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Biomedical subjects

A Spector

Publications and source records attributed to A Spector.

At least 55 records · Page 3Linked to original sources
PubMed1994

The redox active components H2O2 and N-acetyl-L-cysteine regulate expression of c-jun and c-fos in lens systems.

Hydrogen peroxide (H2O2) is implicated in human cataract development. At the molecular level H2O2 has been observed to cause damage to DNA, protein and lipid. It is now demonstrated, for the first time in a lens system, that H2O2 at concentrations found in cataract patients induces expression of both c-jun and c-fos. At optimal concentrations of H2O2, mRNA accumulation of c-jun and c-fos in the rat lenses is induced 20- and 18-fold above normal levels respectively, but with distinct kinetics. This induction occurs at the transcriptional level. H2O2 also induces transactivation by activating protein-1 (AP-1) in rabbit lens epithelial cells. The antioxidant N-acetyl-cysteine (NAC) has a dual effect on the induction of c-jun and c-fos. Preincubation of rat lenses with 5 mM NAC inhibits the induction by H2O2, while 30 mM and 50 mM NAC induce expression of these genes and mask the H2O2 effect. H7 (50 microM), genistein (2 microM) and okadaic acid (20 nM), all block the induction of c-jun and c-fos mRNA accumulation in the H2O2-treated rat lenses. These results suggest that H2O2 activates protein kinase and phosphatase dependent signal transduction pathways to induce c-jun and c-fos expression which may regulate lens crystallin genes and other genes containing AP-1 binding sites.

Acetylcysteine↗
PubMed1993

Photochemically induced cataracts in rat lenses can be prevented by AL-3823A, a glutathione peroxidase mimic.

Oxidative stress is known to cause cataracts in lens culture systems and is believed to be an important factor contributing to human cataracts. In this communication, it is demonstrated that cataract development of cultured rat lenses produced as a result of photochemically induced oxidation in a 4% oxygen atmosphere similar to the native environment of the lens can be blocked by the transition metal complex AL-3823A. In this system, riboflavin is added to the medium as a photosensitizer. AL-3823A acts primarily as a glutathione peroxidase mimic, which catalytically metabolizes H2O2 and also has low superoxide dismutase-like activity. Measurements of H2O2, O2.-, and OH. indicate that appreciable levels of the first two of these oxidants and low levels of OH. are produced by this photochemical stressing system. The H2O2 concentrations are similar to those found in some patients with cataracts. The development of cataracts was followed over a 96-hr period. Transparency, hydration, glyceraldehyde-3-phosphate dehydrogenase activity, and protein and nonprotein thiol were monitored. All parameters show marked changes during the 96-hr period. However, in the presence of 200 microM AL-3823A, no difference between control and light-exposed lenses was observed with respect to these parameters. The results suggest that in vivo human cataract development caused by oxidative stress may be prevented by compounds of this type.

Animals↗
PubMed1993

Growth and physiological functions of vascular endothelial cells in a new serum-free medium (SFM).

Endothelial cells grown in vitro are widely used for the study of vascular physiology and pathology. The high levels of serum supplementation normally used in endothelial cell culture may create problems in experimental design or in interpretation of results. A serum-free medium (SFM) has been designed specifically for culture of nonhuman vascular endothelial cells. Growth as well as retention of various physiological markers in this medium was demonstrated in large-vessel endothelial cells from bovine, porcine, ovine, and canine species. Cells from all species tested took up DiI-acetylated LDL. Factor VIII-related antigen was demonstrated in cultures of bovine and canine cells. Bovine and porcine cells were shown to have retained the ability to produce prostacyclin. Further supplementation of the SFM with various lipid-containing additives failed to stimulate either growth or prostacyclin production. PDGF produced by bovine, porcine, and canine endothelial cells was measured and found to be present at lower concentration than in serum supplemented cultures. All cells grown in SFM exhibited the histiotypic "cobblestone" morphology normally associated with cultured endothelial cells. While not optimized for culture of human endothelial cells, short-term growth < or = 4 passages) of human umbilical vein endothelial cells was achieved by supplementing the SFM with hydrocortisone, bovine pituitary extract, and epidermal growth factor.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

The prevention of cataract caused by oxidative stress in cultured rat lenses. II. Early effects of photochemical stress and recovery.

Previous work has demonstrated that photochemically induced oxidative stress generated with 4 microM riboflavin in a 4% oxygen atmosphere utilizing daylight type radiation is capable of causing cataract in cultured rat lenses. Such cataract is prevented by the GSH peroxidase type mimic, AL-3823A. Examination of the early stages of cataract formation produced by short-term oxidative stress and recovery is now reported. A 24-hr oxidative stress, under the above conditions, causes loss of transparency, particularly in the equatorial region, increased hydration, loss of glyceraldehyde-3-PO4 dehydrogenase activity, oxidation of non-protein thiol and a decrease in 86Rb and [14C]choline uptake and ATP levels. Examination of recovery of these parameters during a 72-hr period indicates, in most cases, little or no reversal of oxidative damage. Hydration and loss of non-protein thiol continued during the recovery period. The presence of AL-3823A during the stress period prevented change in all parameters. Transport systems appear to be particularly vulnerable to this type of oxidative stress losing 50% or more activity within 4 hr. Even after a 2-hr stress, choline transport did not recover even though, under these conditions, ATP levels had only decreased slightly. Cytosolic components such as non-protein thiol and glyceraldehyde-3-PO4 dehydrogenase also showed little change after a 4-hr insult. 86Rb efflux experiments indicated no change in permeability during a 24-hr stress period. The overall conclusion from these studies is that a 24-hr oxidative stress which appears to reflect physiological conditions existing during cataract development, causes extensive, irreversible damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The prevention of cataract caused by oxidative stress in cultured rat lenses. I. H2O2 and photochemically induced cataract.

H2O2 stress is shown to produce cataract in cultured rat lenses. The loss of transparency begins in the equatorial region within 24 hours and the entire superficial cortex is opaque by 96 hours. No involvement of the nuclear region is observed. However after an additional 48 hours, the nuclear region becomes opaque. The loss of transparency is accompanied by a large uptake of H2O which occurs gradually over the 96 hour period, complete loss of glyceraldehyde phosphate dehydrogenase (GPD) activity, almost complete loss of non-protein thiol and a slight decrease in protein thiol. Control lenses show no change other than the establishment of a new non-protein thiol base line approximately 60% lower than 0 time levels. The Alcon glutathione peroxidase type mimic, AL-3823A, completely eliminates almost all of the H2O2 induced effects and the lens remains transparent. Utilizing a more severe photochemical model than may be anticipated physiologically with 10 microM riboflavin and exposure to daylight fluorescent lamps, significant concentrations of superoxide and low levels of OH. are produced as well as extraordinarily high concentrations of H2O2 ranging from about 400 to 1000 microM. As with the H2O2 model, opacification begins at the equator but the cataract develops more rapidly, the lens being completely opaque by 68 hours. Hydration, GPD activity, non-protein and protein thiol all decrease more rapidly than in the H2O2 model. AL-3823A prevents loss of transparency until approximately 92 hours and markedly decreases changes in other parameters. At 92 hours, slight loss of transparency is observed. Catalase is somewhat less effective. AL-3823A is shown to also significantly decrease superoxide levels. The marked delay in the onset of changes in lens biochemistry and physiology in the severe photochemical stress model and the maintenance of normal parameters in the H2O2 model in the presence of AL-3823A suggests that such compounds may prevent cataract caused by oxidative stress under physiological conditions.

Animals↗
PubMed1993

DNA single strand breaks in human lens epithelial cells from patients with cataract.

In recent years, there has been renewed interest in the association between DNA damage to the lens epithelium and the development of lens opacities. Although a number of in vitro studies have indicated that lens epithelial cells are susceptible to a variety of DNA damaging insults and that these cells possess the capacity to repair such damage, no previous studies have directly addressed whether DNA damage is associated with human cataract in vivo. Utilizing samples of lens epithelial cells obtained from patients undergoing cataract surgery, the percentage of cells containing DNA single strand breaks was directly determined by the single-cell gel assay (SGA) method. Non-cataractous human Eye Bank lenses of similar ages to the cataractous samples and calf lenses were used as controls. In approximately 50% of the cataractous samples analyzed, the proportion of cells containing DNA single strand breaks was significantly higher than in control lenses. No relationship between age and DNA damage was noted. These findings are consistent with the hypothesis that in some human patients with cataract, DNA damage in the lens epithelial cell population may be related to the development of lens fiber cell opacity.

Aged↗
PubMed1992

Na,K-ATPase of cultured bovine lens epithelial cells: H2O2 effects.

Na,K-ATPase function was studied in cultured bovine lens epithelial cells under confluent and non-confluent conditions. The affinity of the Na,K-ATPase for the cardiac glycoside, ouabain, differs between the confluent and non-confluent cultures. The confluent cells have a higher affinity for ouabain than do the non-confluent cells. The ouabain affinity of the confluent cells is similar to that for the Na,K-ATPase isolated from the bovine axolemma and the bovine lens cortex. The ouabain affinity of the non-confluent cells is similar to that for the Na,K-ATPase of the renal medulla and bovine lens epithelium. Similar results are not found with confluent and non-confluent MDCK cells. H2O2 treatment of confluent and non-confluent lens epithelial cell cultures has differing effects on the Na,K-ATPase function. In the confluent cell preparations, H2O2 affects K(+)-dependent dephosphorylation of the intermediate phosphoenzyme. In the non-confluent preparations. H2O2 appears to inhibit K(+)-occlusion.

Animals↗
PubMed1991

Phosphorylation of alpha-crystallin B in Alexander's disease brain.

The phosphorylation of alpha-crystallin B was studied in homogenates of autopsy samples of brain tissue from patients with Alexander's disease, a condition characterized by over-expression of this protein. After incubation in the presence of [gamma-32P]ATP and cAMP the homogenates were analyzed by two-dimensional electrophoresis, (isoelectric focusing followed by SDS-PAGE). Three major polypeptides having the same molecular weight as bovine lens alpha-crystallin B and pIs 7.1, 6.9 and 6.7 were detected in the Coomassie blue stained gels. These three polypeptides were recognized by an alpha-crystallin B-specific antiserum in Western blots. The polypeptides with pIs 7.1 and 6.7 co-migrated in isoelectric focusing gels with bovine lens alpha B and its phosphorylated form alpha Bp, respectively. Radioautography of the two-dimensional gels demonstrated the presence of 32P in the most acidic polypeptide. The results demonstrate the occurrence of alpha B phosphorylation in Alexander's disease brain tissue.

Blotting, Western↗
PubMed1991

The calf gamma crystallins--a Raman spectroscopic study.

The solution structures of the four major components of bovine lens gamma-crystallin, gamma s, gamma II, gamma III and gamma IV are compared using Raman spectroscopy. The spectral region sensitive to the vibrational frequencies of aromatic and sulfur containing residues and to the backbone skeletal stretching modes (500-1000 cm-1), and that reflecting secondary structure (1,000-1,700 cm-1) are strikingly similar in all four gamma-crystallin fractions. These similarities are indicative of the dominant anti-parallel beta sheet structure common to all the gamma-crystallins. A comparison of the ratios of the Raman intensities at 850 cm-1 and 830 cm-1 (I850/I830), an empirical measure of the degree of hydrogen bonding of phenolic hydroxyl groups, suggests that the tyrosine residues in all the gamma-crystallin fractions are moderately hydrogen bonded. Distinct differences in the solution structures of the gamma-crystallins were observed in the higher energy end of the vibrational Raman spectra. The sulfhydryl stretching frequencies for the gamma-crystallins exhibit complex splitting patterns in the 2,500-2,600 cm-1 region. These patterns are due to the competing effects of hydrogen bonding and S-pi interactions with neighboring aromatic residues. All five proteins exhibit multiple, but distinct, thiol frequencies, suggesting that the microenvironments of the cysteine residues in these proteins are significantly different.

Animals↗
PubMed1991

Traumatic osteoarthrosis of the ankle joint secondary to ankle fractures.

Arthritis, in one form or another, is one of the most common disorders that affect man. Whether the etiology is systemic, neurologic, or traumatic, the one common finding is the destruction of joint cartilage. Although there have been massive amounts of literature concerning osteoarthritis, little has been written about arthritis of the ankle joint. Current concepts in the pathology, terminology, symptomatology, and treatment of the arthritic ankle are discussed.

Ankle Injuries↗
PubMed1990

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues.

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.

Animals↗
PubMed1990

Some aspects of Dr Kinoshita's contributions to lens protein chemistry.

A review of some of Dr Kinoshita's contributions to our understanding of lens protein and glutathione biochemistry is presented. Particular emphasis is placed on Dr Kinoshita's work involved with the relationship of carbohydrate metabolism and the maintenance of reduced glutathione, the question of the biological function of glutathione in the lens, the effect of oxidative stress provided by diamide and azoester on glutathione, membrane pump function and protein and also ascorbate and H2O2 effects on Na+, K(+)-ATPase. The importance of oxidative stress was recognized early by Dr Kinoshita and he has continued to make significant contributions in this area as illustrated by his work with Dr Zigler on posterior subcapsular cataracts and with Drs Garland and Zigler on mixed function oxidation. It is concluded that Dr Kinoshita's overall contributions in the areas mentioned above have been broad and of considerable importance.

Animals↗
PubMed1990

Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells.

The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide. Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C. The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2. The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH. scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2. Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+. DNA-protein cross-linking was not observed. In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E. coli endonuclease III or bacteriophage T4 endonuclease V, respectively. No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution. Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures. Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells. In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2. The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.

Animals↗
PubMed1990

Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells.

DNA damage caused by UV-B and UV-A irradiation and the rate of repair of such damage was quantitated in bovine lens epithelial cell cultures using a modified alkaline elution methodology. Two enzymes, bacteriophage T4 endonuclease V, which cleaves at the site of pyrimidine dimers, and E. coli endonuclease III, which cleaves at the site of thymine glycols, were utilized. Pyrimidine dimers were not detected after UV-A irradiation of lens cultures with up to 400 J/m2. In contrast, after exposure to as little as 2 J/m2 of UV-B irradiation, large numbers of pyrimidine dimers were observed. At higher fluences, thymine glycols were also found. Significant levels of DNA-DNA crosslinking were suggested by reduced rates of elution of DNA from cells treated with both UV-B irradiation and H2O2 in comparison to treatment with H2O2 alone. Protein-DNA crosslinks, in contrast, were not observed. The rate of repair of UV-B induced DNA damage was quantitated by harvesting cells at various times after the UV-B exposure. Single-strand breaks were never observed immediately after UV-B exposure but appeared later during the repair phase. In contrast to the repair of H2O2 induced DNA damage, which is largely completed within 30 min of exposure, more than 50% of the UV-B light induced DNA damage remained unrepaired five hours after exposure. This difference between the rate of repair of H2O2 and UV-B induced DNA damage could provide valuable insights into the nature of DNA damaging agents in the lens environment and may reflect underlying differences in the potential for epithelial cell DNA mutation in response to various DNA damaging insults.

Animals↗
PubMed1990

Prevalence of age-related cataract in Ganzi and Shanghai. The Epidemiological Study Group.

It has been reported that the prevalence of cataract is much higher in plateau than that in plain regions. But the data are incomplete and can not be compared because of different examination methods and different diagnostic criteria. A small group of ophthalmologists was sent to Ganzi, Sichuan Province at the altitude of 3.393 m, and to Qinpu in the suburb of Shanghai which is at the sea level. In Ganzi 3,905 (91.97%) people including 822 Hans and 3,083 Zang and in Qinpu 3,101 (93.20%) were studied. Age-related cataract was diagnosed when there are cortical or nuclear, or mixed opacities; and aphakia due to previous extraction of age-related cataract. Direct method was used to standardize the data. The prevalence of age-related cataract in Ganzi is about 82% higher than that in Qinpu in all age groups; from age 16-60, the prevalence of age-related cataract in Ganzi is about 180% (in Hans) and 160% (in Zangs) higher than that in Qinpu. There is no significant difference in the prevalence of age-related cataract between the ethnic groups in Ganzi. In Ganzi, the sunshine hours per year are 1.28 times that in Qinpu, and the global radiation in Ganzi is 1.47 times that in Qinpu. The higher prevalence of cataract in Ganzi than in Qinpu may be due to the greater exposure to the sunshine on the Tibetan plateau. The differences in prevalence between males and females and between indoor and outdoor work are discussed.

Adolescent↗
PubMed1989

The dephosphorylation of lens alpha-crystallin A chain.

The present communication reports the presence of a phosphoprotein phosphatase activity in bovine lens preparations which dephosphorylates alpha Ap, the phosphorylated form of alpha A, one of the alpha-crystallin polypeptides, in a Ca2+/calmodulin dependent manner. The activity was found in soluble preparations from epithelial cells but it could not be detected in similar preparations from fiber cells. A 60,000 Mr calmodulin binding polypeptide and a 15,000 Mr polypeptide found in the epithelial cell preparations comigrated in SDS-PAGE with the A and B subunits of bovine brain calcineurin (phosphoprotein phosphatase 2B) respectively. The 15,000 Mr was specifically recognized by an anti-bovine brain calcineurin antiserum. Bovine brain calcineurin was as effective in dephosphorylating alpha Ap as the lens preparations. Thus, it is likely that the activity present in the lens is related to this enzyme. The results indicate that the lens specific polypeptide alpha A may be subject to metabolic control through phosphorylation and dephosphorylation pathways regulated by cAMP and calcium respectively. Changes in the activities of these pathways appear to occur during differentiation of the lens epithelial cell and may be related to gene regulation during the differentiation process.

Animals↗