Proximal locking screw repositioning in reconstruction femoral nails

Chard (Beta vulgaris L. var. cicla) is one of the plants used as hypoglycaemic agent by diabetics in Turkey and it has been reported to reduce blood glucose. The purpose of this study was to investigate the effect of feeding chard on diabetes induced impairments in rat skins. Uncontrolled induced diabetes caused significant increases in nonenzymatic glycosylation of skin proteins, lipid peroxidation and blood glucose. Administration of chard extract inhibited these effects except the increase in lipid peroxidation. SDS-polyacrylamide gel electrophoresis revealed no significant differences in any protein bands between any of the groups. The data indicate that the use of chard may be effective in preventing or at least retarding the development of some diabetic complications.     

Glutathione levels of the human crystalline lens in aging and its antioxidant effect against the oxidation of lens proteins

This paper reports the role of glutathione (GSH) in the crystalline lens as an antioxidant against the oxidation of lens protein. GSH levels in normal lenses decreased gradually with increasing age, from approximately 5 mumol per g lens (wet weight) to 3 mumol per g lens (wet weight). On the other hand, levels of oxidized GSH in the lenses increased until the age of 40. After that, it remained almost constant at the level of approximately 0.9 mumol per g lens. Protein-bound GSH levels in both soluble and insoluble lens proteins dropped noticeably in the 50-year and older age groups, although there were significant differences in levels between both fractions. A decrease of tryptophan and tyrosine residues in lens proteins was proportional to a decrease in GSH levels in the lens as a result of aging. Those residue levels in the cataractous lenses were approximately half those in the normal lens proteins, and GSH levels in such lenses were almost one-tenth that in the normal lens. This study revealed that GSH may play an important role in preventing the oxidation of lens proteins from various oxidants. Furthermore, it is conceivable that these normal changes in GSH levels in the lenses increase the vulnerability of the lens to senile cataractogenesis.     

Role of the Maillard reaction in aging of tissue proteins. Advanced glycation end product-dependent increase in imidazolium cross-links in human lens proteins

Dicarbonyl compounds such as glyoxal and methylglyoxal are reactive dicarbonyl intermediates in the nonenzymatic browning and cross-linking of proteins during the Maillard reaction. We describe here the quantification of glyoxal and methylglyoxal-derived imidazolium cross-links in tissue proteins. The imidazolium salt cross-links, glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), were measured by liquid chromatography/mass spectrometry and were present in lens protein at concentrations of 0. 02-0.2 and 0.1-0.8 mmol/mol of lysine, respectively. The lens concentrations of GOLD and MOLD correlated significantly with one another and also increased with lens age. GOLD and MOLD were present at significantly higher concentrations than the fluorescent cross-links pentosidine and dityrosine, identifying them as major Maillard reaction cross-links in lens proteins. Like the N-carboxy-alkyllysines Nepsilon-(carboxymethyl)lysine and Nepsilon-(carboxyethyl)lysine, these cross-links were also detected at lower concentrations in human skin collagen and increased with age in collagen. The presence of GOLD and MOLD in tissue proteins implicates methylglyoxal and glyoxal, either free or protein-bound, as important precursors of protein cross-links formed during Maillard reactions in vivo during aging and in disease.     

The refractive index and protein distribution in the blue eye trevally lens

BACKGROUND: The relationship between structure (crystallin distribution) and function (refractive index) in the lens is not understood and can be studied by comparing biochemical and optical properties. Such a comparison has been made using a blue eyed trevally lens. METHODS: The optical parameter of refractive index distribution was determined using a nondestructive ray tracing technique. The distributions of the various classes of proteins in the lens were determined by dissolving lenses in concentric layers and using biochemical protein assay. HPLC and SDS-PAGE electrophoresis were used to investigate the proportion of proteins in each layer. RESULTS: The refractive index distribution, from center to edge, follows a second order polynomial. The proteins do not vary in their proportions over most of the lens; only in the inner-most regions is there a rapid increase in insoluble protein and a concomitant decrease in the soluble protein classes. The smallest proteins (gamma crystallins) become insoluble later than the alpha- and beta-crystallins. CONCLUSIONS: There are no similarities in the distributions of any of the protein classes to that of the refractive index in the fish lens. This result indicates that a quantitative relationship cannot be derived by comparing protein to refractive index distributions. However, the findings are consistent with those made in other species: a high content of gamma-crystallins is always found in lenses which have steep refractive index gradients and high index magnitudes.     

Calcium-induced changes on crystallins in organ-cultured porcine lens

In this study, intact porcine lenses were cultured in vitro for 7 days supplemented with commercial balanced salt solution (BSS) which is usually used as an irrigation solution during intraocular surgery, and the lenses were maintained under various culture conditions, e.g. temperature and CO2 concentration. The intact porcine lenses after 7 days culture were analyzed with optical density scanner, gel permeation chromatography on TSK HM-55 column and SDS-PAGE (polyacrylamide gel electrophoresis). It was found that lenses exhibited the least opacity when lenses were cultured with Ca(+2)-free BSS buffer, CO2-free incubator and maintained at a temperature of 25 degrees C. After the lenses were cultured with Ca(+2)-free BSS or BSS medium, the composition of crystallins in lenses was separated with TSK HM-55 column. It was indicated that the percentage of high molecular weight (HMW) protein and (alpha-crystallin increased, and gamma-crystallin decreased in lenses incubated with BSS medium compared with lenses incubated with Ca(+2)-free BSS medium. Following an increase in the concentration of calcium in the medium from 4.3 mM, 20 mM, 50 mM, 100 mM to 200 mM, the opacity of the lens was measured with a densitometer. The changed percentage of various crystallins was similar to lenses with BSS media that increased in HMW protein and alpha-crystallin, decreasing in gamma-crystallin. In the case of lens protein pattern, the crystallin washed from TSK HM-55 gel was separated with SDS-PAGE (polyacrylamide gel electrophoresis). It was indicated that some of proteins disappeared when lenses were incubated with various concentrations of calcium. The vanished pH proteins were 20.5 kDa at 50 mM calcium, 20.5 kDa and 21 kDa at 100 mM, 20.5 kDa, 21 kDa, 22 kDa and 23 kDa at 200 mM which were compared with the protein bands in the presence of 20 mM calcium in BSS medium. This study indicates that the commercial balanced salt solution (BSS) which is usually used as an irrigating solution during intraocular operations may increase the risk for lens opacity because of the calcium contained in the solution.     

Protein associated with human lens 'native' membrane during aging and cataract formation

Plasma membrane contains extrinsic as well as intrinsic proteins. Changes in the extrinsic proteins of lens membrane during human aging and cataract formation have not been investigated in detail. Unlike previous studies which examined lens membrane after being stripped of extrinsic proteins by treatment with chaotropic agents, we have isolated whole or 'native' lens membrane on a sucrose gradient by ultracentrifugation of the total water-insoluble protein. Essentially all of the water-insoluble protein from young to aged to cataractous human lens appeared membrane associated. In young lens (20-37 years old), most of the membrane banded at the 25/45% sucrose interface fraction. This fraction contained relatively little urea-soluble protein and likely represents fiber-cell plasma membrane with its physiologically associated extrinsic and intrinsic proteins. With aging (62-80 years old), about one-third of the membrane, as judged by the distribution of cholesterol, banded at a much higher density (50/58% sucrose fraction). The higher density was due to a great increase in the membrane's relative protein content (protein/cholesterol). Although this extra protein was composed of both urea-insoluble and -soluble fractions, the urea-soluble protein predominated in all lenses. Cataractous lens differed from aged-clear lens in that much more of the total membrane (70-75%) had shifted to the high density and participated in this massive binding of cytosolic proteins. Although alpha-crystallin was the principal extrinsic-membrane protein in young lens, high molecular weight aggregate of modified (acidic) crystallins accounted for the increased extrinsic protein in aging. The extrinsic proteins bound to both clear-aged and cataractous lens membrane were aggregated. In conclusion, examination of human lens native membrane fractions revealed that the association of crystallins with membrane in both aging and cataracts was much greater than previously recognized and most of this increased protein was non-covalently bound to the membrane. Much more of the lens total membrane from cataractous than clear-aged lens was involved in this massive protein association and the protein bound to cataract membrane appeared more highly aggregated.     

Enhanced nonenzymatic glycosylation of blood proteins in stored blood

Significantly enhanced nonenzymatic glycosylation of hemoglobin, plasma, and erythrocyte membrane proteins was demonstrated following storage of whole blood in the liquid state under conventional blood bank conditions. Among the blood components studied, the proteins of the erythrocyte membrane were mainly involved, although the levels of glycosylated hemoglobin and plasma proteins were also significantly increased. In contrast to the nonenzymatic glycosylation observed in vivo in patients with diabetes, the in vitro process is less intensive and most probably results in less functional alteration.     

Benzisothiazole-1,1-dioxide alkanoic acid derivatives as inhibitors of rat lens aldose reductase

Derivatives of 4-substituted 1,2-benzisothiazole-1,1-dioxide alkanoic acids were prepared and their in vitro aldose reductase inhibitory activity was tested in rat lens enzyme. The acetic derivatives 10, 12, and 16a-d proved to be much more potent inhibitors than the propionic derivatives 11, 13, and 17a-d. The presence of an acyl moiety on the amino group in position 4 of the acetic derivatives 16a-d led to a significant increase in activity with respect to the parent compound 14. One of the most active compounds in vitro, 10, was also evaluated in vivo as an inhibitor of glutathione lens depletion in galactosemic rats, but it did not show any activity in maintaining the rat lens glutathione level, probably due to problems of ocular bioavailability or metabolism.     

Tissue ascorbic acid and polyol pathway metabolism in experimental diabetes

Previous studies demonstrating reduced plasma concentrations of ascorbic acid (AA) in diabetes and interactions between this vitamin and biochemical mechanisms such as synthesis of structural proteins, oxidative stress, polyol pathway and nonenzymatic glycation of proteins suggest that disturbed AA metabolism may be important in the pathogenesis of diabetic microangiopathy. However, limited information is available on the concentration of AA in tissues which develop diabetic complications. This study demonstrates reduced renal but not sciatic nerve or plasma AA concentration in two animal models of insulin-dependent diabetes mellitus, namely the STZ-diabetic rat and the spontaneously diabetic BB rat. Decreased lens AA concentration was also observed in STZ-diabetic rats. Improvement of glycaemic control by insulin treatment (albeit insufficient to achieve normoglycaemia) partially corrected lens and renal AA concentration in STZ-diabetic rats. AA treatment increased kidney and lens AA concentrations of STZ-diabetic and non-diabetic rats and corrected the abnormalities observed for untreated diabetic rats. Sciatic nerve AA concentration was not increased by AA treatment in any group. Tissue ratios of dehydroascorbic acid (DHAA)/AA, one index of oxidative stress, were not different between the diabetic and non-diabetic groups and were unaltered by AA supplementation. AA treatment of STZ-diabetic rats had no effect on elevated tissue concentrations of glucose, sorbitol and fructose or reduced myo-inositol concentration. The effect of reduced tissue AA levels in diabetes on either collagen synthesis or ability to combat increased free radical production is not known. However, correction of abnormal kidney and lens AA concentrations in experimental diabetes by AA supplementation suggests that if AA does have a role in the development or progression of the renal and ocular complications of diabetes, this treatment could be beneficial.     

Diabetes-induced changes in lens antioxidant status, glucose utilization and energy metabolism: effect of DL-alpha-lipoic acid

The study was aimed at evaluating changes in lens antioxidant status, glucose utilization, redox state of free cytosolic NAD(P)-couples and adenine nucleotides in rats with 6-week streptozotocin-induced diabetes, and to assess a possibility of preventing them by DL-alpha-lipoic acid. Rats were divided into control and diabetic groups treated with and without DL-alpha-lipoic acid (100 mg x kg body weight(-1) x day(-1), i.p.). The concentrations of glucose, sorbitol, fructose, myo-inositol, oxidized glutathione, glycolytic intermediates, malate, alpha-glycerophosphate, and adenine nucleotides were assayed in individual lenses spectrofluorometrically by enzymatic methods, reduced glutathione and ascorbate--colorimetrically, and taurine by HPLC. Free cytosolic NAD+:NADH and NADP+:NADPH ratios were calculated from the lactate dehydrogenase and malic enzyme systems. Sorbitol pathway metabolites were found to increase, and antioxidant concentrations were reduced in diabetic rats compared with controls. The profile of glycolytic intermediates (increase in glucose 6-phosphate and fructose 6-phosphate, decrease in fructosel,6-diphosphate, increase in dihydroxyacetone phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and no change in lactate), and 5.9-fold increase in alpha-glycerophosphate suggest diabetes-induced inhibition of glycolysis. Free cytosolic NAD+:NADH ratios, ATP levels, ATP/ADP x inorganic phosphate (Pi), and adenylate charge were reduced in diabetic rats while free cytosolic NADP+:NADPH ratios were elevated. Diabetes-induced changes in the concentrations of antioxidants, key glycolytic intermediates, free cytosolic NAD+:NADH ratios, and energy status were partially prevented by DL-alpha-lipoic acid, while sorbitol pathway metabolites and free cytosolic NADP+:NADPH ratios remained unaffected. In conclusion, diabetes-induced impairment of lens antioxidative defense, glucose intermediary metabolism via glycolysis, energy status and redox changes are partially prevented by DL-alpha-lipoic acid. The findings support the important role of oxidative stress in lens metabolic imbalances in diabetes.     

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There is strong evidence to show that diabetes is associated with increased oxidative stress. However, the source of this oxidative stress remains unclear. Using transgenic mice that overexpress aldose reductase (AR) in their lenses, we found that the flux of glucose through the polyol pathway is the major cause of hyperglycemic oxidative stress in this tissue. The substantial decrease in the level of reduced glutathione (GSH) with concomitant rise in the level of lipid peroxidation product malondialdehyde (MDA) in the lens of transgenic mice, but not in the nontransgenic mice, suggests that glucose autoxidation and nonenzymatic glycation do not contribute significantly to oxidative stress in diabetic lenses. AR reduction of glucose to sorbitol probably contributes to oxidative stress by depleting its cofactor NADPH, which is also required for the regeneration of GSH. Sorbitol dehydrogenase, the second enzyme in the polyol pathway that converts sorbitol to fructose, also contributes to oxidative stress, most likely because depletion of its cofactor NAD+ leads to more glucose being channeled through the polyol pathway. Despite a more than 100% increase of MDA, oxidative stress plays only a minor role in the development of cataract in this acute diabetic cataract model. However, chronic oxidative stress generated by the polyol pathway is likely to be an important contributing factor in the slow-developing diabetic cataract as well as in the development of other diabetic complications.--Lee, A. Y. W., Chung, S. S. M. Contributions of polyol pathway to oxidative stress in diabetic cataract. FASEB J. 13, 23-30 (1999)     

Diabetic retinopathy: a review

In the United States, diabetic retinopathy is the leading cause of new blindness in those of occupational age. We present an overview of risk factors, including renal disease, uncontrolled blood pressure, pregnancy, poor glucose control, elevated glycosylated hemoglobin, duration of disease, and age at time of diagnosis; pathogenesis, addressing the involvement of aldose reductase, nonenzymatic glycosylation of proteins, vasoproliferative factors, ischemia and vasodilation, systemic growth factors, and platelets and blood viscosity; pathology, including nonproliferative, preproliferative, and proliferative retinopathy; and the management of this condition.     

Thiolation of the gammaB-crystallins in intact bovine lens exposed to hydrogen peroxide

Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H2O2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H2O2. The H2O2 treatment decreased the glutathione and increased the protein-glutathione and protein-cysteine disulfides in the lens. The molecular mass of the gammaB-crystallin isolated from lenses, not treated with H2O2, agreed with the published sequence (Mr 20,966). Some lenses also had a less abundant gammaB-crystallin component 305 Da higher (Mr 21,270), suggesting the presence of a glutathione adduct. The gammaB-crystallins from H2O2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified gammaB-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of gammaB-crystallins from different lenses indicated that the +305 Da modifications were not at a specific cysteine. For the lenses incubated without H2O2, there was evidence of adducts at Cys-41 and in peptide 10-31, which includes 3 cysteines. Analysis of modified peptide 10-31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, gammaB-crystallins from H2O2-treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.     

Further characterization of a rat hepatoma-derived aldose-reductase-like protein--organ distribution and modulation in vitro

A protein detected in N-methyl-N-nitrosourea-initiated rat hepatomas by two-dimensional electrophoresis at 35 kDa/pI 7.4 was identified in a previous study by internal amino acid micro sequencing as an aldose-reductase-like protein [Zeindl-Eberhart, E., Jungblut, P. R., Otto, A. & Rabes, H. M. (1994) Identification of tumor-associated protein variants during rat hepatocarcinogenesis, J. Biol. Chem. 269, 14589-14594]. Two-dimensional electrophoresis of rat lens proteins revealed a spot at 37 kDa/pI 6.8 that showed a high degree of identity (98.5%) with rat lens aldose reductase after amino acid sequencing and 80% sequence identity to the rat-hepatoma-derived aldose-reductase-like protein. This suggests that hepatoma-derived aldose-reductase-like protein and rat lens aldose reductase are related proteins encoded by different genes. A different expression profile of these proteins was found in various rat organs. Rat lens aldose reductase is present, in addition to in lens, in heart, brain, muscle, lung, duodenum, kidney, spleen and bone marrow, while the hepatoma-derived aldose-reductase-like protein is found preferentially in hepatomas and in embryonic liver. Though different in organ expression, an identical response was found for both proteins after stimulation with fibroblast growth factor-1 and after exposure to increased glucose concentrations. Since rat hepatoma-derived aldose-reductase-like protein is expressed in embryonic, but not in adult liver, it is assumed that it is expressed in hepatomas as a functionally active embryonal type of aldose reductase during hepatocarcinogenesis. Immunohistochemistry revealed that the hepatoma-derived aldose-reductase-like protein is expressed already in the preneoplastic stage of hepatocarcinogenesis and might potentially serve as a marker enzyme in early hepatic neoplasia.     

Pathogenesis of diabetic microangiopathy

Several are the recent experimental data regarding the pathogenesis of diabetic microangiopathy, even if the comprehensive picture of this condition is still rather incomplete. The functional and structural alterations of several organs involved (first of all kidney and retina) are especially dependent on the activation of the polyol pathway and on the increase of the nonenzymatic glycosylation. An important determinant of diabetic microangiopathy is the increase of permeability, at first charged especially to the haemodynamic alterations and partly reversible, and later supported by irreversible variations of the cellular and extracellular components of the vascular wall. Genetic factors certainly contribute to the explanation of the diverse gravity of microvascular damage in diabetic patients, even if the mechanisms by which they interfere are only partly known. Furthermore, the links between arterial hypertension and diabetic nephropathy, and also the reduction in glycosaminoglycans in the basal membranes are perhaps genetically originated.     

Bovine and human alpha-crystallins as molecular chaperones: prevention of the inactivation of glutathione reductase by fructation

With no measurable protein synthesis occurring in the centre of the lens, structural proteins and enzymes there will need to be stable for many years, if not decades, in order to maintain lens integrity and function. Recent work has indicated that alpha-crystallin, which is sequentially related to heat shock proteins, has chaperone-like properties in that it is capable of preventing heat-induced aggregation of various proteins, including other crystallins. Thus this universal vertebrate lens protein may contribute to maintenance of lens integrity by protecting other lens proteins from non-enzymic insults or the consequences thereof. We previously showed that the enzyme glutathione reductase was inactivated in a time-dependent manner when incubated with various sugars, suggesting glycation was responsible for this effect. In this paper we confirmed that this was the case. Using this enzyme model system, the inclusion of either bovine or human alpha-crystallin protected against the inactivation of glutathione reductase by fructation. This action was specific, with control proteins displaying no such protection. Use of high performance liquid chromatography supported the fact that alpha-crystallin did not act simply by mopping up free sugar but rather maintained the activity of the modified enzyme. Dose-dependent experiments indicated that human alpha-crystallin was more effective than its bovine counterpart, which might be expected considering the much longer lifespan of humans. The stoichiometry of the protection by both alpha-crystallins indicated that alpha-crystallin with glutathione reductase was not acting like GroEL as a large complex with a hydrophobic pore, but rather that individual subunits may be capable of acting as chaperones.     

Lipid advanced glycosylation: pathway for lipid oxidation in vivo

To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.     

The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress

alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction.