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Douglas Borchman

Publications and source records attributed to Douglas Borchman.

18 recordsLinked to original sources
PubMed2006

Oxidation-induced changes in human lens epithelial cells. 1. Phospholipids.

Lipid compositional changes in lens epithelial cells (HLE B-3) grown in a hyperoxic atmosphere were studied to determine if oxidation could cause changes in the amount and type of phospholipid similar to those found in vivo with age and cataract. The phosphatidylcholines in HLE B-3 cells were 8 times more unsaturated than the sphingomyelins. Cell viability was the same for cells grown for up to 48 h in a normoxic or hyperoxic atmosphere. Lipid oxidation was about three times higher after growth in a hyperoxic atmosphere compared with cells grown in a normoxic atmosphere. The lack of change in the relative amount of sphingomyelin and the decrease in phosphatidylcholine coupled with the increase in lysophosphatidylcholine support the idea that similar mechanisms may be responsible for the lipid compositional changes in both lens epithelial and fiber cells. It is postulated that lipases eliminate oxidized unsaturated glycerolipids, leaving a membrane increasingly composed of more ordered and more saturated sphingolipids. Oxidative stress leads to changes in membrane composition that are consistent with those seen with age in human epithelial cells. Oxidation-induced epithelial phospholipid change is an area of research that has gone virtually unexplored in the human lens and could be relevant to all cell types and may be important to lens clarity.

Aging↗
PubMed2006

Regulation of sarco/endoplasmic and plasma membrane calcium ATPase gene expression by calcium in cultured human lens epithelial cells.

Since Ca(2+)-ATPase is a major determinant of calcium homeostasis in the lens, we examined the expression of Ca(2+)-ATPase by calcium. An immortalized human lens epithelial cell line, HLE B-3, was treated with thapsigargin to inhibit sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) releasing calcium from intracellular stores. Isoforms of the plasma membrane Ca(2+)-ATPase (PMCA) and SERCA were quantified by Western blot and quantitative real time reverse transcription polymerase chain reaction. We showed that both PMCA1 and SERCA3 isoform protein and mRNA are upregulated two- to three-fold in thapsigargin-treated HLE B-3 cells in a time and dose-dependent manner. Thapsigargin did not change the protein or mRNA levels of PMCA2, 3, 4 or SERCA2b. Considering the harmful effects of increased intracellular calcium levels, the upregulation of both SERCA and PMCA pumps suggests it is a compensatory mechanism to restore the calcium concentration to the physiological resting level.

Calcium↗
PubMed2006

Oxidation-induced changes in human lens epithelial cells 2. Mitochondria and the generation of reactive oxygen species.

The relationships among reactive oxygen species (ROS) generation, lipid compositional changes, antioxidant power, and mitochondrial membrane potential were determined in a human lens epithelial cell line, HLE-B3. Cells grown in a hyperoxic atmosphere grew linearly for about 3 days, and then progressively died. Total antioxidant power and ROS generation increased by 50 and 43%, respectively, in cells grown in a hyperoxic atmosphere compared to those cultured in a normoxic atmosphere. By specifically uncoupling the mitochondrial proton gradient, we determined that the mitochondria are most likely the major source of ROS generation. ROS generation correlated inversely with mitochondrial membrane potential and the amount of cardiolipin, factors likely to contribute to loss of cell viability. Our results support the idea that hyperoxic damage to HLE-B3 cells derives from enhanced generation of ROS from the mitochondrial electron transport chain resulting in the oxidation of cardiolipin. With extended hyperoxic insult, the oxidants overwhelm the antioxidant defense system and eventually cell death ensues.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed2006

In vitro and in situ tracking of choline-phospholipid biogenesis by MALDI TOF-MS.

The quantitative monitoring of newly synthesized species of phosphatidylcholines (PCs) and sphingomyelins (SMs) has been achieved in cultured human lens epithelial cells, both in situ and in vitro, with the use of MALDI TOF-MS. As the cells were cultured with deuterated choline-d(9), new peaks that differed from the hydrogenated species by 9.06 Da appeared in the mass spectra. The initial rates of appearance of all deuterated species of PCs were comparable and 4 times higher than those for SMs. After 12 h, those rates began to decrease for PCs but not for deuterated SMs, whose relative contents continued to increase throughout the 72 h of the experiment. The differences in initial rates are consistent with the reported initial generation of PCs, their subsequent degradation, and transfer of their headgroup, phosphorylcholine, to SMs. To further test the ability of MALDI TOF-MS to quantify changes in phospholipid (PL) metabolic pathways, myriocin, an inhibitor of SM synthesis, was added to the cells. In vitro and in situ results revealed a decrease in SMs and an unexpected increase in some PCs. With the use of other deuterated precursors and in combination with postsource decay or tandem MS/MS, this approach could allow the simultaneous tracking of the biosynthesis of multiple PL classes while providing details on their acyl chains.

Cell Line↗
PubMed2006

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity.

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.

Adolescent↗
PubMed2005

alpha-Crystallin binding in vitro to lipids from clear human lenses.

The association of alpha-crystallin to lens membranes increases with age and cataract. Lipid compositional changes also occur with age, cataract, and diabetes. In this study we determined the influence of lipid compositional differences on the binding capacity of alpha-crystallin to lipid vesicles in vitro. Lipids were extracted from pools of human lenses from younger (22+/-4 y, n=30) and older (69+/-3 y, n=26) nondiabetic donors as well as from diabetics taking insulin (60+/-9 y, n=26) and diabetics not taking insulin (58+/-9 y, n=20). Diabetics were insulin dependent for an average of 6 years. Extracted lipids were extruded into large unilamellar vesicles. alpha-Crystallin was mixed with the lipid at 36 degrees C, allowed to bind for about 12 h, and centrifuged at 14,000 g. This centrifugal force was low enough to not pellet free alpha-crystallin but high enough to pellet the lipid and bound alpha-crystallin. alpha-Crystallin-lipid binding was characterized by comparing the amount alpha-crystallin in the pellets of samples with and without lipid. Protein was measured using an assay that minimized interference from lipids. Lipid composition was determined by 31P-NMR spectroscopy. The binding capacity of alpha-crystallin to lipids was 12, 19, 8.9, 17 microg bound/mg lipid for lens lipids extracted from younger, older, insulin-treated and nontreated diabetic donors, respectively. The amount of alpha-crystallin in the pellet (bound alpha-crystallin) was significantly lower for the lipids from the younger group of lenses, p=0.033 and insulin-treated group, p=0.006, compared with the older group of lenses. Higher binding capacity was associated with a higher relative amount of sphingolipid and lower relative amounts of phosphatidylethanolamine-related lipid and phosphatidylcholine. The binding capacity of alpha-crystallin to lens lipids, measured in vitro, increases with age and decreases in diabetic donors that were treated with insulin. Our data support the idea that with age and perhaps certain types of diabetes, more alpha-crystallin is bound to the membrane and serves as a condensation point to which other crystallins bind and then become oxidized.

Adult↗
PubMed2005

Human lens phospholipid changes with age and cataract.

PURPOSE: To determine the phospholipid changes responsible for the increase in membrane lipid hydrocarbon chain order, or stiffness, with age and cataract in the human lens. METHODS: Clear human lenses were pooled into four groups, with donors ranging in age from 15 to 29, 30 to 49, 50 to 64, and 65 to 74 years. Whole human cataractous lenses were obtained from donors after extracapsular cataract extraction. Cataractous lenses were grouped into four classifications: mature, mixed cortical and nuclear, immature nuclear sclerotic, mature posterior subcapsular, and mature nuclear. Lipids were extracted and quantified gravimetrically. The relative phospholipid composition was determined by (31)P-nuclear magnetic resonance spectroscopy. RESULTS: The relative and absolute amount of sphingolipids, including dihydrosphingomyelin and sphingomyelin, increased with age, whereas glycerolipids, including phosphatidylcholine and two phosphatidylethanolamine-related phospholipids, decreased. These changes were exacerbated by the presence of cataract and were substantial, greater than the changes in lipid levels reported in any organ in association with any disease. CONCLUSIONS: The changes in the amount of lipids with age and cataract support the idea that glycerolipids are selectively oxidized over lipids with fewer double bonds, such as sphingolipids. As a result of the elevation of sphingolipid levels with species, age, and cataract, lipid hydrocarbon chain order, or stiffness, increases. Increased membrane stiffness may increase light-scattering, reduce calcium pump activity, alter protein-lipid interactions, and perhaps slow fiber cell elongation.

Adolescent↗
PubMed2004

Direct perturbation of lens membrane structure may contribute to cataracts caused by U18666A, an oxidosqualene cyclase inhibitor.

Induction of cataracts in experimental animals is a common toxic feature of oxidosqualene cyclase (OSC) inhibitors. U18666A has been shown to produce irreversible lens damage within a few weeks of treatment. Drug actions, besides reducing the availability of cholesterol, could contribute to cataract formation. Cholesterol added to cultures of lens epithelial cells could only partially overcome the growth-inhibiting effects of U18666A. In view of this finding and the fact that U18666A and other OSC inhibitors are highly lipophilic cationic tertiary amines, we tested the hypothesis that the cataractogenic effect of U18666A is related to direct perturbation of lens membrane structure and function. Based on changes in the anisotropy of fluorescent probes, U18666A incorporated into bovine lens lipid model membranes increased membrane structural order and, using small-angle x-ray diffraction, U18666A was shown to intercalate into the lens lipid model membranes and produce a broad condensing effect on membrane structure. Also, exposure of cultured lens epithelial cells and intact rat lenses to U18666A induced apoptosis. Induction of apoptosis may begin by intercalation of U18666A into cell membranes. By increasing membrane structural order, U18666A may also increase light scatter, thus directly contributing to lens opacification.

Androstenes↗
PubMed2004

Sphingolipids in human lens membranes: an update on their composition and possible biological implications.

The unique nature of the most abundant phospholipids in human lens membranes remained overlooked until the 1990s when it was possible to discern dihydrosphingomyelins (DHSMs) from the more common sphingomyelins (SMs). Unlike in other mammalian membranes, DHSMs comprise nearly half of the phospholipids in adult human lenses. Compared to SMs with a trans double bond between carbons 4 and 5 of the sphingoid backbone, the absence of this unsaturation site in DHSMs allows the participation of the OH group on C3 in intermolecular H-bonds and leads to stronger interlipid interactions with both neighboring DHSMs and cholesterol. Phospholipid compositional changes with age and lens region observed in mammals with various life spans and lens growth rates, suggest that the highest levels of DHSMs along with the lowest amounts of phosphatidylcholines and SMs are found in lenses with the lowest growth rate, namely human lenses. The participation of phospholipid metabolites in the control of mitosis and elongation of lens cells is plausible and deserves investigation.

Adult↗
PubMed2004

Lens lipids and maximum lifespan.

Unlike in most organs, the lipid composition of lenses varies dramatically among species and with age. The focus of this study is to assess how these changes relate to lifespan. Studies on cataract suggest that the lens may serve as a window into the processes leading to accelerated mortality. As a first step toward elucidating cellular processes in the lens that may serve as markers for accelerated mortality, we examined the correlation between species-dependent and age-related lens lipid compositional differences and maximum life span. We included data from camels, which, even in old age, rarely develop cataracts although they live under adverse conditions. Camel lens lipids were mainly composed of sphingolipids (77%) and phosphatidylcholines (23%). Bovine lens lipid composition was comparable to a previous study, and both bovine lens sphingolipids, phosphatidylcholines and camel lens phosphatidylcholines content fit well (within the 95% confidence limits) in the curve obtained by plotting maximum life spans of other species with sphingolipids and phosphatidylcholines. Lifespan was directly related to lens sphingolipid content and indirectly related to lens phosphatidylcholine content. The camel lens sphingolipid value was significantly above the curve for other species. Except for the camel lens nucleus, lipid order and sphingolipid content were linearly related, p < 0.005 with a slope of 0.85+/-0.07, and intercept of 6.9+/-3.8. Lipid phase transition temperature and sphingolipid content were also linearly related, p = 0.01 with a slope of 0.20+/-0.07, and intercept of 21.7+/-5.3. Our data support the hypothesis that humans have adapted so that their lens membranes have a high sphingolipid content that confers resistance to oxidation, allowing these membranes to stay clear for a relatively longer time than is the case in many other species. Age-related changes in human lens lipid composition may serve as a marker for oxidative stress and may reflect systemic oxidative insult, providing a window into the health of an individual.

Animals↗
PubMed2003

Light scattering of human lens vesicles in vitro.

In passing through the lens, light crosses thousands of cell membranes. To explore the possible contribution of lipids to the scattering properties of the lens, we have carried out in vitro studies with lipids extracted from human lenses 1-90 years of age. Sphingomyelin and human lens lipids were extruded into large unilamellar vesicles (LUVs). The intensity of light scattered by human lens LUVs increased with age and lipid hydrocarbon chain order. Hydrocarbon chain order also correlated with light scattering intensity by sphingomyelin LUVs. Light scattered by LUVs composed of sphingomyelin (1-30 mg ml(-1)) was 20 to 100 times more intense than that scattered by the same concentration of alpha-crystallin in aqueous media. Increased lipid hydrocarbon chain order as well as variations in the headgroup and interfacial region of bilayers resulting from lipid compositional changes can influence membrane light scattering properties. In vitro measurements suggest that the contribution to light scattering by lipids may be significant and should not be disregarded in the investigation of factors and components that lead to the increase in light scattering by human lenses with age and cataract.

Adolescent↗
PubMed2003

Glycero- versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth.

The human lens differs from other mammalian lenses in its very slow growth and unusual phospholipid composition of its cell membranes. Dihydrosphingomyelins (DHSMs) make up about half of all phospholipids in adult human fiber membranes. In all other membranes, sphingomyelins(SMs) with a trans double bond in their backbone, are prevalent. In our quest to understand the biological implications of such elevated DHSM levels, we analyzed membranes from various regions of human, elephant, giraffe, polar bear, pig and cow lenses. The levels of DHSMs were minor in non-human lens membranes. A strong correlation was observed between growth rate and relative contents of phosphatidylcholines(PCs) in epithelia and outer cortical fibers. Sphingomyelins became increasingly predominant in differentiated fibers and this increase was age dependent. Indeed, nuclear fiber membranes of aged non-human mammals were composed, almost exclusively, of (SMs). Although human lens membranes followed comparable compositional trends, the magnitude of the changes was much smaller. We postulate that the high relative contents of DHSMs provide a biochemically inert matrix in which only small amounts of PCs and SMs and their metabolites, known to promote and arrest growth, respectively, are present. This compositional difference is proposed to contribute to the slow multiplication and elongation of human lens cells.

Adolescent↗
PubMed2003

Influence of age, diabetes, and cataract on calcium, lipid-calcium, and protein-calcium relationships in human lenses.

PURPOSE: Calcium is elevated in most cataractous human lenses and may contribute to cataractogenesis. In this study, age-related changes were examined in the total calcium content of clear human lenses and the binding of calcium to lens lipids and proteins. METHODS: Total lens calcium was determined by atomic absorption spectroscopy. Calcium binding was measured by light scattering and measurement of calcium by atomic absorption spectroscopy in bound and unbound fractions. RESULTS: The calcium content of clear human lenses decreased between 18 and 55 years of age and increased between 55 and 75 years, as well as in the presence of cataract. Total calcium levels in clear lenses from subjects with insulin-dependent diabetes did not differ from that in lenses of age-matched control subjects. In vitro binding studies have shown that lens lipids can bind nearly all the calcium present in the human lens. Age and cataract diminished the capacity of lens lipids to bind calcium. Calcium-induced light-scattering, measured in vitro for lens proteins, correlated with increasing age and cataract. CONCLUSIONS: The data support the hypothesis that increased intracellular calcium concentrations and a diminished capacity of lens lipids to bind to calcium initiate a cascade of events that culminates in increased light-scattering from lipids and especially proteins. Calcium binding to lipid membranes cannot directly contribute to light-scattering in cataractous lenses. It has been suggested that most of the diffusible calcium in the lens is in the intercellular spaces and that lens lipids in the outer leaflet of the bilayer bind to that calcium. If so, this could account for the 150-fold difference between free and bound calcium levels in the lens.

Adolescent↗
PubMed2003

Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.

PURPOSE: Human lens membranes contain unusually high levels of cholesterol and sphingolipids, lipids known to segregate into liquid-ordered domains. The current study was conducted to pursue the determination and characterization of these domains in membranes of clear and cataractous human lenses. METHODS: Cortical and nuclear regions of aged clear and cataractous lenses were obtained. After lysis with Triton X-100 at 4 degrees C and sucrose linear-density centrifugation, sedimenting and nonsedimenting fractions (when present) were collected. Phospholipids were analyzed by (31)P-nuclear magnetic resonance (NMR) and mass spectrometry. Caveolae and raft markers were tested by Western blot analysis. RESULTS: Only samples from clear lenses exhibited a nonsedimenting band. Phospholipid contents were comparable for sedimenting fractions of clear and cataractous membranes. Cholesterol to phospholipid molar ratios in light-density bands were nearly 7, three times greater than in sedimenting fractions. The portion of total cholesterol present in nonsedimenting fractions increased from 5.5% in the cortex to 14% in the nucleus. Two lysophospholipids comprising approximately 10% of all phospholipids in total membranes were undetectable in nonsedimenting fractions. Caveolin-1 was enriched in these fractions. CONCLUSIONS: Phospholipid compositional differences between lighter and heavier fractions from clear lenses were relatively minor and could not, alone, account for the substantial enrichment of cholesterol in the lighter fractions. Specific proteins, such as caveolin-1, must recruit cholesterol and induce clustering. Undetectable amounts of light-density domains in cataractous membranes suggest either disruption of these aggregates and thus the function of proteins within them, possibly relevant to lens transparency, and/or greater density of these clusters due to stronger binding of insoluble crystallins to membranes.

Aged↗
PubMed2002

31P NMR quantification and monophasic solvent purification of human and bovine lens phospholipids.

Most lipid extraction procedures [Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497-509; Bligh, E.G., and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification, Can. J. Biochem. Physiol. 37, 911-917] employ biphasic solvent mixtures designed to dissolve the lipids in an organic phase and remove impurities in an aqueous phase. However, when applying these protocols to biological matrices such as that of the ocular lens, the formation of an emulsion layer between the organic and aqueous phases causes poor reproducibility in extraction yields and gives only a small amount of the lipid-containing chloroform phase. In this study, we quantified phospholipids at each step of the Folch et al. extraction protocol and compared the yield of human and bovine lens phospholipids obtained by the Folch-based approach and a novel monophasic methanol extraction method designed to circumvent the problems associated with biphasic extraction protocols. A monophasic methanol extraction coupled with 31P NMR spectroscopy was found to be the simplest, quickest, and most effective method for quantifying the phospholipid content of the lens.

2-Propanol↗
PubMed2002

Interactions of Ca(2+) with sphingomyelin and dihydrosphingomyelin.

The changes induced by Ca(2+) on human lens sphingolipids, sphingomyelin (SM), and dihydrosphingomyelin were investigated by infrared spectroscopy. Ca(2+)-concentration-dependent studies of the head group region revealed that, for both sphingolipids, Ca(2+) partially dehydrates some of the phosphate groups and binds to others. Ca(2+) affects the interface of each sphingolipid differently. In SM, Ca(2+) shifts the amide I' band to frequencies lower than those in dehydrated samples of SM alone. This could be attributed to the direct binding of Ca(2+) to carbonyl groups and/or strong tightening of interlipid H-bonds to levels beyond those in dehydrated samples of SM only. In contrast, Ca(2+) induces relatively minor dehydration around the amide groups of dihydrosphingomyelin and a slight enhancement of direct lipid-lipid interactions. Temperature-dependent studies reveal that 0.2 M Ca(2+) increases the transition temperature T(m) from 31.6 +/- 1.0 degrees C to 35.7 +/- 1.1 degrees C for SM and from 45.5 +/- 1.1 degrees C to 48.2 +/- 1.0 degrees C for dihydrosphingomyelin. Binding of Ca(2+) to some phosphate groups remains above T(m). The strength of the interaction is, however, weaker. This allows for the partial rehydration of these moieties. Similarly, above T(m), Ca(2+)-lipid and/or direct inter-lipid interactions are weakened and lead to the rehydration of amide groups.

Animals↗
PubMed2002

UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects.

The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5 mWcm(-2), 340-410 nm wavelength, peak at 365 nm) for 4-5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70-90% of 340-400 nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30-35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein-thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, alpha- and beta- tubulin, vimentin and alpha-actinin (60-100%). The results indicate that a 4-5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O(2) present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.

Animals↗