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Biomedical subjects

M F Lou

Publications and source records attributed to M F Lou.

At least 37 records · Page 2Linked to original sources
PubMed1997

Relationship of protein-glutathione mixed disulfide and thioltransferase in H2O2-induced cataract in cultured pig lens.

It has been previously shown in H2O2-induced cataract model in the rat lens that protein-GSH (PSSG) formation precedes protein-protein disulfide (PSSP) conjugation and lens opacity. This elevated PSSG spontaneously reduces to a normal level when H2O2 is removed. To verify if thioltransferase (TTase), an enzyme that is known in other tissues to dethiolate PSSG, takes part in this recovery process, we examined the relationship of PSSG and TTase in this cataract model. To ensure enough tissue would be available for various biochemical studies, H2O2 induced cataract in pig lens was established and validated with the rat lens model. The study was divided into two parts. One part was to examine the effect of H2O2 concentration, ranging from 0.1 mM-10 mM, during 24 hr. Another part was to study the H2O2 (1.5 mM) induced cataract progression and recovery, parallel to the long-term study in rat lenses reported previously. These lenses were compared for transparency, wet weight, GSH, PSSG levels and the activity of two redox regulating enzymes, glutathione reductase (GR) and TTase. For the most part, pig lens responded to oxidation parallel to the rat lens except that a higher concentration of H2O2 was needed to achieve the same results. Damage induced by H2O3 was concentration dependent. In general TTase activity and GSH level were depleted with a concomitant increase in PSSG. The D50 (50% damage) for GSH in pig lens was 1.5 mM H2O2 (0.5 mM for rat lens) which was chosen for further studies in cataract progression and recovery. At 1.5 mM H2O2, pig lens showed superficial opacity within 24 hr and deeper cortical opacity in 48 hr. The pre-exposed lens became less cloudy when H2O3 was removed from the medium. Incubation of the lens in 1.5 mM H2O2 for one day also induced 50% GSH depletion and four fold PSSG elevations. This accumulated PSSG was dethiolated spontaneously in the absence of H2O2, similar to the findings in the rat lens and human lens models. In contrast protein-cysteine (PSSC) showed little change and did not respond to the recovery condition. TTase lost 50% activity in these lenses during 24-hr H2O3 exposure but regained most of it under recovery. The study on rat lens showed similar results as before, therefore only data on the relationship of TTase activity to PSSG level during cataract development and recovery is reported here. It was found that in the H2O2 (0.5 mM)-exposed rat lenses, the TTase activity was depleted but PSSG accumulation was accelerated within 8 hr. Both recovered quickly (within 8 hr) as soon as the oxidant was removed. Therefore, protein thiolation and dethiolation processes in the cultured rat or pig lenses display a mirror image with the activity pattern of TTase. Based on the close relationship between lens TTase and PSSG indicated above, it is speculated that TTase may regulate PSSG and maintain it at a low concentration in situ. This repair process may contribute to the improved transparency during recovery. Further studies are planned to substantiate this hypothesis.

Animals↗
PubMed1997

[Body image concerns and behavioral responses in a patient with cerebellopontine angle tumor].

This case report investigates the behavioral responses of body image change of a cerebellopontine angle tumor patient. Twelve process recordings obtained from a field study comprised the data for analysis. The dimensions of the subject's body image change were body structure, body function, body sensation, and social function change. Two types of behavioral response were delineated and categorized as the following: (1) Identifying the individual's body image change, such as seeking information, seeking reassurance, and comparative behavior. (2) Maintaining the integrity of individual's body image, such as rationalizing behavior, maintenance behavior, cooperative behavior and seeking religious support. This paper reports how the investigators used nursing interventions to help the subject gradually get through the process and adapt to the body image changes properly.

Adaptation, Psychological↗
PubMed1996

Evidence for the presence of thioltransferase in the lens.

Thioltransferase (TTase) activity was identified and partially purified from the ocular tissue for the first time. The enzyme activity depended on the presence of reduced glutathione (GSH), glutathione reductase (GR) and NADPH to reduce the disulfide bond in a synthetic substrate, hydroxyl ethyl disulfide (HEDS). Maximum activity was obtained in a pH 7.4 phosphate buffer at 30 degrees C. This enzyme distinguishes from other reducing enzymes such as thioredoxin that do not require GSH and GR for their catalytic activity. It also differs from the 52 kDa enzyme, protein disulfide isomerase by its smaller molecular size and its stability against heat treatment. TTase activity was higher in the epithelial layer but distributed evenly in the rest of the lens also, TTase showed similar activity in the lenses obtained from rats, pigs, bovine, guinea pigs, chick embryos and humans. The molecular weight of this enzyme was estimated to be 11.5 kDa on a SDS-PAGE system. Western blot analysis showed the protein reacted positively to the antibody raised by the purified pig liver TTase. Similarly the antibody raised by the partially purified lens enzyme reacted positively with the purified pig lever TTase. The presence of TTase in the lens was confirmed further with the slot blot analysis where it demonstrated a 32P-labeled cDNA from pig liver TTase hybridizing with the RNA in the pig lens or rabbit lens epithelium cells. Based on the above information it was concluded that the lens TTase is comparable to TTase from other tissues in its functional and structural properties. It is hypothesized that the lens TTase has a significant physiological role in sulfhydryl homeostasis in the lens by protecting the SH groups of the proteins from S-thiolation. It is speculated that, lens TTase may be primary antioxidant in the lens along with GSH and GR by protecting the vulnerable lens proteins against oxidative damage.

Animals↗
PubMed1996

Inhibition of naphthalene cataract in rats by aldose reductase inhibitors.

Naphthalene-induced cataract in rat lenses can be completely prevented by AL01576, an aldose reductase inhibitor (ARI). In an attempt to understand the mechanism of this inhibition, several ARIs were examined to compare their efficacies in preventing naphthalene cataract, using both in vitro and in vivo models. Two classes of ARIs were tested: One group including AL01576, AL04114 (a AL01576 analog) and Sorbinil contained the spirohydantoin group, while Tolrestat contained a carboxylic acid group. Furthermore, to clarify if aldose reductase played a role in naphthalene-induced cataractogenesis in addition to its role in sugar cataract formation, a new dual cataract model was established for ARI evaluations. This was achieved by feeding rats simultaneously with high galactose and naphthalene or incubating rat lenses in culture media containing high galactose and naphthalene dihydrodiol. Under these conditions, both cortical cataract and perinuclear cataract developed in the same lens. It was found that at the same dosage of 10 mg/kg/day, both AL01576 and AL04114 completely prevented all morphological and biochemical changes in the lenses of naphthalene-fed rats. Sorbinil was less efficacious, while Tolrestat was inactive. AL01576 showed a dose-response effect in preventing naphthalene cataract and at 10 mg/kg/day, it was also effective as an intervention agent after cataractogenesis had begun. With the dual cataract model, Tolrestat prevented the high galactose-induced cortical cataract but showed no protection against the naphthalene-induced perinuclear cataract. AL01576, on the other hand, prevented both cataract formations. Results for dulcitol and glutathione levels were in good agreement with the morphological findings. AL04114, and ARI as potent as AL01576 but without its property for cytochrome P-450 inhibition, displayed similar efficacy in preventing naphthalene cataract. Based on these results, it was concluded that the prevention of the naphthalene cataract probably results from inhibition of the conversion of naphthalene dihydrodiol to 1,2-dihydroxynaphthalene and that the effect of the ARIs cannot be explained by their inhibition of the dihydrodiol dehydrogenase activity of aldose reductase.

Aldehyde Reductase↗
PubMed1995

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen.

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Further studies on the dynamic changes of glutathione and protein-thiol mixed disulfides in H2O2 induced cataract in rat lenses: distributions and effect of aging.

To further investigate the role of protein-thiol mixed disulfides in cataractogenesis, an in vitro H2O2 cataract model was used with rat lenses to study the effect of aging, and the dynamic changes in the cortex, nucleus and the lens protein fractions. A group of lenses was exposed to H2O2-containing media (0.6 mM) for 1 to 3 days so that cortical cataract was induced gradually. Another group of lenses was first subjected to H2O2 exposure for one day and then recovered in the oxidant-free media for one or two days. These lenses were examined for the distribution of free glutathione and protein-thiol mixed disulfides (protein-glutathione and protein-cysteine) in the cortical and nuclear regions as well as in the water soluble and water insoluble fractions. Similar to the results reported earlier, the glutathione depletion in the whole lens occurred immediately and extensively during the 3-day H2O2 exposure. This loss was evenly distributed in the cortical and nuclear fractions. The level of protein-glutathione increased rapidly and continued throughout the 3 days. Most of the accumulation was found in the cortex and in both lens protein fractions. The protein-cysteine modification responded more slowly and less to oxidative stress. The delayed formation occurred mainly in the nucleus and in both lens protein fractions. In the recovery group, glutathione depletion was less drastic in the cortical and nuclear regions, but the elevated protein-glutathione in both regions and both protein fractions spontaneously decreased to its respective basal level within 1 day. Protein-cysteine on the other hand remained quite high, and in some cases it continued to rise in the absence of oxidation. Aging showed little effect on the response of rat lenses to oxidative stress. Similar patterns in glutathione and protein-thiol mixed disulfides occurred in both age groups (1, 23 months) and in both chronic oxidative stress and recovery conditions.

Aging↗
PubMed1995

The culture of rat lenses in high sugar media: effect on mixed disulfide levels.

Lens proteins are long lived proteins with those in the center of the lens predating the birth of the individual. As a result, they are subject to a host of modifications and damage through a variety of mechanisms. Two such modifications have been proposed as primary events which could cause conformational changes potentiating further modifications. These are non-enzymatic glycation and mixed disulfide formation. Human lenses accumulate protein-thiol mixed disulfides of three kinds throughout the lifespan. The presence of one of these, protein-glutathione (PSSG) mixed disulfide has been shown to be intimately involved in protein aggregation. We have utilized ex vivo lens culture and in vitro incubations of purified gamma-crystallin to evaluate the following hypotheses. A) Lenses cultured with a high sugar media will form higher mixed disulfide levels than controls; B) glycation of lens proteins will be dependent on initial mixed disulfide level. Xylose levels in the cultured lens rise rapidly (to 23 mM by 4 h), and the level of glycation after one week is elevated 6-7% over control values. Mixed disulfide levels are also substantially increased but not more than for lenses cultured in control media. gamma-Crystallin modified with 0, 1, or 5 equivalents of GSH was differentially glycated by radioactive fructose. The amount of fructose bound by the protein was found to be inversely related to the extent of mixed disulfide formation. These results indicate that 1) protein modification of one kind may influence further modifications of other types; 2) glycation of lens proteins has no effect on mixed disulfide formation in this system; 3) the sulfhydryl status of lens proteins can affect the potential for protein glycation.

Animals↗
PubMed1995

Ascorbic acid mediated alteration of alpha-crystallin secondary structure.

Glycation, the non-enzymatic addition of sugar or other carbonyl compounds to the amino groups of a protein, has been shown to occur with a variety of sugars and a diverse group of proteins. This type of alteration is believed to be an important component of aging for lens proteins and perhaps in cataractogenesis. Glycation has been shown to alter function and spectroscopic techniques have shown that in many cases conformational changes have occurred. Circular dichroism spectroscopy has documented modifications to alpha-crystallin tertiary structure induced by glucose and glucose 6-phosphate but generally no change to secondary structure. Ascorbate and is oxidized derivative dehydroascorbate have been shown to be powerful glycating agents as well as forming cross-links between peptide chains. In this study, alpha-crystallin incubated with ascorbic acid for one or two wk shows significant incorporation of ascorbate, non-reducible cross-links between the protein chains and altered CD spectra in the far UV region indicative of secondary structure modification.

Animals↗
PubMed1993

A new mixed disulfide species in human cataractous and aged lenses.

The process of ageing in the normal human eye lens is unique among tissues due to the absence of turnover in the structural proteins. These proteins accumulate a variety of modifications throughout their lifetime. Significantly, the cysteine residues are subject to disulfide formation with the low molecular weight thiol compounds present in the lens. It has been shown that accumulation of glutathione and cysteine mixed disulfides in the proteins of normal human lens is a function of age. In this report a third mixed disulfide species gamma-glutamylcysteine (gamma-Glu-Cys), has been identified by comparison with standards which were produced through two distinct methods. This new mixed disulfide is only prominent in old lenses (> 60 years) and cataractous lenses. In these situations its level may approach those of cysteine mixed disulfide. The appearance of gamma-Glu-Cys may be coincident with biochemical abnormalities preceding cataract formation. This protein modification may be a result of changes in the GSH biosynthetic pathway within the lens.

Adult↗
PubMed1993

The effect and recovery of long-term H2O2 exposure on lens morphology and biochemistry.

Oxidative stress has long been speculated to play an important role in cataractogenesis. In the H2O2-induced cataract model, rat lens showed extensive biochemical damage but very mild morphological changes after being exposed to H2O2 (0.5 mM) for 24 hr in culture. This damage included reduced glutathione (GSH) depletion, protein-GSH mixed disulfide (PSSG) elevation but not protein-protein disulfide (PSSP) formation. In order to understand the role of protein-thiol mixed disulfide formation in relation to the sequence of events during cataract induction, we conducted a long term H2O2 exposure study for up to 96 hr to monitor the dynamic changes in GSH and PSSG levels, the formation of PSSP aggregate, protein solubility, and the progression in lens opacity. Rat lenses were cultured in 0.5 mM H2O2 and harvested at intervals of 24, 48, 72 and 96 hr for the examination of morphological and biochemical changes. Contralateral lenses cultured in H2O2-free media were used as controls. It was found that the lenses had only patchy opacity at the equator after 24 hr, but became hydrated suddenly at 48 hr (31% heavier than the control), with an opacity which involved the entire outer cortical region. By 72 hr incubation, the nucleus was opacified. Lens GSH progressively decreased with time of H2O2 exposure, 40% was lost by 24 hr and over 95% by 48 hr. There was a concomitant elevation of PSSG, 16-fold over the controls by 24 hr and 45-fold by 48 hr followed by a decline to 34-fold after 72 hr. In addition, the level of protein-cysteine mixed disulfide (PSSC) was elevated after 48 hr incubation in H2O2. At this time point, PSSP aggregates began to appear both in water soluble (WS) and urea soluble (US) fractions along with a drastic reduction in protein solubility. Western blot analysis of the protein fractions identified beta and gamma, but not alpha-crystallin in the disulfide-containing aggregates. The lens clarity and biochemical changes partially recovered if the oxidant was removed within 24 hr, indicating a potential therapeutic role for antioxidants. The complete normalization of PSSG level under this recovery condition signifies that cells may have a natural defense system for controlling PSSG elevation.

Animals↗
PubMed1993

Distribution and activity of glutathione-S-transferase in normal human lenses and in cataractous human epithelia.

The distribution of glutathione-S-transferase (GST) activity was determined in frozen normal human lenses. The highest activity of GST was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. Western blot showed that both mu and pi isoenzymes of GST were present in human lenses. This result is similar to that found in rat lenses. In addition, GST activity was analyzed in 50 lens epithelia which were obtained during cataract surgery. Twenty-seven lens epithelia showed no activity. Statistically significant association was found between cortical and mixed cortical--nuclear cataract and loss of GST activity. No association was found between pure nuclear cataract and loss of epithelial GST activity.

Adult↗
PubMed1992

Protein-thiol mixed disulfides in human lens.

Protein-thiol mixed disulfide formation has been implicated as a possible mechanism for the protein-protein aggregation in cataractogenesis. Previously we have found that two species of thiols are bound to proteins: GSH (PSSG) and cysteine (PSSC). In this study we found these molecules are ubiquitous in animal lenses with the highest levels in human, dog and rat, and lowest in monkey. However, the relative amount of PSSG to PSSC is quite different in each animal species. The ratio of PSSG/PSSC was 1/10 in rat lens, 4/1 in human and dog lenses and 2/1 in monkey lens. We also studied the effect of aging on the protein-thiol mixed disulfide levels in human donor lenses between 3 months and 88 years. Lens GSH levels were inversely related to age, similar to earlier reports, but PSSC levels increased linearly with age. PSSG levels showed a triphasic pattern with an initial sharp and linear increase from a low content in infants to a highest level at age 20; fell back about 50% to a new steady state level that was maintained for four more decades; finally, above 60 years, the levels in some lenses were two to three-fold higher while some lenses remained at the same low value. PSSC in human lens appeared to concentrate in the nuclear region and in the water insoluble proteins while PSSG was more evenly distributed. Besides the aging effect on the protein-thiol mixed disulfides, oxidative stress also potentiated protein modification in the human lens.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

The possible mechanism of naphthalene cataract in rat and its prevention by an aldose reductase inhibitor (ALO1576).

The naphthalene-induced cataract in rats has been studied for many years as a possible model of human aging-related cataract. While the molecular mechanism of this cataract is unclear, it has recently been demonstrated that the aldose reductase inhibitor ALO1576 can prevent lens opacification in this system. The present study was undertaken to investigate the molecular basis for the effects of naphthalene on the lens and the role of pigmentation in the cataractogenic mechanism. Cataracts were induced in five strains of rats (two pigmented, three albino) by oral administration of naphthalene. Initial lens changes were observed after 1 week by slit-lamp; by 3 weeks a distinct shell-like opacity was present in the deep cortex. Little difference in the course of opacification was found between the pigmented and albino strains. Major biochemical effects were a decrease of 20-30% in glutathione (GSH) by 1 week of feeding, disulfide cross-linking of lens proteins present by 3 weeks, and a nearly 20-fold increase in the content of protein-GSH mixed disulfide. No effect was seen in the ability of the affected lenses to accumulate activity [3H]choline or 86Rb from the medium in organ culture nor in the activity of the Na+/K(+)-ATPase. ALO1576 (10 mg kg-1 day-1) completely prevented all morphological and biochemical changes in the lenses of the naphthalene-fed rats in both pigmented and non-pigmented strains. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats. Naphthalene dihydrodiol was found in the aqueous humor and lens of naphthalene-fed rats. It is proposed that naphthalene dihydrodiol produced in the liver reaches the aqueous humor and penetrates the lens where it is further metabolized ultimately to form the toxic species, naphthoquinone.

Aldehyde Reductase↗
PubMed1992

Establishment of a naphthalene cataract model in vitro.

In the past, almost all studies on naphthalene cataract were based on in vivo experiments. Such studies are laborious and time-consuming and are complicated by systemic toxicity arising from the metabolites of naphthalene. In order to study the direct effects of naphthalene metabolites on the lens, we established an in vitro 'naphthalene cataract' model system by exposing rat lens to naphthalene dihydrodiol (2.5 x 10(5) M) containing medium for 48 hr. Under these conditions, we analysed several biochemical parameters including the glutathione level, protein mixed disulfides, protein patterns on SDS-gels, active transport, NA+/K(+)-ATPase activities and the measurement of naphthalene metabolites in the cultured lenses. The results showed that both the morphological and biochemical changes were very similar to those observed in lenses of rats fed naphthalene (1 g kg-1 day-1). Furthermore, ALO1576 completely blocked the in vitro changes as it did in vivo. Therefore, this model system can be used as a new tool to investigate the mechanism of naphthalene cataract formation. Other naphthalene metabolites such as 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene and 1,2-naphthoquinone were also studied in vitro and the results showed that the effects of these naphthalene metabolites were very different from those observed in naphthalene cataracts in vivo.

Aldehyde Reductase↗
PubMed1991

Spectroscopic detection of lipid peroxidation products and structural changes in a sphingomyelin model system.

The peroxidation induced by tert-butyl hydroperoxide in sphingomyelin from bovine brain was investigated in detail. The lipid peroxidation products resulting from oxidation of lipid acyl chains were detected, identified and characterized by optical absorption. Fourier transform infrared, fluorescence and NMR spectroscopies. The extent of hydrocarbon chain degradation in vitro was quantified by measuring the relative change in absorbance of the peak at 241 nm characteristic of conjugated double bond or diene absorption band. FTIR data revealed that the lipid peroxidation of sphingomyelin disrupted the acyl chain and head group regions resulting in derangement of the ordered membrane.

Fourier Analysis↗
PubMed1991

Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo.

The effects of ambient exposure to near-ultraviolet (near-UV) radiation (300-400 nm) on the ocular lens of the diurnal squirrel (Sciurus carolinensis) are reported. Gray squirrels lived in cages illuminated for 12 hr a day with near-UV light (6 mW/cm2, 365 nm) for 1 yr. The non-UV-exposed controls were housed separately. In the lenses of UV-exposed animals, anterior pole changes occurred. Central epithelial cells swelled, disappeared, or underwent proliferation. A band of disoriented degenerating fiber cells was seen in the midcortex, with a degree of liquefaction. When lens protein compartments were separated by centrifugation, water-insoluble but urea-soluble fractions were enhanced in the outer and inner cortex and the nucleus. Both high-performance liquid chromatography and polyacrylamide gel electrophoresis revealed that proteins mainly in the midcortex and nucleus were altered considerably. Evidence of a loss of sulfhydryl compounds (by chemical and Raman spectroscopic analyses) and an increase of protein-thiol mixed disulfides (chemically) was also observed. These data prove that repetitive ambient exposure of diurnal animals to near-UV radiation at subsolar levels damages the lens by interfering with the maintenance of epithelial cells and altering the structural proteins; some of this may be due to the conversion of sulfhydryls to mixed disulfides.

Animals↗