Biomedical subjects

T R Shearer

Publications and source records attributed to T R Shearer.

At least 19 recordsLinked to original sources

PubMed1992

Superior prevention of calcium ionophore cataract by E64d.

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.

Animals↗

PubMed1992

Involvement of calpain in diamide-induced cataract in cultured lenses.

Lenses cultured in diamide first developed outer cortical opacities followed by nuclear cataract. Lens hydration and total calcium were markedly increased by diamide. Proteolysis of crystallins were observed in nuclear cataract lenses. Calpain in the soluble fraction of lenses cultured with diamide was decreased, while calpain in the insoluble fraction was increased. Co-culture with E64d, an inhibitor of cysteine protease such as calpain, especially prevented nuclear opacities and proteolysis of crystallins, indicating that calpain was involved in cataract formation by diamide.

Animals↗

PubMed1992

Calpain II induced insolubilization of lens beta-crystallin polypeptides may induce cataract.

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca2+, it was limited to 6% of the soluble protein present and resulted from precipitation of proteolyzed beta-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble beta-crystallin polypeptides produced by calpain II were similar to insoluble beta-crystallin polypeptides found in cataractous lenses. Trypsin also caused insolubilization of beta-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from beta-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.

Animals↗

PubMed1992

Association of calpain with insoluble pellet of rat lens.

Calpains are calcium-activated neutral proteases found in many tissues including the lens. The purpose of this research was to localize calpain in various biochemical fractions of the rat lens. Lenses were homogenized (with and without added calcium) and separated into water-soluble and -insoluble fractions, which were further extracted with urea, NaOH, and SDS. Of the total calpain 10% was insoluble. In the lens calpain was found to be both insoluble and associated with the membrane. Extraction of calpain from the insoluble fraction suggested calpain was loosely and tightly associated with the membrane. Calpain associated with membrane-rich fractions was obtained from discontinuous sucrose gradients, confirming the above. Calcium increased the amount of calpain associated with the insoluble fraction up to 30% of the total calpain. When the calcium was chelated, this calpain once again became soluble, and its specific activity was higher than water-soluble calpain. The translocation of calpain from the water-soluble fraction to insoluble fractions by calcium may be important because: (1) it may bring calpain into proximity with its substrates; and (2) it may activate calpain, since membrane phospholipids lower the protease's calcium requirement.

Animals↗

PubMed1992

Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses.

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.

Amino Acid Sequence↗

PubMed1992

Hydration and elevated calcium alone do not produce xylose nuclear cataract: role of proteolysis by calpain.

The purpose of this experiment was to determine the contribution of calpain proteolytic enzyme (EC 3.4.22.17) in the formation of nuclear cataract during lens culture in xylose. Increased lens calcium was found to be required for formation of xylose nuclear cataract in our culture system. Inhibition of calpain by the cysteine protease inhibitor E64 was effective in slowing the formation of nuclear cataract, even though lens calcium and hydration were markedly elevated. These results showed that hydration and elevated calcium alone do not produce xylose nuclear cataract, and they indicated that calpain proteolysis may be necessary for xylose nuclear cataract in the rat lens.

Animals↗

PubMed1992

Selenite nuclear cataractogenesis: a scanning electron microscope study.

The sequential changes during selenite nuclear cataractogenesis were examined with a scanning electron microscope (SEM) and correlated with slit lamp observations. A posterior opacity, visible with the slit lamp 1-2 days after injection of sodium selenite, was found to consist of masses of vacuoles in the superficial posterior cortex by SEM. 2-3 days post injection, a biomicroscopic refractile ring around the nucleus was represented by SEM abnormalities suggesting membrane damage and possible loss of cytosol in the perinuclear region. All normal structure in this region was lost by 5 days after injection when the central nucleus had become opaque. SEM also showed evidence for damage in areas which were still clear by slit lamp examination. Changes, characteristic of aging, were found near selenite induced damage in peripheral (younger) fibers.

Animals↗

PubMed1992

Review of selenite cataract.

Recent advances in understanding the mechanism of selenite cataract have resulted from locating the cleavage sites on proteolyzed beta-crystallins from the cataract, mimicking the insolubilization of crystallins found in the cataract in an in vitro system, studying cataract produced in lenses cultured in selenite, and permanently or temporarily reducing the rate formation of selenite cataract by use of various inhibitors. The present review discusses the selenite cataract as a useful model for understanding the role calcium-induced proteolysis in cataract formation.

Amino Acid Sequence↗

PubMed1992

Calpain in rat cornea.

This study was conducted to provide a description of calpain proteolytic enzyme (EC 3.4.22.17) in normal rat cornea and to document immunohistochemical changes in calpain distribution during maturation. Corneal soluble proteins were fractionated by diethylaminoethyl chromatography on high-performance liquid chromatography. Fractions were analyzed for calpain by enzyme-linked immunosorbent assay, immunoblotting, and caseinolytic enzyme activity with fluorescein isothiocyanate-labeled casein. Calpain II from the soluble fraction of 2-week-old and 3-month-old rat corneas eluted at a similar NaCl concentration (220-240 mmol/l) as calpain II from other tissues, was inhibited by E64, contained an 80-kilodalton subunit in immunoblots, and was present at specific activity of 473 units per gram of protein in 3-month-old rats and 801 units per gram of protein in 2-week-old rats. Calpain antigen also was present in the ethylenediaminetetraacetic acid and EGTA washed insoluble fraction of cornea. Calpain was found (by immunohistochemical analysis) in all layers of the epithelium but not in the stroma. Enzyme-linked immunsorbent assay, immunoblots, and immunohistochemical analysis showed that calpain in the whole cornea did not change with corneal maturation. It was hypothesized that calpain in the cornea may be involved in the turnover of epithelial cells during normal maturation.

Animals↗

PubMed1992

Induction of elongation in cultured rat lens epithelial cells by FGF and inhibition by selenite.

The purposes of this experiment were to: (1) test if fibroblast growth factor (FGF) induced elongation of cultured rat lens epithelial cells (LEC) and (2) determine if selenite affected elongation of LEC. FGF (125-500 ng/ml) reduced the number of colonies of LEC, but it did not induce elongation when cells were cultured on plastic dishes. One hundred micromolar and, to a lesser extent, 10 mumol/l selenite also reduced the number of colonies of LEC. Coculture of FGF and selenite on plastic caused a synergistic reduction in the number of colonies. FGF (125-1000 ng/ml) induced a dramatic morphologic change in LEC. Elongated processes radiated from stellate-like cell aggregates when cells were cultured on reconstituted basement membrane matrix (Matrigel). Again, 100 mumol/l selenite and, to a lesser extent, 10 mumol/l selenite reduced the number of cell aggregates with processes on Matrigel. These results indicated that an inhibitory effect of selenite on the elongation of LEC may be a factor in the development of selenite cortical cataract.

Animals↗

PubMed1991

Cysteine protease inhibitor E64 reduces the rate of formation of selenite cataract in the whole animal.

The purpose of this experiment was to test the effectiveness of E64 in prevention of selenite nuclear cataract in the whole animal. E64 is an inhibitor of cysteine proteases such as calpain (EC.3.4.22.17). In the whole animal, daily intraperitoneal injection of E64 was mildly effective in slowing the rate of formation of selenite nuclear cataract, although prevention was not permanent. Frequency of the nuclear cataract in selenite group at 5 days post selenite injection was significantly decreased from 40% to 17% in the selenite + E64 group, and the density of cataract in the Se + E64 group was reduced. However, crystallins and calpain were still degraded in the selenite + E64 group. E64 was more effective against selenite cataract when present continuously during lens culture, where it slowed the rate of formation of nuclear opacity. Amelioration of cataract occurred both in vitro and in vivo even though lens calcium concentrations were elevated. The results supported the idea that application of calpain inhibitor is beneficial in prevention of rodent selenite cataracts.

Animals↗

PubMed1991

Amelioration of cataracts and proteolysis in cultured lenses by cysteine protease inhibitor E64.

Cataracts were produced in cultured rat lenses by either 10 microM calcium ionophore A23187, 25 microM sodium selenite, or 30 mM xylose. E64, an inhibitor of cysteine proteases, such as calpain (EC, 3.4.22.17), reduced severity of cataract and proteolysis of crystallins when included at a 500 microM concentration in the culture medium along with cataractogenic agents. Calpain II enzyme activity and the amount of calpain antigen were decreased in the cytosol of cataractous lens. However, E64 caused an increase in the amount of an 80-kD calpain subunit associated with the ethyleneglycol-bis-(beta-aminoethylether) tetraacetic acid/ethylenediaminetetraacetic acid-washed insoluble proteins when lenses were incubated with cataractous agents. These data indicate that E64 was at least partially effective in inhibiting lens calpain, and that activation of lens calpain may involve binding to the insoluble fraction. These results provide strong evidence for the activation of calpain in rodent cataracts and suggest testing inhibitors of calpain as anticataract drugs.

Animals↗

PubMed1990

Calpain II in two in vivo models of sugar cataract.

Cataracts were produced in rat lenses by either feeding a diet containing 50% galactose or by inducing diabetic condition by intravenous injection of streptozotocin. Proteolysis of crystallins, protease activity of calpain II enzyme (EC 3.4.22.17), and presence of calpain molecule (antigen) were determined at four cataract stages--I, cortical vacuoles, II, vacuoles plus hazy cortex, III, nuclear cataract, and IV, mature cataracts. Calpain activity was normal or moderately elevated at early stages of galactose and diabetic cataracts. Later stages III and IV showed proteolysis of lens crystallins, increased proportion of insoluble proteins, loss of calpain enzyme activity and calpain molecule from the soluble fraction, and reduced amounts of calpain associated with insoluble pellet. In galactose cataract, the largest increase in lens calcium were found when proteolysis was present. These results provide evidence for calpain-induced proteolysis of lens crystallins in two in vivo models of sugar cataracts in rodents.

Animals↗

PubMed1990

Calpain and calpastatin in rabbit corneal epithelium.

The purpose of this study was to provide a direct assay for calpain and its endogenous inhibitor calpastatin in normal rabbit epithelium. Corneal epithelial extracts were fractionated by DEAE (1) chromatography on HPLC. Fractions were analyzed for calpain by ELISA, immunoblotting, and caseinolytic enzyme activity with FITC-labeled casein. Results demonstrated immunoreactive peaks for calpains I and II. Calpain II from the soluble fraction of corneal epithelium eluted at a similar NaCl concentration (260 mM) as calpain II from other tissues, was inhibited by both E64 and the removal of Ca, contained an 80 kDa subunit in immunoblots, and was present at specific activity of 220 units/g protein (in a crude homogenate). Calpain antigen was also present in the EDTA/EGTA washed insoluble fraction of corneal epithelium. Calpastatin in corneal epithelium eluted at 130 - 160 mM NaCl on DEAE, coeluted with calpain I, and was present at 330 units/g protein (crude homogenate). The results demonstrated a calpain/calpastatin system in corneal epithelium, where it is speculated to play a role in epithelial cell turnover and wound healing.

Animals↗

PubMed1990

Changes in crystallin concentration in rat aqueous and vitreous humors after selenium-induced reversible cortical cataract.

Five months after selenite injection, 58% of the rats that had developed cataract earlier underwent a reversal of the cortical opacity. The purpose of this study was to determine if lenses undergoing recovery from cortical opacity reestablish their ability to retain crystallins. By direct ELISA method, the aqueous humor (AH) of control rats was found to contain 18, 39 and 10 ng/ml alpha-, beta- and gamma-crystallin, respectively, while vitreous humor (VH) contained 43, 98 and 23 ng/ml of alpha-, beta- and gamma-crystallin, respectively. In rats with mature cataracts which did not recover by 5 months after selenite injection, there was an approximately 10-fold greater crystallin concentration in the AH and about 20 times greater crystallin concentration in the VH than in the controls. In contrast, rats undergoing recovery from cortical cataract showed almost normal concentration of crystallins in the AH. While crystallins were still elevated in the VH of the rats undergoing recovery from cortical cataract, the crystallin content was lower than in the rats which did not recover. Higher crystallin concentrations in the VH could be explained by either a greater loss through the posterior capsule, or a slower turnover of VH. Decreased crystallin loss from lenses undergoing recovery suggested that the recovered lens at least partly reestablishes its ability to retain crystallins. These data may demonstrate that the lens fiber permeability is lowered while lens repair is occurring.

Animals↗

PubMed1989

Age-related changes in calpain II and calpastatin in rat lens.

The purpose of these experiments was to determine how the activity and regulation of calpain in rat lens changed during aging. Calpain II enzyme activity and immunoreactivity decreased with both chronological and anatomical age. Two pieces of data suggested that loss of soluble calpain II was a result of both autolysis and insolubilization during aging: (i) proteolytic fragments of calpain were detected in lenses with molecular weights similar to fragments produced during incubation of purified calpain II with calcium; (ii) the water-insoluble fraction of lens cortex contained increasing amounts of calpain antigen during aging both the 75-kDa calpain subunit and a unique high-molecular-weight immunoreactive protein. The regulation of calpain II also appeared to change with age. The activity of calpain II in vivo may be regulated by the relative concentrations of calpain II and its endogenous inhibitor calpastatin. Calpain II concentrations decreased in the rat lens with age, whereas levels of the endogenous inhibitor calpastatin were maintained. Assays of calpain II and calpastatin indicated that upon aging there was insufficient activity of calpain II to overcome the inhibition of calpastatin in the nucleus. These findings were confirmed by incubation of crude lens homogenates of 2-week- and 7-month-old rat lens regions with calcium. It is hypothesized that binding of calpain II to membrane may be important for calpain II activation, especially in older lens regions, because it may allow escape from the inhibitory action of calpastatin.

Aging↗

PubMed1989

Calpain II in human lens.

The purposes of the current study were to: determine if human lenses contain calpain II (EC.34.22.17) activity, measure the effect of aging and anatomical location on lens calpain II activity, and determine if human lenses contain the endogenous calpain inhibitor calpastatin. Both enzymatic and immunologic assays indicated that human lenses contained calpain II activity. Calpain II activity was highest in the cortex of lenses from young donors, and lowest in the nucleus of aged lenses, where it was sometimes nondetectable. In some cases, calpain II activity persisted in the nucleus of lenses from donors greater than 70 years of age. Human lenses also contained endogenous calpain inhibitor (calpastatin) in excess over calpain enzymatic activity. Calpastatin activity did not decrease during aging. Although human lenses contained approximately 3% of the calpain activity found in rat lenses, calpain II may still be a major endopeptidase in human lenses. Demonstration of calpain II in human lenses suggested that calpain II could be involved in both lens maturation and cataract formation.

Aged↗

PubMed1989

Role of proteolysis in lenses: a review.

It has been suggested that proteases are involved in removal of damaged or obsolete proteins and/or that the activation of proteases could contribute to cataract formation. This review summarizes the properties of several recently studied lens endopeptidases including: trypsin-like protease, multicatalytic endopeptidase complex, membrane bound proteases, and calpain. Properties discussed include composition, substrate specificity, distribution, changes in activity during aging, and regulation. Additionally, properties of the lens ubiquitin conjugation system are reviewed. When possible, an attempt was made to relate these findings to whether the lens proteolytic activity was involved in clearing damaged proteins, or whether it could contribute to cataract formation. Clearing of damaged or obsolete lens proteins may involve the participation of several protease activities. Findings suggest that lens protease activities are lost at variable rates during aging, and differ in concentration between species. It was concluded that the consequence of proteolytic activity in the lens may depend closely on the compliment of proteolytic activities found. For instance, proteases causing only partial degradation of lens proteins may predominate in lenses undergoing cataract formation, while proteases assisting in the removal of partially degraded proteins are lost. The partially degraded lens proteins, as well as other denatured lens proteins, may then accumulate and lead to cataract formation.

Aging↗