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1.
Sci Adv ; 5(10): eaax6525, 2019 10.
Article in English | MEDLINE | ID: mdl-31633028

ABSTRACT

Functionally related genes tend to be chromosomally clustered in eukaryotic genomes even after the exclusion of tandem duplicates, but the biological significance of this widespread phenomenon is unclear. We propose that stochastic expression fluctuations of neighboring genes resulting from chromatin dynamics are more or less synchronized such that their expression ratio is more stable than that for unlinked genes. Consequently, chromosomal clustering could be advantageous when the expression ratio of the clustered genes needs to stay constant, for example, because of the accumulation of toxic compounds when this ratio is altered. Evidence from manipulative experiments on the yeast GAL cluster, comprising three chromosomally adjacent genes encoding enzymes catalyzing consecutive reactions in galactose catabolism, unequivocally supports this hypothesis and elucidates how disorder in one biological phenomenon-gene expression noise-could prompt the emergence of order in another-genome organization.


Subject(s)
Models, Genetic , Cluster Analysis , Galactitol/analysis , Galactokinase/genetics , Galactokinase/metabolism , Gas Chromatography-Mass Spectrometry , Gene Expression , Multigene Family , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
2.
Phytother Res ; 27(5): 753-60, 2013 May.
Article in English | MEDLINE | ID: mdl-22826152

ABSTRACT

The polyol enzyme aldose reductase (AR) and advanced glycation endproducts (AGEs) play an important role in diabetic complications such as cataracts. The purpose of this study was to investigate four standardized plant extracts used for the treatment of diabetes and related diseases, and their principal components for AR inhibitory activity and to find out their influence in diabetic complications. Thus, Boswellia serrata Triana & Planch. (Burseraceae), Lagerstroemia speciosa (L.) Pers. (Lythraceae), Ocimum gratissimum (L.) (Lamiaceae) and Syzygium cumin (L.) Skeels. (Myrthaceae) and their respective major constituents, boswellic acid, corosolic acid, ursolic acid and ellagic acid, were studied for their inhibitory activity against rat lens AR, rat kidney AR, human recombinant AR and generation of AGEs. In addition, in vivo inhibition of lens galactitol accumulation by the major constituents of the plants in galactose-fed rat has been studied. The results revealed that all the tested extracts and their active ingredients possess significant AR inhibitory actions in both in vitro and in vivo assays with urosolic acid showing the most potent effect. Furthermore, the study indicates the potential of the studied plants and their major constituents as possible protective agents against long-term diabetic complications.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Glycation End Products, Advanced/analysis , Plant Extracts/pharmacology , Triterpenes/pharmacology , Animals , Boswellia/chemistry , Ellagic Acid/pharmacology , Galactitol/analysis , Humans , Kidney/drug effects , Kidney/enzymology , Lagerstroemia/chemistry , Lens, Crystalline/drug effects , Lens, Crystalline/enzymology , Male , Ocimum/chemistry , Plants, Medicinal/chemistry , Rats , Rats, Wistar , Recombinant Proteins/antagonists & inhibitors , Syzygium/chemistry , Ursolic Acid
3.
J Pharm Biomed Anal ; 56(4): 836-40, 2011 Dec 05.
Article in English | MEDLINE | ID: mdl-21840664

ABSTRACT

A simple method for the extraction and determination of ginsenoside Rb(1), astragaloside IV and dulcitol in sugar-free "Fufangfufangteng Heji" was developed using an ultrasonic-assisted liquid-liquid extraction (UALLE) coupled with Hydrophilic Interaction Liquid Interface Chromatography and Evaporative Light Scattering Detector (HILIC-ELSD) analysis. Good chromatographic separation was achieved using a Phenomenex Luna HILIC column (250mm×4.6mm i.d., 5µm), and a mobile phase consisting of acetonitrile-water at a flow rate of 1.0ml/min with a gradient elution within 25min was also used. Compared to the conventional analysis method, the proposed method had the advantages of a longer column life, shorter analysis time, lower baseline noise, short sample pretreatment time and low consumption of organic solvent. The linear ranges for ginsenoside Rb(1), astragaloside IV and dulcitol were 0.0256-0.179, 0.110-0.770, 0.105-0.630mg/ml, respectively. The recoveries of ginsenoside Rb(1), astragaloside IV and dulcitol during the pharmaceutical preparation were within the range of 97.2-100.3%, and their relative standard deviations were 1.2-3.1%.


Subject(s)
Chromatography, Liquid/methods , Galactitol/analysis , Ginsenosides/analysis , Plant Preparations/analysis , Saponins/analysis , Triterpenes/analysis , Calibration , Cardiotonic Agents/analysis , Cardiotonic Agents/chemistry , Drug Combinations , Galactitol/chemistry , Ginsenosides/chemistry , Light , Liquid-Liquid Extraction/methods , Plant Preparations/chemistry , Reproducibility of Results , Saponins/chemistry , Scattering, Radiation , Sound , Triterpenes/chemistry
4.
Clin Chem ; 56(7): 1177-82, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20489133

ABSTRACT

BACKGROUND: Duarte galactosemia (DG) is frequently detected in newborn-screening programs. DG patients do not manifest the symptoms of classic galactosemia, but whether they require dietary galactose restriction is controversial. We sought to assess the relationships of selected galactose metabolites (plasma galactose, plasma galactitol, erythrocyte (RBC) galactitol, RBC galactonate, and urine galactitol and galactonate) to RBC galactose 1-phosphate (Gal-1-P), dietary galactose intake, and neurodevelopmental/clinical outcomes in DG children. METHODS: We studied 30 children 1-6 years of age who had DG galactosemia and were on a regular diet. All participants underwent a physical and ophthalmologic examination and a neurodevelopmental assessment. RBC galactitol, RBC galactonate, RBC Gal-1-P, plasma galactose, plasma galactonate, and urine galactitol and galactonate concentrations were measured. RESULTS: RBC galactitol and galactonate concentrations were about 2 and 6 times higher, respectively, than control values. Plasma galactose and galactitol concentrations were also about twice the control values. The mean values for RBC Gal-1-P and urine galactitol were within the reference interval. We found a relationship between plasma and urine galactitol concentrations but no relationship between RBC galactose metabolites and urine galactitol. There was a significant relationship between galactose intake and RBC galactose metabolites, especially RBC galactitol (P < 0.0005) and RBC galactonate (P < 0.0005). Galactose intake was not related to the urine galactitol, plasma galactose, or plasma galactitol concentration. RBC galactitol, RBC galactonate, plasma galactose, plasma galactitol, and urine galactonate concentrations showed no relationship with clinical or developmental outcomes. CONCLUSIONS: DG children on a regular diet have RBC Gal-1-P concentrations within the reference interval but increased concentrations of other galactose metabolites, including RBC galactitol and RBC galactonate. These increased concentrations correlate with galactose intake and neither cause any developmental or clinical pathology during early childhood nor oblige a lactose-restricted diet.


Subject(s)
Galactitol/analysis , Galactose/analysis , Galactosemias/blood , Galactosemias/urine , Galactosephosphates/analysis , Sugar Acids/analysis , Child , Child, Preschool , Dietary Carbohydrates/administration & dosage , Erythrocytes/metabolism , Female , Galactitol/blood , Galactitol/urine , Galactose/administration & dosage , Galactose/blood , Galactose/urine , Galactosemias/physiopathology , Galactosephosphates/blood , Galactosephosphates/urine , Humans , Infant , Male , Monitoring, Physiologic , Reference Values , Sugar Acids/blood , Sugar Acids/urine
5.
Zhong Yao Cai ; 32(6): 972-4, 2009 Jun.
Article in Chinese | MEDLINE | ID: mdl-19764338

ABSTRACT

OBJECTIVE: To optimize the process of extracting melampyrit from Euonymus fortunei by central composite design-response surface methodology. METHODS: The independent variables were the solvent fold and extractive time, and the dependent variable was the extraction rate of melampyrit from Euonymus fortunei. Then different mathematic models were used to estimate the relationship between the independent and dependent variables. The response surface methodology was used to optimize the process of extraction and the prediction was carried out through comparing the observed and predicted values. RESULTS: The regression coefficient of binomial fitting complex model was 0.9515, and the optimum conditions of extraction process were 12-fold volume of solvent, 1.5 hours for decoction and 2 times for extraction. The bias between the observed and predicted values was -5.37%. CONCLUSION: It shows that this method is convenient and the optimum model is highly predictive.


Subject(s)
Euonymus/chemistry , Galactitol/isolation & purification , Plants, Medicinal/chemistry , Technology, Pharmaceutical/methods , Chromatography, High Pressure Liquid/methods , Galactitol/analysis , Plant Components, Aerial/chemistry , Reproducibility of Results , Solvents/chemistry , Temperature , Time Factors , Water/chemistry
6.
Curr Protoc Hum Genet ; Chapter 17: Unit 17.5, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18428423

ABSTRACT

Galactose metabolism occurs through an evolutionarily conserved pathway in which galactose and uridine diphosphoglucose are converted to glucose-1-phosphate and uridine diphosphogalactose through the action of three sequential enzymes: galactokinase (GALK, EC 2.7.1.6), galactose-1-phosphate uridyltransferase (GALT, EC 2.7.7.12), and uridine phosphogalactose 4'-epimerase (GALE, EC 5.1.3.2). Inborn errors of galactose metabolism occur with impaired activity for each of the enzymes. Classical galactosemia is the most common and the most severe of these diseases and is caused by deficiency of the GALT enzyme, affecting from approximately 1 in 10,000 to 1 in 30,000 live births. Deficiency of GALE is the rarest of the three diseases. Assays for galactitol and galactose-1-phosphate and methods for assaying enzyme activities of GALT, GALK, and GALE are provided here. Interpretation of diagnostic results for screen-positive newborns or symptomatic patients, as well as therapeutic interventions based on biochemical phenotype and molecular genotype, are also included as decision trees.


Subject(s)
Carbohydrate Metabolism, Inborn Errors/diagnosis , Carbohydrate Metabolism, Inborn Errors/metabolism , Galactose/metabolism , Carbohydrate Metabolism, Inborn Errors/genetics , DNA Mutational Analysis , DNA Primers , Galactitol/analysis , Galactokinase/analysis , Galactokinase/deficiency , Galactokinase/genetics , Galactosemias/diagnosis , Galactosemias/genetics , Galactosemias/metabolism , Galactosephosphates/analysis , Genetics, Medical , Humans , Infant, Newborn , Neonatal Screening , Polymerase Chain Reaction , UDPglucose 4-Epimerase/analysis , UDPglucose 4-Epimerase/deficiency , UDPglucose 4-Epimerase/genetics , UTP-Hexose-1-Phosphate Uridylyltransferase/analysis , UTP-Hexose-1-Phosphate Uridylyltransferase/deficiency , UTP-Hexose-1-Phosphate Uridylyltransferase/genetics
7.
Carbohydr Res ; 342(18): 2757-65, 2007 Dec 28.
Article in English | MEDLINE | ID: mdl-17935705

ABSTRACT

A new class of galactooligosaccharides has been identified from the terrestrial cyanobacterium Nostoc commune by MS and NMR techniques. These consist of beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]n-beta-d-1,4-anhydrogalactitols with n ranging from 2 to 8, corresponding to compounds designated 1 through 7. In total these saccharides amounted to approximately 0.35% of the dry thallus of N. commune, while in several other cyanobacteria they were not detected. Possibly they play some role in protection from damage by heat and desiccation as suggested by experiments with heterologous systems. For example, phosphoglucomutase (EC 2.7.5.1) from rabbit muscle was protected against heat inactivation by these oligosaccharides, and alpha-amylase (EC 3.2.1.1) from porcine pancreas by the oligosaccharides 6 and 7. The homologues of lower molecular mass, however, enhanced heat sensitivity of alpha-amylase. The viability of Escherichia coli was completely abolished by desiccation, whereas in the presence of 4 survival rates were approximately 50% of controls not subjected to desiccation. The newly identified saccharides are compared with known galactofuranose-based oligo- and polysaccharides and possible biological functions of them are discussed.


Subject(s)
Furans/chemistry , Galactitol/analogs & derivatives , Galactose/analogs & derivatives , Nostoc commune/chemistry , Oligosaccharides/analysis , Oligosaccharides/pharmacology , Animals , Carbohydrate Sequence , Chromatography, Ion Exchange , Chromatography, Thin Layer , Desiccation , Escherichia coli/cytology , Escherichia coli/drug effects , Furans/analysis , Galactitol/analysis , Galactitol/chemistry , Galactose/analysis , Galactose/chemistry , Hot Temperature , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Oligosaccharides/chemistry , Phosphoglucomutase/metabolism , Rabbits , Sucrose/pharmacology , Temperature , Trehalose/pharmacology , alpha-Amylases/metabolism
8.
Zhongguo Zhong Yao Za Zhi ; 32(13): 1289-91, 2007 Jul.
Article in Chinese | MEDLINE | ID: mdl-17879727

ABSTRACT

OBJECTIVE: To establish the processing method of Cistanche tubulosa decoction pieces. METHOD: The orthogonal test of four factors and three levels was used to optimize the main factors in the process of fresh C. tubulosa decoction pieces processing, including the thickness, temperature, and the time for inactivation of the enzyme in the plant. The result showed that the optimized condition was that fresh C. tubulosa was cut into 4 mm thickness, and heated at 70 degrees C for inactivation the enzyme in the plant for 6 min. Moreover, the optimized method was compared with the method of insolation and traditional dried method. RESULT: The content of echinacoside in the C. tubulosa decoction piece by the optimized method was 7.3 times of that dried by insolation, and 12.8 times of that by traditional dry method; the content of verbascoside was 6. 5 and 14. 9 times of that dried by insolation and by traditional dry method, respectively; the content of galactitol was 7.1 and 13.2 times of that dried by insolation and by traditional dry method, respectively. CONCLUSION: The quality of C. tubulosa decoction pieces could be improved by this method, and its crud drug could be saved, which would protect the source of the mild Herba Cistanche, and produced the better economic and ecological benefits.


Subject(s)
Cistanche/chemistry , Drugs, Chinese Herbal/isolation & purification , Plants, Medicinal/chemistry , Technology, Pharmaceutical/methods , Desiccation/methods , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/standards , Galactitol/analysis , Glucosides/analysis , Glycosides/analysis , Hot Temperature , Phenols/analysis , Quality Control
9.
AJNR Am J Neuroradiol ; 27(1): 204-7, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16418384

ABSTRACT

We describe how proton MR spectroscopy ((1)H-MR spectroscopy) was useful in elucidating the diagnosis of galactosemia in an undiagnosed 6-month-old infant. In vivo (1)H-MR spectroscopy of the brain showed a doublet at 3.7 parts per million, which was identified as galactitol (Gal-ol) by in vitro (1)H-MR spectroscopy of the urine. Galactosemia was subsequently confirmed by laboratory tests and treatment was initiated. A follow-up brain MR imaging and (1)H-MR spectroscopy study revealed resolution of white matter lesions and disappearance of Gal-ol peaks.


Subject(s)
Brain Chemistry , Galactitol/analysis , Galactosemias/diagnosis , Magnetic Resonance Spectroscopy , Brain/pathology , Female , Galactitol/urine , Galactosemias/diet therapy , Humans , Infant , Magnetic Resonance Imaging
10.
Mol Microbiol ; 51(4): 1015-25, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14763977

ABSTRACT

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.


Subject(s)
Cellulase/biosynthesis , Galactokinase/genetics , Galactokinase/metabolism , Galactose/metabolism , Hypocrea/enzymology , Lactose/metabolism , Amino Acid Sequence , Binding Sites/genetics , Cellulase/genetics , Cloning, Molecular , Conserved Sequence/genetics , Conserved Sequence/physiology , DNA, Fungal/chemistry , DNA, Fungal/isolation & purification , Enzyme Induction , Galactitol/analysis , Galactitol/metabolism , Galactokinase/chemistry , Gene Expression Regulation, Fungal , Genes, Fungal , Glucans/metabolism , Hypocrea/genetics , Hypocrea/growth & development , Hypocrea/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Molecular Sequence Data , Molecular Weight , Sequence Alignment , Sequence Analysis, DNA , UDPglucose-Hexose-1-Phosphate Uridylyltransferase/genetics , UDPglucose-Hexose-1-Phosphate Uridylyltransferase/metabolism
11.
Exp Eye Res ; 77(1): 51-7, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12823987

ABSTRACT

Apoptosis of lens epithelial cells (LECs) is implicated in the pathogenesis of several types of cataract formation. The high intracellular levels of polyol induce histological change in the LECs, which is considered the earliest event in sugar cataractogenesis. This study was designed to investigate whether high galactose exposure induces apoptosis in LECs during the development of sugar cataract. The effect of an aldose reductase inhibitor, SNK-860, was also examined. We induced sugar cataract in Sprague-Dawley rats by feeding them a 50% galactose-containing diet with or without SNK-860. The percentage of LECs undergoing apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) method, and DNA fragmentation analyses were performed. Galactitol levels in the lens epithelium were quantified by gas chromatography. The number of TUNEL-positive cells gradually increased throughout the period of galactose exposure, up to 5 days. DNA fragmentation analysis in LECs of rats fed a galactose-rich diet demonstrated an apparent ladder pattern. SNK-860 reduced the percentage of TUNEL-positive cells, the amount of intracellular galactitol, and the levels of DNA laddering. To explore the mechanism of the apoptotic process, the expression of p53, a potent mediator of apoptosis, was examined. Based on Western blot and real-time reverse transcription-polymerase chain reaction results, the amount of p53-expression increased at both the protein and mRNA levels after galactose exposure, and the increase in p53-expression was inhibited by SNK-860. Based on these results, we concluded that apoptosis occurs in rat lens epithelial cells following galactose exposure. Furthermore, the reduction of apoptosis by aldose reductase inhibitor suggests that this apoptosis is associated with the accumulation of sugar alcohols. It is probable that the mechanism of apoptosis during sugar cataract formation involves the increased expression of p53.


Subject(s)
Cataract/pathology , Epithelial Cells/pathology , Imidazolidines , Lens, Crystalline/pathology , Aldehyde Reductase/antagonists & inhibitors , Animals , Apoptosis , Blotting, Western/methods , Cataract/metabolism , DNA Fragmentation , Epithelial Cells/metabolism , Galactitol/analysis , Galactose , Genes, p53 , Imidazoles/pharmacology , In Situ Nick-End Labeling , Lens, Crystalline/metabolism , Male , Models, Animal , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/analysis
12.
NMR Biomed ; 14(3): 192-8, 2001 May.
Article in English | MEDLINE | ID: mdl-11357184

ABSTRACT

The development of tools to follow and quantitate the fate of galactose in mammalian cells is crucial to the study and understanding of the inherited disorders of galactose metabolism. In this study we incubated normal human lymphoblasts with 1- or 2-(13)C galactose for 2.5 or 5 h and prepared TCA extracts of the cells. The various galactose metabolites were identified and quantified using a combination of proton, carbon and phosphorus NMR spectra. Galactose-1-phosphate (gal-1P), uridine diphosphogalactose, uridine diphosphoglucose and galactitol were present in the extracts. Average levels for gal-1P were around 10 nmol/mg protein and for uridine diphosphoglucose, uridine diphosphogalactose and galactitol in the range of 0.5-2 nmol/mg protein. Galactonate was never found in any conditions. Percentage labeling could be estimated for gal-1P and for the ribose carbons of AMP. The labeling agrees with a conversion of galactose to glucose through the Leloir pathway.


Subject(s)
Galactose/metabolism , Lymphocytes/metabolism , Magnetic Resonance Spectroscopy/methods , Carbon Isotopes , Cell Extracts/chemistry , Cells, Cultured , Galactitol/analysis , Galactitol/metabolism , Galactose/analysis , Galactosephosphates/analysis , Galactosephosphates/metabolism , Humans , Hydrogen , Phosphorus Isotopes , Uridine Diphosphate Galactose/analysis , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate Glucose/analysis , Uridine Diphosphate Glucose/metabolism
14.
Diabetologia ; 42(12): 1404-9, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10651257

ABSTRACT

AIMS/HYPOTHESIS: Although increased polyol pathway activity has been implicated in the pathogenesis of diabetic microangiopathy, the relation with diabetic macroangiopathy remains unclear. Galactose feeding is known to stimulate the polyol pathway and to develop abnormalities similar to those in diabetic microangiopathy. Our study was conducted to investigate whether an activation of polyol pathway by long-term treatment with galactose produced morphological changes in coronary arteries of dogs and the effect of an aldose reductase inhibitor, epalrestat, was also studied. METHODS: Dogs received either normal chow or chow containing 30% galactose with or without epalrestat given orally (20 or 50 mg x kg(-1)). After 44 months, morphometric analyses of coronary arteries were carried out and the galactitol contents in aortas were measured. RESULTS: The ratio of areas of the intimal layer to those of the medial layer, an indicator of intimal thickening, was statistically significantly increased in galactose-fed dogs compared with control dogs. Galactose-fed dogs had a remarkable accumulation of galactitol in their aortas. These morphological and biochemical deficits were reduced by treatment with epalrestat. CONCLUSION/INTERPRETATION: This report morphologically shows diabetes-like macrovascular abnormalities in galactosaemic animals, suggesting that polyol pathway hyperactivity is closely related to the development of diabetic macroangiopathy, which could be prevented by aldose reductase inhibition.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Coronary Vessels/pathology , Enzyme Inhibitors/pharmacology , Galactose/administration & dosage , Polymers/metabolism , Rhodanine/analogs & derivatives , Animals , Aorta/chemistry , Blood Glucose/analysis , Body Weight , Coronary Vessels/drug effects , Diabetic Angiopathies/chemically induced , Diabetic Angiopathies/pathology , Diabetic Angiopathies/prevention & control , Dogs , Enzyme Inhibitors/therapeutic use , Erythrocytes/chemistry , Galactitol/analysis , Galactitol/blood , Glycated Hemoglobin/analysis , Male , Rhodanine/pharmacology , Rhodanine/therapeutic use , Thiazolidines
15.
NMR Biomed ; 8(3): 133-8, 1995 May.
Article in English | MEDLINE | ID: mdl-8580001

ABSTRACT

We report 13C NMR measurements of the flux through aldose reductase in isolated rat sciatic nerve, and its inhibition by an aldose reductase inhibitor of the sulphonylnitromethane class. [1-13C] galactose was used as substrate, and the rate of production of [1-13C] dulcitol was measured. Quantitation required the use both of internal extracellular, and external, standards. The mean net forward flux (+/- SD) was 20 +/- 11 nmol/(mL nerve water)/min (n = 10). In the presence of the inhibitor, flux was reduced significantly (p < 0.001) to 13% of control. Since dulcitol is symmetrical, an estimate of the backward flux, to [6-13C] galactose, is also possible; under our conditions, this was negligible.


Subject(s)
Aldehyde Reductase/metabolism , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Nitroparaffins/pharmacology , Sciatic Nerve/enzymology , Sulfones/pharmacology , Aldehyde Reductase/antagonists & inhibitors , Animals , Carbon Isotopes , Galactitol/analysis , Galactitol/metabolism , Galactose/metabolism , In Vitro Techniques , Male , Rats , Rats, Wistar
16.
Exp Eye Res ; 58(2): 207-18, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8157113

ABSTRACT

It was reported previously that dietary ascorbate (ASC) delays the development of galactose-induced cataract in guinea pigs compared to the rate which is observed in ASC-deficient animals. Experiments were conducted to explore the possible mechanism of this phenomenon. Guinea pigs were fed for a period of up to 4 weeks either a normal diet (1 g ASC/kg diet) or a scorbutic diet (< 0.04 g ASC/kg diet) combined with 10% galactose in the drinking water. After 2 weeks, levels of ASC in animals on the scorbutic diet decreased by 95% in the aqueous humor and by 78% in the lens. Slit lamp examination showed that galactose-induced vacuoles in the lens equator formed at a significantly faster rate in the scorbutic animals. However, examination of biochemical parameters in whole lenses of the two groups of animals after 2 weeks showed no significant differences with regard to accumulation of galactose and galactitol, decreases in the levels of myoinositol, taurine and GSH or changes in cation concentrations. In order to examine possible regional changes in the lenses, various parameters were studied in the lens capsule-epithelium. On day 4, the capsule epithelia of scorbutic animals on a galactose diet had a content of galactitol two-and-a-half times higher than that of normal galactose-fed animals. Scorbutic conditions also intensified the loss of Na(+)-K+ ATPase activity in the lens capsule-epithelium caused by galactose feeding. Oxidized glutathione was not detectable in the lens capsule epithelia of any of the animals studied. Hexose monophosphate shunt activity was elevated in lenses of normal galactose-fed animals during the first hour of culture after death whereas lenses of scorbutic galactose-fed animals were not. Consistent with the in vivo findings, galactitol accumulation in dog lens epithelial cells exposed to 30 mM galactose was significantly inhibited by the presence of either ASC or dehydroascorbate (DHA) in the medium. Hexose monophosphate shunt activity in the cells was stimulated to two-and-a-half times its initial level by either 1 mM DHA or 30 mM galactose and slightly more than three-fold by a combination of the two challenges. The results suggest that decreased polyol accumulation in the lens epithelium of the normal galactose-fed guinea pig, which has a high level of ASC in the aqueous humor, accounts for the delay in onset of cataract compared to that for the ASC-deficient animal.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Ascorbic Acid/therapeutic use , Cataract/prevention & control , Animals , Aqueous Humor/chemistry , Cataract/chemically induced , Cells, Cultured , Dehydroascorbic Acid/pharmacology , Dogs , Female , Galactitol/analysis , Galactose , Glutathione/analysis , Guinea Pigs , Hydrogen Peroxide/analysis , Inositol/analysis , Lens, Crystalline/chemistry , Lens, Crystalline/drug effects , NADP/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism
17.
Biochem J ; 291 ( Pt 3): 889-94, 1993 May 01.
Article in English | MEDLINE | ID: mdl-8489515

ABSTRACT

Keratan sulphate chains from bovine articular cartilage were fully digested with keratanase from Pseudomonas sp. and the products were reduced with alkaline borohydride. The resultant fragments were fractionated on a Nucleosil 5SB column and the earliest eluting fucose-containing oligosaccharides were isolated. Structural analysis using 1H n.m.r. spectroscopy (600 MHz) showed the two least-charged species to have the following structure: GlcNAc(6S) beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S) beta 1- 3Gal beta 1-4GlcNAc(6S) beta 1-3Gal-ol and GlcNAc(6S) beta 1-3Gal beta 1- 4(Fuc alpha 1-3)GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S) beta 1-6(Gal beta 1- 3)GalNAc-ol. Both galactoses adjacent to the fucosylated N-acetylglucosamine residue are unsulphated. Therefore, it can be deduced from these structures that the presence of fucose on N-acetylglucosamine residues in keratan sulphates protects both of the adjacent unsulphated galactose residues from keratanase cleavage. This result has implications for the interpretation of keratanase fingerprints, because in articular cartilage keratan sulphates the keratanase-resistant blocks are not solely those with fully sulphated galactose residues, but also include the fucosylated sequences, which have unsulphated galactoses. It is, therefore, not possible to estimate their galactose sulphation or the size of the fully sulphated disaccharide-repeat sequences from keratan sulphates that contain fucose.


Subject(s)
Cartilage, Articular/chemistry , Fucose/analysis , Glycoside Hydrolases , Keratan Sulfate/metabolism , Magnetic Resonance Spectroscopy , Oligosaccharides/chemistry , beta-Galactosidase/metabolism , Acetylglucosamine/analysis , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cattle , Galactitol/analysis , Galactose/analysis , Molecular Sequence Data , Oligosaccharides/analysis , Oligosaccharides/metabolism , Pseudomonas/enzymology
18.
Diabetes ; 39(11): 1347-52, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2227109

ABSTRACT

In conflict with a previous report, we find that phenolic inhibitors of lipid peroxidation (butylated hydroxytoluene [BHT] and butylated hydroxyanisole [BHA]) do not have significant inhibitory effect on galactosemic cataract formation. This is consistent with the lack of enhancement of stable products of lipid peroxidation (measured by the thiobarbituric acid assay) in the lenses of galactosemic rats. This does not imply that oxidative stress plays no role in galactosemic cataract formation (indeed, we find that galactosemic lens homogenates contain increased amounts of an Fe2+ oxidant, possibly a peroxide), but rather that BHT- and BHA-inhibitable lipid peroxidation specifically has no role to play. In instances where drugs appear to inhibit galactosemic cataract formation, other effects caused by the drugs, e.g., inhibition of feeding or induction of general detoxification pathways, must be considered.


Subject(s)
Cataract/metabolism , Lipid Peroxidation/physiology , Animals , Antioxidants/pharmacology , Butylated Hydroxyanisole/pharmacology , Butylated Hydroxytoluene/pharmacology , Cataract/chemically induced , Cataract/prevention & control , Diabetic Retinopathy/chemically induced , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/prevention & control , Galactitol/analysis , Galactose/metabolism , Galactose/pharmacology , Glutathione/antagonists & inhibitors , Lens, Crystalline/chemistry , Oxidation-Reduction , Rats , Rats, Inbred Strains
19.
J Chromatogr ; 490(1): 1-8, 1989 May 05.
Article in English | MEDLINE | ID: mdl-2503529

ABSTRACT

The three commonly found hexitols mannitol, sorbitol and galactitol are well separated from each other and from myoinositol by gas chromatography as their butylboronate derivative on Dexsil-400, on a 1:1 mixture of OV-1 and OV-17, or on a DB-17 fused-silica capillary column. The method allows all four substances to be measured by autosampling electron ionization gas chromatography-mass spectrometry (GC-MS) in small tissue samples at organ concentrations as small as 5 mumol/kg wet mass in less than 4 min. Comparisons were made to determine the relative sensitivity of GC-MS and other detection methods. The order of sensitivity was electron ionization GC-MS greater than chemical ionization GC-MS greater than flame photometric detection using a boron-selective filter greater than hydrogen flame ionization detection.


Subject(s)
Galactitol/analysis , Inositol/analysis , Mannitol/analysis , Sorbitol/analysis , Sugar Alcohols/analysis , Animals , Chromatography, Gas , Erythrocytes/analysis , Galactitol/blood , Gas Chromatography-Mass Spectrometry , Inositol/blood , Mannitol/blood , Rats , Retina/analysis , Sciatic Nerve/analysis , Sorbitol/blood
20.
Curr Eye Res ; 8(5): 449-57, 1989 May.
Article in English | MEDLINE | ID: mdl-2500302

ABSTRACT

The cataractogenic effect of a galactose diet was studied in male and female pigs in relation to the daily galactose intake. Two experiments were performed. In the first, 10 male and 10 female pigs were maintained on a 5% galactose diet for 30 days; galactose was given either as pure sugar or as hydrolyzed whey. In the second experiment, 18 castrated male and 21 female pigs were fed a 25% galactose diet for 49 days. Galactose was given as whey, hydrolyzed whey or as an alternating diet. Lenses were analyzed for sugar-alcohols, glutathione and protein glycation, and compared to lenses of pigs on a control standard diet. The 5% galactose diet induced large accumulation of dulcitol in the lens, which was similar whether galactose was ingested alone or as hydrolyzed whey. Glutathione and inositol contents were slightly below control values only in males on the galactose alone diet, perhaps indicating initiation of a cataractogenic process. No change was observed in females. The lenses of females on control diet were different from those of control males: having decreased glutathione, inositol and sorbitol contents and higher protein glycation. The 25% galactose diet resulted in an approximately 10 times higher lens dulcitol accumulation and advanced lens damage as measured by loss of inositol and increase in protein glycation. These changes were more severe in males than in females. The data indicate that there is a relationship between total galactose intake and dulcitol accumulation.


Subject(s)
Cataract/etiology , Galactitol/analysis , Galactose/pharmacology , Glutathione/analysis , Lens, Crystalline/metabolism , Sugar Alcohols/analysis , Animals , Chromatography, Gas , Chromatography, High Pressure Liquid , Diet , Female , Inositol/analysis , Lysine/analogs & derivatives , Lysine/analysis , Male , Sex Factors , Sorbitol/analysis , Swine , Time Factors
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