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PubMedDate not supplied

Arylsulfatase-cytochemical localization in lenses of normal and galactose-fed rats.

Our laboratory is involved in studying the mechanism of repair in the ocular lens. As lysosomal enzymes have been shown to play an important role in tissue repair, we have been investigating the status of lysosomal enzymes, such as acid phosphatase and arylsulfatases, in the normal and injured lens. In the present investigation, we have examined the presence, distribution, and possible role of arylsulfatases (E.C. 3.1.6.1) in lenses of normal and galactose-fed rats. Arylsulfatases were localized in lenses using the cytochemical procedure described by Hopsu-Havu and Helminen (1974) using p-nitrocatechol sulfate as a substrate and then examined at the ultrastructural level. The reaction product resulting from arylsulfatase activity was mainly localized in the epithelial cells with very little activity in the cortical fibers. The intracellular activity was confined to lysosomes. Some extracellular activity was visible in the intercellular regions in both the epithelium and superficial cortex. With the progression of galactose-induced lesion in the epithelium the number of lysosomes exhibiting enzyme reaction product was found to have increased, and the lysosomes closely abutted the capsule. The biochemical assay indicated a considerable increase in the activity of arylsulfatases with the continuation of a galactose diet. The possible role of arylsulfatases in the normal and cataractous lens is discussed.

Animals↗
PubMed1987

Incorporation of 14C-galactose into gangliosides of rabbit lens.

Rabbit lenses were incubated in organ culture with 14C-galactose for 6, 12 and 20 hours. Gangliosides were extracted using the Folch-Suzuki method, purified by dialysis and analyzed by thin-layer chromatography. Six radioactive bands, including the origin, were observed. Tentative identification of these bands as N-acetylneuraminylgalactosylglucosylceramide (GM3), N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM2), galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)- galactosylglucosylceramide (GM1), N-acetylneuraminylgalactosyl-N- acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD1a), N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N- acetylneuraminyl-N-acetylneuraminyl)-galactosylglucosylceramide (GT) was made by comparison with authentic standard gangliosides. Galactose incorporation into GM3 and GM2 increased during the first 12 hours but decreased during the period from 12 to 20 hours. GD1a and GT incorporated the greatest amount of label during the period from 12 to 20 hours. Incorporation of labeled galactose into GM1 was nearly constant during this time period. Specific activities for GM1, GM3 and GT were nearly the same at 6 hours and were about half those of GM2 and GD1a for the same time period.

Animals↗
PubMed1998

Inhibition of insulin release by alpha- and beta-D-galactose pentaacetate.

Several monosaccharide polyacetate esters display higher biological efficiency than the corresponding unesterified carbohydrates, this being attributable to their capacity to cross the plasma membrane without requiring the intervention of a specific carrier system and to their subsequent intracellular esterase-catalyzed hydrolysis. Some of these esters, however, also exert a direct effect upon a receptor system apparently displaying analogies with that involved in the identification of bitter compounds by taste buds. For instance, under suitable experimental conditions, esters of non-nutrient monosaccharides, such as L-glucose, D-mannoheptulose or 2-deoxy-D-glucose, unexpectedly stimulate insulin release. The present work reveals that alpha-D-galactose pentaacetate and, to a lesser extent, beta-D-galactose pentaacetate both inhibit leucine-induced insulin release in rat pancreatic islets. This indicates that the postulated receptor system displays anomeric specificity and may participate in a negative modulation of insulin secretion. It is proposed that advantage could be taken of the negative insulinotropic action of D-galactose pentaacetate esters to prevent excessive insulin release in pathological situations.

Animals↗
PubMed1978

Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.

Animals↗
PubMed1975

Sulfate and galactose metabolism in differentiating ciliary body and iris epithelia: autoradiographic and ultrastructural studies.

Immature and adult rat eyes were bisected and incubated with 35-SO4 and 3-U-galactose in short-term pulse-chase experiments. Autoradiographs (ARG) of the tissue revealed that very little sulfate is incorporated by the peripheral neural retina, the pigment epithelia of the retina, ciliary body, or iris. The inner, inverted optic cup cells at the ora serrata, i.e., those that are undergoing differentiation into the unpigmented epithelium of the ciliary body, incorporate large amounts of 35-sulfate into "fixable" macromolecules. The sulfate label is chased from the apically located Golgi apparatus to the basal surface of these cells within one hour. Ultrastructurally, these cells are beginning to develop lateral and basal invaginations of the plasma membrane characteristic of the adult secretory epithelial cells. Electron microscopic ARG show label associated with the plasma membranes. The sulfated macro-molecules at this site appear to be glycolipids and glycoproteins rather than glycosaminoglycans. The preferential synthesis of these macromolecules and their placement at the cellular site of aqueous humor production suggests a role for these sulfated substances in establishing, and perhaps maintaining, that secretory process. 3-H-galactose was incorporated into "fixable" macromolecules to some degree by all the neuroepithelial cells. After chase incubation, ARG showed a high concentration of label in differentiated retinal pigment epithelium (RPE), but not in undiffenentiated peripheral RPE. Ciliary body unpigmented and pigment epithelium, and iris muscle cells incorporate galactose, but to a lesser degree than either RPE or corneal endothelial cells.

Age Factors↗
PubMed1975

[Effect of various coumarins on the intestinal absorption of galactose in vivo (author's transl)].

The effect of various coumarins on the active transport of galactose by small intestine in chick and rat was studied, using the in vivo technique of sucessive absorptions. A 10(-4) M concentration of the different coumarins inhibits the absorption of galactose in the chick. This effect persists in successive absorptions without coumarin. In rat, inhibition of galactose active transport by coumarins was observed at 10(-3) M concentration.

Animals↗
PubMed1976

Transformation of human peripheral lymphocytes by galactose oxidase.

Human peripheral lymphocytes can be transformed by treatment with galactose oxidase alone. Prior treatment with neuraminidase enhances this effect. The aldehyde blocking agents thiocarbohydrazide, hydroxylamine, dimedone, and sodium borohydride block transformation when they follow, but not when they precede, galactose oxidase treatment. Thus, as is the case for periodate-induced lymphocyte transformation, the formation of free aldehyde at the cell surface would seem to be a critical event in the triggering of transformation by this agent. The degree of transformation is highly variable from individual to individual, and also for the same donor at different times. However, the lymphocytes of some people give a consistently poor response to galactose oxidase. Similar results have been obtained for periodate-induced transformation of human lymphocytes, but to this date this is unexplained.

Alcohol Oxidoreductases↗
PubMed1976

Test for alcoholic cirrhosis by conversion of [14C]- or [13C]galactose to expired CO2.

After ingestion of galactose (10 g per m2) labeled with 14C or 13C, breath was collected from subjects at intervals for 4 hr followed by measurement of 14CO2 by liquid scintillation counting or of 13CO2 by mass spectrometry. Nine subjects without liver disease and 21 "cirrhotic" patients were tested with 14C; 8 control subjects and 4 patients with diagnosis of cirrhosis were tested with 13C. The mean rates of expiration of labeled CO2 by the patients with "cirrhosis" were one-third to one-half of mean normal rates during the first 90 min. The time of peak concentration of tracer CO2 for cirrhotic patients (150 to 180 min) was later than for normal subjects (90 to 120 min). There was distinctly greater separation between control and liver disease groups by test of 14CO2 radioactivity at 1 hr than by serum alkaline phosphatase, total bilirubin, and transaminase, but only slightly better separation than by serum albumin concentration (which was highly correlated with 14CO2 output). The [14C]galactose test is simpler than the standard intravenous galactose tolerance test, and , like the latter, appears superior to some other tests for recognition of cirrhosis. The use of 13C provides an example of a new direction for clinical application of this stable, nonradioactive nuclide.

Adult↗
PubMed2003

The N314D polymorphism of galactose-1-phosphate uridyl transferase does not modify the risk of ovarian cancer.

It has been proposed that high levels of galactose consumption increase the risk of ovarian cancer. Galactose levels are determined, in part, by the galactose-1-phosphate uridyl transferase gene (GALT). The N314D allele of the GALT gene has been associated with low GALT activity and with an increased risk of ovarian cancer. We screened for the presence of the N314D GALT allele in 891 incident cases of epithelial ovarian cancer and in 364 unaffected female controls. No significant difference in the prevalence of the N314D allele was observed between the cases (18.1%) and the controls (18.7%). The odds ratio associated with the presence of one N314D allele was 0.94 (95% confidence interval (CI), 0.68-1.3; P = 0.70), and the odds ratio associated with two N314D alleles was 1.62 (95% CI, 0.34-7.7; P = 0.54). Subanalyses of the cases by histological type, by age, by ethnic group, by family history, and by BRCA1/2 mutation status did not reveal any significant associations. We conclude that the GALT N314D allele does not predispose to epithelial ovarian cancer.

Adult↗
PubMed1992

Detection of the tumor marker D-galactose-beta-(1-->3)-N-acetyl-D-galactosamine in colonic cancer and precancer.

The enzyme D-galactose oxidase (GO) oxidizes the carbon-6 position of the hydroxyl groups of galactose-N-acetyl galactosamine, which are commonly present in colon cancer cells and in rectal mucin of patients with colon cancer. We have studied the marker disaccharide galactose and N-acetylgalactosamine on tissue sections by the GO-Schiff reagent in normal, preneoplastic, and neoplastic human colorectal epithelial and compared it with peanut agglutinin reactivity. Fifty-seven (81.4%) of 70 carcinomas, 83.3% (10/12) of precancerous lesions, 50% (10/20) of the mucosa remote from cancer, and 58.1% (25/43) of the mucosa immediately adjacent to cancer showed a positive reaction with GO-Schiff, but the normal control mucosa was nonreactive. The GO-Schiff reagent showed an intense reactivity with mucinous adenocarcinomas and poorly differentiated adenocarcinomas. An intense reactivity was also seen in the intracellular mucus of abnormal dilated crypts (polyps, five of five cases; colitis, four of seven cases; and remote mucosa, 10 of 20 cases). Comparison of peanut agglutinin and GO-Schiff reactivity showed that the nonmucinous (glandular) adenocarcinomas less frequently reacted with the GO-Schiff sequence. Our results showed that the carbohydrate moiety detected by the two techniques may not necessarily be the same, warranting further biochemical analysis. Meanwhile, the data suggested that, like peanut agglutinin, the GO-Schiff sequence has the potential to identify the tumor marker either at the tissue level or by a mucin test for screening colorectal cancer or precancer.

Adenocarcinoma↗
PubMed2003

Inhibiting effects of Achyranthes bidentata polysaccharide and Lycium barbarum polysaccharide on nonenzyme glycation in D-galactose induced mouse aging model.

OBJECTIVE: To investigate the inhibiting effects and mechanism of achyranthes bidentata polysaccharide (ABP) and lycium barbarum polysaccharide (LBP) on nonenzyme glycation in D-galactose induced mouse aging model. METHODS: Serum AGE levels were determined by AGE-ELISA, MTT method was used to determine lymphocyte proliferation, IL-2 activity was determined by a bioassay method. Spontaneous motor activity was used to detect mouse's neuromuscular movement, latency of step-through method was used to examine learning and memory abilities of mouse, colormetric assay was used to determine hydroxyproline concentration in mouse skin, pyrogallol autoxidation method was used to determine superoxide dismutase (SOD) activity of erythrocytes. RESULTS: Decreased levels of serum AGE, hydroxyproline concentration in mouse skin and spontaneous motor activity in D-galactose mouse aging model were detected after treated with ABP or LBP, while lymphocyte proliferation and IL-2 activity, learning and memory abilities, SOD activity of erythrocytes, were enhanced. CONCLUSIONS: ABP and LBP could inhibit nonenzyme glycation in D-galactose induced mouse aging model in vivo and ABP has a better inhibiting effect than LBP.

Achyranthes↗
PubMed1992

Galactose oxidase of Dactylium dendroides. Gene cloning and sequence analysis.

The gaoA gene, encoding the secreted copper-containing enzyme galactose oxidase, has been isolated from the Deuteromycete fungus Dactylium dendroides. Degenerate oligonucleotide primers were designed from amino acid sequence data for use in the polymerase chain reaction. A 1.4-kilobase DNA fragment amplified from genomic DNA was used to screen a genomic library constructed in ZAP. A strongly hybridizing clone was rescued as a pBluescript derivative, pGAO9, by in vivo excision. The sequence of 3466 nucleotides of pGAO9 insert DNA was determined by progressively designing sequencing primers. The translation product of the single long open reading frame matches the available galactose oxidase peptide sequence data, which represents 42% of the residues in the protein. The mature enzyme has 639 residues, which have been assigned to a 1.7-A electron density map (Ito, N., Phillips, S. E. V., Stevens, C., Ogel, Z. B., McPherson, M. J., Keen, J. N., Yadav, K. D. S., and Knowles, P. F. (1991) Nature 350, 87-90). The gene lacks introns and encodes an mRNA of approximately 2.5 kilobases with three transcription initiation start points at least 324 nucleotides upstream of the translation start site. Multiple ATG codons are present between the transcription initiation region and the start of the mature protein; two in-frame ATGs could encode the initiating Met residue to give proteins with 89 or 41 residue N-terminal leader peptides. The shorter potential leader has N-terminal features characteristic of a secretion signal sequence and may also contain a pro-sequence processed by an enzyme specific for a monobasic (arginine) cleavage site, as proposed for other fungal genes. The codon bias of gaoA is characteristic of other filamentous fungal genes. No significant homologies exist between galactose oxidase and other protein sequences available in data bases.

Amino Acid Sequence↗
PubMedDate not supplied

[Influence of glucose and galactose on the morphology and biological properties of Yersinia pseudotuberculosis].

When cultivated in the presence of glucose, irrespective of temperature and the degree of aeration, Y. pseudotuberculosis cells have the ovoid form, constant size and low hydrophobic properties of their surface. Meanwhile the characteristics of the bacteria grown in the medium, carbohydrate-free or with galactose added, essentially depend on the conditions of medium aeration. Under the conditions of intensive stirring at both temperatures these bacteria acquire the coccoid form, not typical for Yersinia, they have a smaller area (approximately 2 times) and more hydrophobic surface in comparison with the cells grown in the presence of glucose. Under stationary conditions the differences between the cells, cultivated in the presence of galactose and glucose, in form and area disappear, but the differences in the hydrophobic properties of the surface are retained. As revealed in this study, the cells grown in the presence of galactose and under the conditions of intensive medium stirring, in contrast to those grown with glucose, have 1.5-fold greater invasive activity, irrespective of aeration conditions, eightfold greater resistance to ampicillin and twofold greater resistance to streptomycin and erythromycin.

Air↗
PubMed2006

Structure of asteroid bodies in the vitreous of galactose-fed dogs.

PURPOSE: Asteroid hyalosis is a condition in which white spherical particles (asteroid bodies) are suspended in the vitreous, usually in the dependent part of the vitreous. These particles seldom cause serious visual symptoms; however, their presence can be a source of irritation. It has been suggested, but not confirmed, that asteroid hyalosis may be associated with systemic diseases such as diabetes, hyperlipidemia, or hypertension. Studies indicate that these particles are composed of lipid material and calcium; however, the specific composition and structure of asteroid bodies remains unknown. We have observed that asteroid hyalosis occurs in galactose-fed dogs, and this represents the first animal model which consistently forms this vitreal condition. The purpose of this study was to identify the main structural component of the asteroid bodies present in the vitreous of these dogs. METHODS: Vitreous humor containing asteroid bodies was collected and frozen from long-term galactose-fed beagles and from age-matched normal controls where asteroid bodies were absent. A portion of the frozen vitreous was sent out for elemental analysis. Thawed vitreous samples were sonicated with HPLC grade water and the aqueous layer was extracted three times with chloroform and then three times with n-butanol. The three organic layers from each extraction were combined and the solvents removed in vacuo. The residue from each extraction was re-dissolved in methanol and analyzed by electrospray ionization mass spectrometry (ESI-MS). RESULTS: Vitreous-containing asteroid bodies had significantly higher levels of calcium and phosphorus. Negative mode ESI-MS analysis of the n-butanol extracts from vitreous samples with and without asteroid bodies were similar with both containing a predominant peak with a mass to charge ratio (m/z) of 538.4. However, similar analyses of the chloroform extracts indicated that three peaks with m/z values of 547.1, 690.5, and 1430.6 were present only in vitreous samples containing asteroid bodies. Subtraction analysis indicated that the m/z of 690.5 peak corresponded to the main component present. This peak was identified and confirmed to be the quasimolecular ion of 1,2-dipalmitoyl-glycero-3-phosphoethanolamine (DPPE). CONCLUSIONS: Based on the current belief that asteroid bodies are composed of lipid-calcium complexes, we propose that the main component of asteroid hyalosis in the galactose-fed dog is a quasimolecular ion of DPPE in which two molecules of DPPE are complexed through their phosphates groups with calcium.

Animals↗
PubMed1992

Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.

Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.

Animals↗
PubMedDate not supplied

Metabolic analysis of galactose toxicity in Escherichia coli with 2-deoxygalactose as the probe.

Biochemical basis of galactose toxicity has been studied in gal T mutants (CGSC 4974) using 2-deoxygalactose, a non-metabolizable analogue of galactose, as the probe. It is found that biochemical features of toxicity in wild type cells either with 2-deoxygalactose or with 2-deoxyglucose are very similar to the picture obtained with gal T mutants and the observed bacteriostasis is probably due to futile phosphorylation and not due to any specific inhibitory effect of phosphorylated galactose.

Escherichia coli↗
PubMed1976

[Screening of newborns for inborn errors of galactose metabolism. Methods and results].

Three inborn errors of galactose metabolism as known today. Only two of them cause illness: the deficiencies of galactokinase and of galactose-l-phosphate uridyltransferase. Both can be detected through mass screening of newborns and are amenable to a simple and successful dietary treatment. Mass screening of newborns for disorders of galactose metabolism can be performed efficiently and inexpensively if it is incorporated in the general newborn screening program.

Biological Assay↗
PubMed1991

Effect of DTNB on rat intestinal galactose transport in vivo.

The effect of the non-penetrating reagent of -SH groups: acid 5,5'-dithiobis (2-nitrobenzoic), (DTNB), on 1 mM galactose absorption in rat intestine in vivo has been studied. DTNB inhibits sugar absorption in about 35%, which is due to an action on the mediated transport component, but without affecting the diffusional passive one. Consequently it does not modify galactose absorption in the presence of 0.5 mM phlorizin or that of the non-transportable sugar 2-deoxy-glucose. Galactose transport inhibition appears after a not longer than 5 min preexposure period and it remains constant at least up to 30 min. The inhibitory effect does not vary between 0.1 and 1 mM DTNB and it reverses completely with 0.5 mM dithioerythritol. Protection by excess of substrate has not been observed. Results show that DTNB affects sulfhydryl groups very probably located at the luminal side and related to the proteins of the cotransport system.

Animals↗