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Biomedical subjects

F J Giblin

Publications and source records attributed to F J Giblin.

At least 55 records · Page 3Linked to original sources
PubMed1987

Disulfide-linked membrane proteins in X-ray-induced cataract.

The formation of membrane protein disulfide was investigated at various stages of development of X-ray-induced cataract in the rabbit. Intermolecular disulfide bonding of lens membrane proteins was detected not only in the mature cataract (occurring 8-9 weeks after the X-ray dose) but also at 1 week prior to maturation, in which no significant increase in lens hydration occurs and where opacification is confined mainly to the posterior subcapsular region. Two-dimensional diagonal electrophoresis revealed that polypeptides with apparent molecular weights of 21, 23, 25, 31, 35, 46 and 53 kilodaltons were involved in cross-linking. The MP26 membrane polypeptide was not significantly involved in the disulfide bonding. The oxidation of membrane proteins in stages other than mature was evident only in the lens nucleus (which remained clear) and not in the cortex. The results of this study indicate that an intermolecular disulfide linkage of cytosolic proteins to membranes occurs prior to formation of mature cataract, and may be a precursor to protein aggregation and insolubilization in the mature nuclear cataract.

Animals↗
PubMed1986

Stimulation of the hexose monophosphate shunt in bovine ciliary body under oxidative stress.

The effect of t-butyl hydroperoxide (TBHP) on the oxidation of C1-radiolabeled glucose and C6-radiolabeled glucose by bovine ciliary body was investigated in the presence and absence of the glutathione reductase inhibitors nitrofurantoin (NF) and 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]. 14CO2 production from 1-14C-glucose was markedly stimulated by TBHP and inhibited by BCNU. Using tissue extracts, BCNU was shown to inhibit both glutathione reductase and glucose-6-phosphate dehydrogenase. These results support the hypothesis that bovine ciliary body has high NADPH regenerating capacity by the hexose monophosphate shunt.

Animals↗
PubMed1985

Detoxification of H2O2 by cultured rabbit lens epithelial cells: participation of the glutathione redox cycle.

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4-6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 10(5) cells)-1 3 hr-1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0.01 mM. Participation of the glutathione redox cycle in the H2O2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1-14C]-labeled glucose. The oxidation of [1-14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0.04-0.05 mM H2O2, while the corresponding increase in oxidation of [6-14C]-labeled glucose was only 1.6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0.03-0.04 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 10(5) epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Further observations on the effect of galactose on the development of X-ray-induced cataract in mice.

The effect of a 30% galactose diet on the progression of X-ray-induced cataract in mice was evaluated by following morphological changes as seen by light and transmission electron microscopy in different regions of the lens. Lens opacities as observed with the slit-lamp biomicroscope developed at a slower rate in galactose-fed animals than in those on a normal diet. The protective effect of galactose on X-ray cataract was seen whether galactose feeding was initiated either 1 week before or after exposure to X-ray. At 4 months after X-ray approximately 50% of galactose-fed animals had mature cataracts, compared to 100% in the control group. Similarly, at two weeks after exposure to X-ray, before any lens opacities were observed, morphological changes were more severe in the control group; cells in the meridional row were more disorganized in the control than in the galactose-fed groups. However, the progression of mature cataracts in the two galactose-fed groups were not significantly different. Since free radicals produced by X-ray are thought to be short-lived, the protective effect of galactose feeding after X-ray was unexpected, raising the possibility that some of the active species may be long-lasting. The nature of such radicals, if any, is unknown and remains to be investigated.

Animals↗
PubMed1984

Metabolism and function of glutathione in the lens.

The mammalian lens contains an unusually high concentration of glutathione (GSH), the highest level being in the epithelium. GSH is present largely in the reduced state. The high concentration of GSH in a normal lens and the decreased concentration in most types of cataracts have led to many hypotheses on its role in cataract formation. These hypotheses are considered in the light of current evidence. GSH is synthesized and degraded in the lens. Both processes require ATP, derived largely from glycolysis. Carbohydrate metabolism is also involved in the maintenance of GSH in the reduced state. There is a direct link between the rate of formation of oxidized glutathione (GSSG) and the stimulation of the hexose monophosphate shunt through the generation of NADPH. One possible function of GSH in the lens is to maintain the thiol (SH) groups of proteins in the reduced state, thus preventing formation of high molecular weight (HMW) protein aggregates. The formation of HMW proteins in X-ray-induced cataracts through disulphide bond formation and the involvement of SH oxidation in HMW proteins isolated from human cataractous lenses suggest a role for GSH in protecting protein SH groups. GSH in the lens may also protect critical SH groups involved in regulating cation transport and permeability. Studies with mammalian lenses indicate that lowering the lens GSH concentration leads to increased permeability to cations and inactivation of Na+,K+-ATPase. A consequence of the changes in ion distribution is the inhibition of protein synthesis, which may explain the cessation of growth in cataractous lenses. GSH may also protect against oxidative damage to the lens. GSH metabolism is intimately involved in detoxification of H2O2, normally present in the aqueous humour. Lenses with impaired shunt activity or inhibited glutathione reductase are more susceptible to oxidative damage by peroxide. This may contribute to the formation of cataract.

Animals↗
PubMed1984

Calcium-induced high molecular weight proteins in the intact rabbit lens.

We have investigated the ability of Ca2+ to induce the formation of high molecular weight (HMW) proteins in the intact lens. Ca2+ cataracts were produced in rabbit lenses by culturing the lenses for either four days in medium containing 20 mM Ca2+ or for three days in medium containing 100 mM Ca2+. Lenses cultured in 20 and 100 mM Ca2+ medium became opaque after 20 hr and contained 30 and 200 times higher levels of Ca2+, respectively, than transparent lenses cultured in medium containing 1 mM Ca2+. Lenses exposed to 100 mM Mg2+ did not lose transparency. The opacification of the lenses extended to a depth of 1 mm into the cortical layer and did not involve the nucleus. No significant differences were found in the concentrations of either soluble or insoluble proteins present in freshly excised lenses and Ca2+ cataracts. Soluble HMW proteins, greater than 1.5 X 10(6) daltons, were in two- and five-fold greater amounts in the 20 and 100 mM Ca2+ cataracts, respectively, compared to controls. HMW protein present in the 100 mM Ca2+ cataract amounted to approximately 3% of the total soluble protein in the lens. The amount of Ca2+ present in the HMW fraction was 1 Ca2+ per 5 X 10(5) daltons, no higher than that present in the unaggregated crystallins. No evidence was found for covalent bonding in the aggregate. Results of polyacrylamide gel electrophoresis and double immunodiffusion indicated the presence of alpha- and beta- but not gamma-crystallin in the HMW protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

In vivo observation of lens protein diffusivity in normal and X-irradiated rabbit lenses.

Using the techniques of quasielastic light scattering spectroscopy we have studied in vivo the effect of X-irradiation on the diffusivity of lens proteins as a function of position in the lens and time after irradiation. The findings were compared with those for normal lenses in vivo. The experimentally observed correlation functions show marked changes in the protein diffusivity well before substantial biochemical changes take place.

Animals↗
PubMed1984

A direct correlation between the levels of ascorbic acid and H2O2 in aqueous humor.

There is evidence that H2O2 present in aqueous humor arises from ascorbic acid which is also present in this fluid, but the extent to which peroxide is derived from ascorbic acid is not known. We have measured the concentrations of H2O2 and ascorbic acid normally present in the aqueous humor of various species and also under conditions in which the level of ascorbic acid in the fluid was experimentally altered. In aqueous humor of rabbit and guinea pig the concentration of ascorbic acid was 10 times higher than that present in aqueous of rat and frog. Similarly, the concentration of H2O2 was four to 10 times higher in rabbit and guinea pig aqueous compared to that in rat and frog. Consistent with the higher concentration of ascorbic acid in posterior compared to anterior aqueous humor in the rabbit, the concentration of H2O2 was also significantly higher in the posterior aqueous. When ascorbic acid in rabbit aqueous humor was elevated by intraperitoneal administration of the compound, there was a significant increase in the level of H2O2 in both anterior and posterior aqueous humor. Moreover, when the level of ascorbic acid was lowered experimentally by placing guinea pigs on an ascorbic acid deficient diet, a 10-fold decrease in the level of both ascorbic acid and H2O2 was observed in the aqueous humor. Upon returning the animals to a normal diet, the concentrations of both compounds returned to control values. The direct correlation between the concentrations of ascorbic acid and H2O2 in aqueous humor suggests that ascorbic acid is the primary source of H2O2 in this fluid.

Animals↗
PubMed1984

Protein synthesis in x-irradiated rabbit lens.

The present study deals with the incorporation of 35S methionine into lens crystallins as a function of time after x-irradiation. Crystallin synthesis is first affected approximately 4 weeks following x-irradiation. This coincides with the time period at which the ratio of the two cations in the lens is affected, as shown in earlier studies. A greater decrease in 35S-methionine incorporation into crystallins is observed between 5-7 weeks following x-irradiation in good agreement with a cation imbalance at these time intervals. These studies also revealed for the first time that the change in cation distribution can affect not only crystallin synthesis, but also the synthesis of certain polypeptides of lens membranes. No alteration in protein synthesis could be detected in lens epithelium even after 7 weeks following irradiation. In addition to the effect of Na+ and K+ levels on protein synthesis, an impaired transport of amino acids into the x-rayed lens was also found to be a factor in the observed reduction in synthesis of the crystallin, cytoskeletal and membrane proteins of the fiber cells. It is concluded that Na+/K+ ratio as well as the availability of amino acids in the lens are important factors in protein synthesis of x-ray cataracts.

Animals↗
PubMed1983

Glutathione oxidation in retina: effects on biochemical and electrical activities.

This study investigates the possible role of glutathione (GSH) in defending the retina against oxidative damage. Freshly excised rat retina was found to contain 1.2 mumol/g wet wt GSH and an undetectable level of oxidized glutathione (GSSG). Whole retinas were either incubated or superfused with various concentrations of the GSH-oxidant diamide in order to study the effects of oxidation of GSH on the activity of the hexose monophosphate shunt (HMS) and on the receptor potential of the retina. It was found that exposure of the retina to diamide produced a stimulation of HMS activity up to 26-times that of the control. Significant changes in GSH content and receptor potential were observed at concentrations of diamide that produced more than a 5.4-fold stimulation of HMS activity. The diamide-induced electrical alterations included an increase in latency and peak time of the receptor potential, a delay in the onset of the off response and an increase in the time required for the potential to return to the baseline. It was found that nearly 80% of GHS could be regenerated and that most of the electrical effects of diamide could be reversed by superfusion with normal medium. The results indicate that the retina possesses an active system for maintaining GSH in the reduced state and that this may be essential for the normal function of this tissue.

Animals↗
PubMed1983

Changes in the distribution of lens calcium during development of x-ray cataract.

The present study was designed to examine the possible role of calcium in the opacification of x-ray-induced cataract in rabbit. The results demonstrate that the concentration of calcium in x-rayed lenses, just prior to lens hydration (7.5 weeks postirradiation), was twice that present in contralateral control lenses. At this stage of immature cataract, the lens nucleus remained transparent and maintained a normal level of calcium, but the lens cortex, containing regions of subcapsular opacification, accumulated a level of calcium that was twice that of the control. In the completely opaque mature cataract, (8-9 weeks postx-ray), both the cortex and nucleus had gained significant amounts of calcium. As the concentration of total calcium increased in the immature x-ray cataract, the amount of the cation bound to membranes and insoluble proteins of the cytosol also increased comparably. However, the relative proportion of calcium in the various fractions remained unaltered in the immature cataract; in both control lenses and immature cataracts, 20% of the total calcium remained in the membrane pellet and 70% was located in the soluble protein fraction. Only in the mature stage of cataract was a shift in the distribution of calcium apparent, as the proportion of calcium in the soluble protein fraction increased to 90%. Although only 7% of the total calcium in a mature cataract was bound to membrane, the amount represented a fivefold increase over the control. The results of this study demonstrate that an elevation in lens calcium accompanies the opacification process in x-ray cataract. The work also suggests that changes in calcium levels are not likely to result from inactivation of Ca-ATPase.

Animals↗
PubMed1983

The effect of inhibition of glutathione reductase on the detoxification of H2O2 by rabbit lens.

Mechanisms by which the lens protects against H2O2 are believed to include the metabolism of glutathione (GSH). In the present study, rabbit lenses were exposed to constant concentrations of H2O2 (0.01 to 0.1 mM) that were maintained in culture media with the use of a peristaltic pump. The rate at which H2O2 entered the lens was proportional to its concentration in the medium and reached 2.6 mumol H2O2/lens/3 hr at 0.1 mM H2O2. Up to 0.06 mM H2O2, a concentration that approximates that present in normal rabbit aqueous humor, the activity of the hexose monophosphate shunt (HMPS) increased linearly with no significant decrease in the concentration of lens GSH. However, at 0.1 mM H2O2, there was indication of oxidative damage to the lens as shown by a sharp decrease in HMPS activity and a coincident drop in the concentration of GSH. Pretreatment of lenses with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), blocked the normal threefold stimulation of HMPS activity occurring in the presence of 0.06 mM H2O2 and resulted in accumulation of oxidized GSH. This result demonstrated the inability of H2O2 to react directly with NADPH in the lens. BCNU was shown not to affect the potential of the HMPS to respond to compounds other than H2O2 since it did not alter methylene blue-stimulation of HMPS activity. The study supports the hypothesis that detoxification of H2O2 in the aqueous humor is linked to the metabolism of GSH in the lens and demonstrates that lenses with impaired GR activity are more susceptible to oxidative damage by peroxide.

Animals↗
PubMed1983

Glutathione in calf trabecular meshwork and its relation to aqueous humor outflow facility.

Previous studies have shown that sulfhydryl reagents can alter the facility of aqueous humor outflow but little is known about the sulfhydryl constituents of the aqueous outflow system or the effect of oxidants upon outflow facility. In the present study the concentration of glutathione (GSH) was measured in excised calf trabecular meshwork (TM) and found to be 0.40 mu mol/g wet wt (0.027 mu mol/mg protein). Oxidized glutathione was not detectable in the tissue. TM was found to have significant hexose monophosphate shunt activity as determined by measurement of the oxidation of 14C-1 and 14C-6-labeled glucose in tissue homogenates. The concentration of GSH in TM of enucleated calf eyes could be totally depleted by infusion of medium containing both diamide, which is an oxidant of GSH, and 1,3bis(2-chlorethyl)-1-nitrosourea (BCNU), which is an inhibitor of the enzyme glutathione reductase. The depletion of GSH was found to have no effect on the facility of aqueous outflow. Experiments were also done in which normal and TM GSH-depleted eyes were perfused with medium containing H202. Exposure to H202 produced no effect on outflow facility in the normal eyes but caused a 33% decrease in facility in eyes with the GSH-depleted TM. The results indicate that GSH may not participate directly in regulating aqueous humor outflow but is able to protect TM against H202-induced oxidative damage that would otherwise lead to a decrease in outflow facility.

Amino Acids↗
PubMed1982

The effect of X-irradiation on Na-K ATPase and cation distribution in rabbit lens.

The Na-K ATPase activity of rabbit lens was measured at various times after exposure to a single dose of 2000 rads of X-ray and was compared with that in contralateral control eyes. A decrease in enzyme activity in both whole lens and in isolated capsule-epithelium was first observed 3 to 4 weeks after irradiation and became increasingly marked at 7.5 weeks after X-ray. These findings are consistent with our earlier observations that active transport of cations is reduced in these lenses and support the view that loss of membrane ATPase is responsible for the impairment of the cation pump in X-irradiated lenses. Despite a significant loss of the enzyme, X-irradiated lenses were able to maintain near normal levels of total cations (Na+ + K+), thus accounting for their normal hydration. The results of the changes in lens Na+ and K+ levels revealed that between 4 and 7.5 weeks after X-ray, the gain in Na+ was compensated by an equivalent loss of K+. A breakdown of this relationship of 1:1 exchange of Na+ for K+ is accompanied by a disproportionate increase in Na+ and water.

Animals↗
PubMed1982

Phase separation of X-irradiated lenses of rabbit.

The phase separation temperature (Tcat) was studied as a function of time (age) after the administration of a single dose of radiation (2000 rad), which induces cataract in the rabbit lens. In the normal unirradiated lens, Tcat decreases linearly with age at a rate (DTcat/dt) approximately 2.2 degrees/week. In the irradiated lens, Tcat initially decreases with age much less than the normal lens, then rises sharply with age at the time of the appearance of opacity in the living rabbit eye. We suggest that the phase separation temperature may serve as a sensitive and early indicator of cataractogenic processes in the lens.

Animals↗
PubMed1982

The role of glutathione metabolism in the detoxification of H2O2 in rabbit lens.

Aqueous humor is known to contain a significant level of H2O2, but the mechanisms by which ocular tissues protect against oxidative damage are not well understood. With the use of C-1-, C-2-, and C-6-labeled glucose, the contribution of glutathione (GSH) metabolism and the hexose monophosphate shunt (HMS) to the detoxification of peroxide in the lens has been evaluated. It was observed that H2O2 in the culture medium disappeared rapidly (0.5 mumol H2O2/lens/hr) upon incubation of a rabbit lens at 37 degrees C. At 0 degrees to 3 degrees C, however, the rate of disappearance of H2O2 was only one fifth of that observed at the higher temperature. In the absence of a lens or after pretreatment of the lens with methyl mercuric hydroxide, the rate of disappearance of peroxide from the medium was reduced to nearly zero. When a nearly constant level of H2O2 (0.05 to 0.07 mM) was maintained in the medium by means of a peristaltic pump, the amount of CO2 liberated by the HMS at 37 degrees C was found to be three times that liberated from lenses cultured in the absence of peroxide. No change was noted in the level of GSH in the H2O2-treated lenses at 37 degrees C. A significant decrease in GSH was observed, however, at 0 degrees to 3 degrees C, suggesting nonenzymatic oxidation of the tripeptide at the lower temperature. The results indicate that GSH metabolism and the HMS pathway contribute significantly to the detoxification of H2O2 in the lens.

Animals↗