Free alanine,aspartic acid,or glutamic acid reduce the glycation of human lens proteins

The amino acids lysine and glycine are reported to react with glucose at physiological pH and temperature and undergo non-enzymic glycation. Three other amino acids present in relatively larger amounts in the lens i.e. alanine, aspartic acid and glutamic acid were also found to undergo non-enzymic glycation as found by incorporation of uniformly labelled (U-[¹⁴C]) glucose into the amino acids. The glucose incorporation was 1.6 to 2.5% for alanine, 35 to 50% for aspartic acid and 2.3 to 3.3% for glutamic acid. Each amino acid of varying concentrations lowered the extent ofin vitro glycation of lens proteins significantly in glucose-treated homogenates of normal lens from humans. The decrease in glycation for alanine was between 32 and 69%, that for aspartate was between 18 and 74%, and for glutamate was between 52 to 74%. Decreased glycation was greater for higher concentrations of glucose. Scavenging of intracellular glucose and decreasing the extent of glycation of lens proteins could be the mechanism of action by which the amino acids alanine, aspartic acid and glutamic acid could exercise a beneficial effect on cataract and diabetic retinopathy.     

Prevention of cataract by pyruvate in experimentally diabetic mice

Previous studies have demonstrated that administration of pyruvate prevents cataract formation in diabetic rats. It is known that the induction of cataractous process in this case is initiated by aldose reductase (AR) catalyzed synthesis and accumulation of excessive sorbitol in the lens fibres and epithelium and their consequent osmotic hydration. Synthesis of this and other polyols is competitively inhibited by pyruvate. The objective of the present investigations was hence to determine whether pyruvate would have a similar protective effect in species where cataract formation is relatively independent of sorbitol synthesis such as in humans where the lens AR activity is extremely low, especially with glucose as a substrate. The Km of AR for glucose is known to be very high. The possible protective effect of pyruvate in the low AR models was conceived on the basis of our previous findings suggesting that it can also exert substantial antiglycating as well as antioxidant effects. The present studies have hence been conducted with mice, a species known to be low in lens AR, similar to that in humans. As stipulated, pyruvate administration has indeed been found to offer a significant protection against development of diabetic cataract in this model also. The effect correlated with the inhibition of protein glycation as well as of oxidative stress. The latter was apparent by the prevention of the loss of glutathione known to be associated with diabetes. Although there was a small but noticeable increment in the sorbitol content of the diabetic lenses, this was osmotically insignificant. Even this increase was prevented by pyruvate. The magnitude of the elevation in the contents of glycated proteins and the depression in the level of glutathione were, on the contrary, highly pronounced, suggesting a more prominent role of the latter factors. In addition, the possibility of a direct metabolic support it could offer to the tissue is also imminent by its effect on the maintenance of ATP, as shown earlier. The present studies are therefore considered more relevant to the pathogenesis of cataract in human diabetics and its possible prevention by endogenous compounds with antiglycating and antioxidant properties. Inhibition of cataract formation by pyruvate in an animal model with low lens AR, similar to that in humans, has been shown for the first time. (Mol Cell Biochem 269: 115–120, 2005)     

Inhibition of protein glycation and advanced glycation end products by ascorbic acid and other vitamins and nutrients

Nonenzymatic glycation, the reaction of glucose and other reducing sugars with protein, reversibly produces Amadori products and over a long period irreversible advanced glycation end products. In diabetes, these reactions are greatly accelerated and are important in the pathogenesis of diabetic complications.

In vitro glycation was studied with bovine albumin as the model protein. A mixture of 25 mM glucose/fructose was used as the glycating agent. The Amadori product was quantitated by thiobarbituric acid colorimetry after hydrolysis. Advanced glycation end products were measured by their intrinsic fluorescence. A number of vitamins and nutrients were found to be potent inhibitors of both the glycation reaction and the subsequent end products. The nutrients were effective at physiological concentrations and exhibited dose-response relationships. The inhibitors included ascorbic acid, tocopherol, pyridoxal, niacinamide, sodium selenite, selenium yeast, and carnosine. A significant correlation was found between the inhibition of glycation and the inhibition of AGE formation (P < 0.001). One of the nutrients, ascorbic acid, was used in a pilot study. Eighteen normal subjects, 7 college age and 10 middle age, were supplemented with 1,000 mg of ascorbic acid in the form of Re-Natured Vitamin C^® for a period of 4 weeks. Serum protein glycation was decreased an average of 46.8% (P < 0.01). These results underline the importance of nutrition in diabetes and indicate the possibility of therapeutic use of these nutrients for the prevention of diabetic complications.     

Plasma protein glycation in Alzheimer's disease

Recent studies have suggested that formation of advanced glycation end-products (AGEs) in some brain proteins could be associated with Alzheimer's disease.These AGEs can be produced by various sugars (hexose, pentose, glyceraldehyde and oxidative products of vitamin C). In this study, we quantified plasma protein glycation specifically derived from glucose in patients with Alzheimer's disease with different grades of cognitive disorders.Two groups of Alzheimer patients were studied: a group with moderate Alzheimer's disease (n=6, 9相似文献   

Analysis and prediction of mammalian protein glycation

Glycation is a nonenzymatic process in which proteins react with reducing sugar molecules and thereby impair the function and change the characteristics of the proteins. Glycation is involved in diabetes and aging where the accumulation of glycation products causes side effects. In this study, we statistically investigate the glycation of epsilon amino groups of lysines and also train a sequence-based predictor. The statistical analysis suggests that acidic amino acids, mainly glutamate, and lysine residues catalyze the glycation of nearby lysines. The catalytic acidic amino acids are found mainly C-terminally from the glycation site, whereas the basic lysine residues are found mainly N-terminally. The predictor was made by combining 60 artificial neural networks in a balloting procedure. The cross-validated Matthews correlation coefficient for the predictor is 0.58, which is quite impressive given the relatively small amount of experimental data available. The method is made available at www.cbs.dtu.dk/services/NetGlycate-1.0.     

The role of glycation in aging and diabetes mellitus

One of the hypotheses trying to explain the process of aging is the idea of glycation of proteins. This reaction, also called the Maillard or browning reaction, may explain age-related symptoms such as cataract, atherosclerosis and modification of collagen-containing tissues. Diabetics, which posses elevated blood sugar levels, show signs of accelerated aging exposing similar complications. The Maillard reaction, which occurs on a large scale in vivo, may play a key role in the initiation of these symptoms.     

Urinary excretion of taurine in epilepsy

Evidence that taurine (2-aminoethanesulfonic acid) is related to the epilepsies is supported by work with both experimental animals and hurmans. It may function as a neurotransmitter or modulator of neurotransmission. Investigators using an automated amino acid analyzer reported lower mean urinary taurine excretion among epileptics. However, Rao et al. reported higher taurine excretion among epileptics using an older method. Analyses of the same epileptic and control urines by both methods coupled with paper and molecular size chromatography indicate that substances in addition to taurine are co-eluted with taurine using the older method, yielding spuriously high values. The resolution of this disparity is important because the urinary excretion of taurine may reflect primarily the influence of taurine transport alleles which may be polygenic components in the idiopathic epilepsies.     

Inhibition of ascorbic acid-induced modifications in lens proteins by peptides.

The effects of three dipeptides L-phenylalanyl-glybine, glycyl-L-phenylalanine,and aspartame (L-aspartyl-L-phenylalanine, methyl ester) as inhibitors of the ascorbic acid-induced modifications in lens proteins were studied. Their efficiency was compared to that of two known inhibitors--aminoguanidine and carnosine. The tested dipeptides diminished protein carbonyl content by 32-58% and most moderated the formation of chromophores, as measured by the absorbency at 325 nm of the glycated proteins. The appearance of non-tryptophan fluorescence (excitation 340 nm/emission 410 nm) was observed for proteins glycated with ascorbic acid. All of the dipeptides examined, as well as aminoguanidine, decreased this glycation-related fluorescence. The potential inhibitors prevented the intensive formation of very high molecular weight aggregates. A competitive mechanism of their inhibitory effect was proposed, based on the reactivity of individual substances toward ascorbic acid. These findings indicate that they have a potential for use as alternatives for aminoguanidine as an anti-glycation agent.     

Formation of advanced glycation end (AGE) products in diabetes: Prevention by pyruvate and a-keto glutarate

Glycation of proteins and their subsequent structural and functional modifications have been ascribed to play a prominent role in the pathogenesis of several secondary complications of diabetes, such as cataract and retinopathy. In addition, it plays a role in the generalized ageing process as well. Investigations have been conducted to explore the possibility of preventing the above process by use of pyruvate and a-keto glutarate as representatives of physiologically compatible keto acids. The results demonstrate that both these compounds are effective in preventing the initial glycation reaction as well as the formation of AGE products. Both these compounds also inhibit the generation of high molecular weight aggregates associated with cataract formation. Mechanistically, the preventive effects appear to be due to (1) competitive inhibition of glycation by the keto acids and (2) the antioxidant (radical scavenging) properties of these compounds. The results are hence considered usefu l from the point of view of developing these and other keto acid derivatives as pharmacological agents useful in preventing glycation related protein changes and consequent tissue pathological manifestations.     

Glycation of human lens proteins from diabetic and (nondiabetic) senile cataract patients

Glycation (nonenzymatic glycosylation) in the human lens (cortex and nucleus) in senile (nondiabetic) and diabetic cataracts was studied by measuring the extent of early and late glycation products, the content of free -amino groups and the formation of disulfide bonds in the soluble lens proteins. There was a significant (p<0.001) increase in early and late glycation in the lens nucleus compared to the cortex in both the senile and diabetic groups. Overall these changes were much larger in the diabetic group. The concentration of free -amino groups was decreased in the senile nucleus as well as in the diabetic nucleus when compared with the senile and diabetic cortex (p<0.001). Disulfide bond content was in the order of diabetic nucleus > diabetic cortex > senile nucleus > senile cortex. Glycation of the lens proteins is a generalized feature which is enhanced in the diabetic lens compared to senile lens proteins and is associated with a decrease in free -amino groups and an increase in disulfide bonds formation in the lens proteins.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Identification of Bilophila wadsworthia by specific PCR which targets the taurine:pyruvate aminotransferase gene

The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.     

Inactivation of cellular enzymes by carbonyls and protein-bound glycation/glycoxidation products

Diabetic plasma contains elevated levels of glucose and various low-molecular-weight carbonyl compounds derived from the metabolism of glucose and related materials. These compounds react with protein side chains (Arg, Lys, Cys, and His) to give glycated materials and advanced glycation end products. In this study, we have examined the effect of glucose and carbonyl compounds (methylglyoxal, glyoxal, glycolaldehyde, and hydroxyacetone), and glycation products arising from reaction of these materials with model proteins, on the activity of three key cellular enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutathione reductase, and lactate dehydrogenase, both in isolation and in cell lysates. In contrast to glucose (1M, both fresh and aged for 8 weeks), which had no effect, marked inhibition of all three enzymes was observed with methylglyoxal and glyoxal. GAPDH was also inhibited by glycolaldehyde and hydroxyacetone. Incubation of these enzymes with proteins that had been preglycated with methylglyoxal, but not glucose, also resulted in significant time- and concentration-dependent inhibition with both isolated enzymes and cell lysates. This inhibition was not metal ion, oxygen, superoxide dismutase, or catalase dependent, suggesting that inhibition is not radical mediated. These effects are suggested to be due to direct adduction of the free- or protein-bound carbonyls with the target enzyme. Such an interpretation is supported by the detection of the loss of thiol groups on GAPDH and the detection of cross-linked materials on protein gels. Though direct comparison of the extent of inhibition induced by free versus protein-bound carbonyls was not possible, the significantly higher concentrations of the latter materials over the former in diabetic plasma and cells lead us to suggest that alterations in the activity of key cellular enzymes induced by glycated proteins may play a significant role in the development of diabetic complications.     

牛磺酸对学习记忆影响的研究现状

牛磺酸作为一种条件必须氨基酸对学习记忆能力的影响受到越来越多的关注,许多研究证明适量牛磺酸的添加可明显提高大鼠的记忆能力。通过牛磺酸对神经细胞、基因、激素、离子、受体、酶类等的作用,对其影响学习记忆方面的研究现状作出综述。     

Nonenzymatic glycation of DNA nucleosides with reducing sugars

Reducing sugars can react with the free amino groups of proteins to form a heterogeneous group of compounds known as advanced glycation endproducts (AGEs) or Maillard reaction products. The objective of this investigation was to monitor the nonenzymatic glycation of DNA nucleosides and to characterize the formation of nucleoside AGEs using capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), UV fluorescence spectroscopy, and mass spectrometry. Deoxyguanosine, deoxyadenosine, deoxythymidine, and deoxycytidine were used as the model nucleosides and were incubated over time with glucose, galactose, or glyceraldehyde. Under increasing concentrations and time, deoxyguanosine exhibited the highest rate of glycation with glyceraldehyde. Deoxyadenosine and deoxycytidine exhibited comparable reactivity with glyceraldehyde and no appreciable reactivity with galactose or glucose. No reactivity was observed between deoxythymidine and the sugars. A combination of CE, HPLC, UV fluorescence spectroscopy, and mass spectrometry provided a convenient method for characterizing nucleoside AGEs and for monitoring the physical factors that influence the formation of sugar adducts of DNA nucleosides.     

高糖对培养大鼠心肌细胞牛磺酸转运的影响及其可能机制

目的：观察不同浓度葡萄糖对细胞牛磺酸(taurine)转运功能的影响。方法：在培养的大鼠心肌细胞上,用^3H标记的牛磺酸测定细胞牛磺酸转运和竞争性定量RTPCR测定细胞牛磺酸转运体(TAUT)mRNA含量。结果：不同浓度葡萄糖(10～30mmol/L)孵育,抑制细胞^3H-牛磺酸转运,呈时间依赖性。与对照组比较,高糖(20mmol／L和30mmol／L)使心肌细胞牛磺酸摄入量显著减少,其^3H-牛磺酸转运的最大速率(Vmax)减少,心肌细胞TAUTmRNA含量较对照组减少。结论：高糖抑制心肌细胞牛磺酸转运,这与TAUT的牛磺酸结合位点减少和TAUT基因转录水平下调有关。     

Neutron scattering by calf lens cytoplasm

Small angle neutron scattering (SANS) was used to compare two models of cataracts: the cold cataract induced in the lens nucleus cytoplasm by lowering the temperature and the opacification induced by calcium in the lens cortex cytoplasm. In both cases opacified cytoplasms display additional scattering at low angles as compared to their clear controls. An analysis of this additional scattering provides quantitative information concerning the size distribution, the number and contrast of the scatterers responsible for lens opacification. The scatterers of cold cataract and of calcium—induced opacification not only have, as shown elsewhere, a different composition but are also found to display completely different sizes (in the thousand Å range for cold-cataract, in the hundred Å range for calcium—induced opacification). These results illustrate the diversity of scatterer types which are able to cause comparable lens opacities.The work reported here was begun as a part of the PhD thesis of D. Laporte who died accidentally in September 1984     

牛磺酸对H2O2诱导的红细胞溶血率的影响

研究牛磺酸对H2O2诱导的红细胞溶血的抵抗作用的影响。实验设对照组(H2O2 生理盐水)和给药组(H2O2 牛磺酸的生理盐水溶液),将绵羊血、小鼠血、家鸡血、家兔血及人血红细胞悬液分别加入对照组和给药组,37℃水浴1h,测定吸光度,计算溶血率。结果显示,给药组的溶血率均明显低于对照组溶血率(p<0,05或p<0.01)。实验证明牛磺酸对H2O2诱导的红细胞溶血有明显的抵抗作用。     

Nonenzymatic glycation of guanosine 5'-triphosphate by glyceraldehyde: an in vitro study of AGE formation

Guanosine 5'-triphosphate (GTP) plays a significant role in the bioenergetics, metabolism, and signaling of cells; consequently, any modifications to the structure of the molecule can have profound effects on a cell's survival and function. Previous studies in our laboratory demonstrated that like proteins, purines, and pyrimidines can nonenzymatically react with sugars to generate advanced glycation endproducts (AGEs) and that these AGEs can form in vitro under physiological conditions. The objective of this investigation was twofold. First, it was to evaluate the susceptibility of ATP, GTP, CTP, and TTP to nonenzymatic modification by D-glucose and DL-glyceraldehyde, and second to assess the effect of various factors such as temperature, pH and incubation time, and sugar concentration on the rate and extent of nucleotide triphosphate AGE formation. Of the four nucleotide triphosphates that were studied, only GTP was significantly reactive forming a heterogeneous group of compounds with DL-glyceraldehyde. D-Glucose exhibited no significant reactivity with any of the nucleotide triphosphates, a finding that was supported by UV and fluorescence spectroscopy. Capillary electrophoresis, high-performance liquid chromatography and mass spectrometry allowed for a thorough analysis of the glycated GTP products and demonstrated that the modification of GTP by dl-glyceraldehyde occurred via the classical Amadori pathway.     

Effect of aminophospholipid glycation on order parameter and hydration of phospholipid bilayer

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.