly lower in the non-cataractous lenses from ARI-treated diabetic rats. Fluorescence measurements of the lens proteins revealed increased lens AGE levels in all diabetic rats, and these were slightly reduced in the aldose reductase inhibitor treated diabetics. With ELISA, immunoreactive AGEs were only detected in cataractous lenses from the untreated diabetic rats. Immunoreactive AGEs were not detected in the clear lenses of the aldose reductase inhibitor treated diabetics or in the non-diabetic controls. These results support the concept that sugar cataract formation is initiated by the aldose reductase catalyzed intracellular accumulation of polyols and that these sugar cataracts can be prevented through inhibition of aldose reductase.

Does glutathione-S-transferase dethiolate lens protein-thiol mixed disulfides?-A comparative study with thioltransferase.

Protein S-thiolation is a process in which under oxidative stress, vulnerable sulfhydryl groups of proteins are conjugated to non-protein thiols such as glutathione (GSH) or cysteine resulting in the formation of protein-thiol mixed disulfides, protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). This process spontaneously disrupts the redox homeostasis of the cells, which in turn leads to functional disturbances in the respective tissue. In the ocular lens, such modification of proteins may trigger a cascade of events starting with the alteration of protein conformation, protein/enzyme deactivation, protein-S-S-protein aggregation and eventually lens opacification or cataract. Generally, the first line of defense system in the cells protects the lens proteins against such damage. Recent studies in our laboratory have shown that in addition to this defense system, lens cells also possess a well developed system to repair the oxidative damage to the lens proteins. We have identified this repair system as thioltransferase (TTase) and have proved that TTase by its dethiolase activity reverses the protein S-thiolation process which returns the oxidatively damaged lens proteins/enzymes to their original reduced state and restores their physiological functions. We investigated if this repair mechanism was mediated by enzymes other than TTase. We studied glutathione S-transferase (GST) and report here for the first time the cloning, high level expression, and purification of human lens mu and pi isoforms of GST. A comparative study of recombinant human lens TTase and GST (mu and pi) on their dethiolating abilities using lens crystallin-thiol mixed disulfides showed that the lens TTase is 60-70% more efficient in the dethiolation/repair process than GST. When TTase and GST were tested in conjunction for the dethiolation of thiol mixed disulfides, there was no significant enhancement of dethiolase activity. These findings suggest that TTase by itself is an efficient enzyme in the dethiolation/repair process and hence can be considered a crucial system to counteract oxidative stress in the lens.

Correlation of nuclear color and opalescence with protein S-thiolation in human lenses.

Human lens nuclei were collected during routine cataract surgery and used to study the role of oxidation in cataract formation and brunescence. This study focused on the comparison of the intensities of nuclear opacity and pigmentation (brunescence) with the changes in free glutathione (GSH) and the three species of protein-thiol mixed disulfides: protein-S-S-glutathione (PSSG), protein-S S-cysteine (PSSC) and protein-S-S-gamma-glutamylcysteine (PSSGC). Eighty-one freshly excised human lens nuclei from a population with a mean age of 77 were used. The nuclear color was graded using the CCRG system, ranging from yellow to dark brown. The nuclear cataract opalescence of these lenses was also graded using the LOCS II system, ranging from LOCS II NO-1 to NO-4. Three normal human lenses (average age of 88 yr) were also included in the study as controls. The nuclear samples were each analyzed for free GSH and protein-thiol mixed disulfides, respectively. It was found that nuclear GSH decreased as the nuclear color increased from yellow to dark brown (from 0.73+/-0.13 to 0.13+/-0.03 micromole g wet wt-1) and as the nuclear opalescence increased from NO.1 to NO.4 (from 0. 80+/-0.19 to 0.20+/-0.01 micromole g wet wt-1). All these values were lower than that of GSH in normal controls (1.43+/-0.59 micromole g wet wt-1). Levels of both PSSG and PSSC progressively increased, however, as the nuclear color intensified. PSSG increased from 0.29+/-0.05 to 0.91+/-0.11 micromole g wet wt-1while PSSC increased from 0.13+/-0.04 to 0.41+/- 0.06 micromole g wet wt-1. PSSGC concentration progressively increased with increases in both nuclear pigmentation (from 0.05+/-0.01 to 0.23+/-0.05 micromole g wet wt-1) and nuclear opacity (from 0.02+/-0.00 to 0.20+/-0.02 micromole g wet wt-1). In comparison, normal controls had lower levels of all three mixed disulfide species: PSSG, 0.22+/-0.06; PSSC, 0.08+/-0.02; PSSGC, 0.02+/-0.06 micromole g wet wt-1, respectively. The correlation of lens nuclear color and opalescence intensity with nuclear protein S-thiolation indicates that protein-thiol mixed disulfides may play an important role in cataractogenesis and development of brunescence in human lenses.

Thiolation of the gammaB-crystallins in intact bovine lens exposed to hydrogen peroxide.

Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment decreased the glutathione and increased the protein-glutathione and protein-cysteine disulfides in the lens. The molecular mass of the gammaB-crystallin isolated from lenses, not treated with H2O2, agreed with the published sequence (Mr 20,966). Some lenses also had a less abundant gammaB-crystallin component 305 Da higher (Mr 21,270), suggesting the presence of a glutathione adduct. The gammaB-crystallins from H2O2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified gammaB-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of gammaB-crystallins from different lenses indicated that the +305 Da modifications were not at a specific cysteine. For the lenses incubated without H2O2, there was evidence of adducts at Cys-41 and in peptide 10-31, which includes 3 cysteines. Analysis of modified peptide 10-31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, gammaB-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.

Evaluation of AL-05712 and AL-05741 as thioltransferase mimics for the prevention and recovery of pre-cataractous changes in lens.

It has been previously shown that during the aging process, the human eye lens accumulates protein-glutathione mixed disulfides (PSSG) and that the reduced glutathione (GSH) level drops. These changes become even more pronounced during cataractogenesis. In this report, the ability of AL-05712 and AL-05741 to lower PSSG and elevate GSH in three separate model systems was evaluated. AL-05741 was able to decrease PSSG in the cell-free system by over 30% at a concentration of 0.1 mM. AL-05712, the ester form of AL-05741, decreased mixed disulfides by about 8% in the same system in the absence of any cellular esterases. Both compounds could partially inhibit the loss of GSH seen in the H2O2 control in cultured rat lenses and in addition, the accumulation of PSSG was substantially decreased. Human lenses incubated in AL-05712 showed a significant elevation of cortical GSH and a decrease in PSSG in three of four sets of cultured human lenses.

Cloning, high level-expression and characterization of human lens thioltransferase.

Polymerase chain reaction (PCR) primers, directed against the nucleotide sequence of pig liver thioltransferase (PLTT) were used to amplify human lens thioltransferase (HLTT) from a pool of human lens cDNA. The 520 bp PCR fragment obtained was cloned unidirectionally into pCR 3.1-Uni vector and sequenced. The cDNA sequence of the lens thioltransferase had 98% and 87% homology to pig liver and human placental thioltransferases (TTase) respectively. Nhe1 and EcoR1 fragment of the recombinant PCR 3.1-Uni vector was subcloned in pET 23a Expression vector. High level expression of HLTT was accomplished in Escherichia coli and the expressed protein was characterized by immunoblot analysis with anti PLTT and N-terminal amino acid sequence analysis. The recombinant enzyme efficiently dethiolated protein thiol mixed disulfides conjugated to both cystine (PSSC) and glutathione (PSSG) and had a significant dehydroascorbate reductase activity. Human lens thioltransferase thus displayed structural and functional characteristics identical to pig liver and human placental thioltransferases.