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PubMed1977

Energetics of galactose, proline, and glutamine transport in a cytochrome-deficient mutant of Salmonella typhimurium.

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the beta-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca2+, Mg2+-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.

Adenosine Triphosphate↗
PubMed2001

Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli.

A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain. The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system. The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25). The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride. The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins. The purified adhesin showed adhesion to human erythrocytes in the presence of Ca(2+) (5 mM). The optimum temperature and pH for the hemadhesion activity was found to be 25 degrees C and 6.5, respectively. The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar. The fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose. The adhesin was found to contain one binding site per monomer for its specific sugar residue. The association constant and the free energy of binding were obtained as 3.98 x 10(5) M(-1) and -31.97 kJ/mol, respectively. The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta-D-galactosidase.

Adhesins, Escherichia coli↗
PubMed1997

Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose.

CAMPATH-1H is a recombinant humanized murine monoclonal immunoglobulin (IgG1) which recognizes the CDw52 antigen of human lymphocytes, and has been the subject of clinical trials for the treatment of non-Hodgkin's lymphoma and rheumatoid arthritis. Peptide mapping by liquid chromatography-mass spectrometry was used to confirm the predicted amino acid sequences and profile glycosylation for two CAMPATH isotypes expressed in a murine myeloma cell line (NS0) and a single isotype expressed in both Chinese hamster ovary (CHO) and NS0 lines. The three major glycoforms identified in CAMPATH are fucosylated biantennary structures, containing zero, one, or two galactose residues. Glycosylation of the IgG1 form of CAMPATH expressed in CHO cells is consistent with normal human IgG. However, IgG1 and IgG4 expressed in NS0 cells include two potentially immunogenic glycoforms which contain either one or two nonreducing terminal alpha-linked galactose residues. Oligosaccharide structures were characterized by a combination of tandem mass spectrometry, methylation analysis, and exoglycosidase digestion. The strategy used here is designed to be widely applicable to recombinant glycoproteins.

Alemtuzumab↗
PubMed1999

Purification and characterization of a lymph node sulfotransferase responsible for 6-O-sulfation of the galactose residues in 2'-fucosyllactose and other sialyl LewisX-related sugars.

A microsomal galactose-6-O-sulfotransferase (Gal-6-O-Stase) from porcine lymph nodes, able to transfer the sulfate group from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) onto 2'-fucosyllactose (2'-FL) and other sialyl LewisX (sLex)-related sugars, has been purified and characterized. The enzyme was purified to about 35,000-fold by a combination of conventional and affinity chromatographic steps. The purified enzyme preparation exhibited two protein bands at around 80-90 and 170 kDa on 7.5% SDS-PAGE under reducing conditions. Both of these protein bands always comigrated in the gel when peak fractions containing Gal-6-O-Stase activity from the 3',5'-ADP-agarose column were subjected to 6% SDS-PAGE under reducing conditions. These protein bands also showed similar binding patterns to WGA (wheat germ agglutinin), Con A (concanvalin A), and EBA (elderberry agglutinin). Similarly, when the enzyme preparation after the hydroxylapatite step was photolabeled with 8-azido-[32P]-PAPS, both 80-90 and 170 kDa protein bands were labeled in a specific manner. These results suggest a possible association of these two protein bands with the enzyme activity. The carbohydrate substrate specificity of this enzyme suggests that it is well suited to catalyze the sulphonation at the C-6 position of the galactose residues of oligosaccharides that are structurally similar to sLex. Furthermore, a survey of several porcine organs revealed that this enzyme was selectively expressed in lymphoid tissues such as lymph nodes (peripheral and mesenteric) and spleen. These findings suggest that this enzyme may be involved in the assembly of 3'-sialyl-6'-sulfo Lewisx, the major capping group of HEV-ligands for L-selectin.

Amino Acid Sequence↗
PubMed1999

A convenient microscale colorimetric method for terminal galactose on immunoglobulins.

A new approach for quantitative determination of terminal galactose (Gal) residues of immunoglobulins was developed by combining exoglycosidase digestion with the classical colorimetric estimation of reducing sugars. The ferricyanide colorimetric method was modified to increase the stability of the chromophore (Prussian blue) and adapted to determine the amount of terminal Gal residues present in immunoglobulins. The method involves the release of covalently bound Gal from immunoglobulins by Diplococcus pneumoniae beta-D-galactosidase (specific for beta(1,4) linked galactose), removal of the glycoprotein and enzyme from the reaction mixture by heat denaturation or ethanol precipitation, followed by colorimetric measurement of the released sugar using the ferricyanide assay. The ferricyanide method was modified to enhance the solubility and stability of the chromophore by increasing the concentration of aqueous sulfuric acid and sodium dodecyl sulfate (SDS). The linear range of the modified method was from approximately 11 to 111 microM Gal. Typical variation in assay results was on the order of 5%. Using the modified method, the terminal Gal content of a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) expressed in Chinese hamster ovary (CHO) cells was determined and evaluated for batch-to-batch consistency. The method was used to optimize pH, time, temperature, and enzyme concentration for beta-galactosidase digestion for maximal release of terminal Gal residues from rIgG.

Animals↗
PubMed1993

Superoxide production by neutrophils from diabetics and normal subjects in response to glucose and galactose.

Glucose added to the medium was found to enhance superoxide production by isolated circulating neutrophils from both diabetic and normal subjects, but quantitatively the enhancement decreased from 4 to 50 mmole/liter. Galactose up to 50 mmole/liter had no effect on superoxide production in cells from the control subjects, but appeared to depress it in those from diabetics. No correlations were found between indices of the degree of hyperglycemia (plasma glucose and hemoglobin A1c) and the magnitude of the respiratory burst in cells from diabetics. When the isolated cells from normal and diabetic subjects were restored to a medium containing glucose at the original concentration in plasma at phlebotomy, the rate of superoxide production was approximately doubled in every case and there was no significant difference between diabetic and normal cells. Preincubation of cells for 1 hr in the presence of 0-50 mmole/liter glucose or galactose prior to activation had no significantly depressant effect on the respiratory burst except at 50 mmole/liter glucose in diabetic cells. It is concluded that circulating neutrophils from the diabetic population under the conditions studied are just as competent as control cells in their ability to sustain superoxide production over a wide range of energy availability.

Adult↗
PubMed1999

Glucose signaling in yeast is partially mimicked by galactose and does not require the Tps1 protein.

Glucose produces multiple effects in Saccharomyces cerevisiae, as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a "sensor" in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed a tps1 hxk2/tps1 HXK2 strain which, at difference of a tps1 strain, grows on glucose, and, at difference of a tps1 hxk2 strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.

Cyclic AMP↗
PubMedDate not supplied

Molecular basis of disorders of human galactose metabolism: past, present, and future.

Molecular cloning and characterization of all three human galactose-metabolic genes have led to the identification of a number of mutations which result in three forms of galactosemia which are caused by kinase (GALK), transferase (GALT), or epimerase (GALE) deficiency. We review here recent developments in the molecular characterization of all three disorders of human galactose metabolism. Recent progress in the biochemical and/or structural analyses of the GALT and GALE proteins has complemented human mutational studies. Interestingly, genotype/phenotype correlations have been modest as in some other Mendelian disorders. We discuss possible reasons for this apparent paradox. Finally, we note the panethnic nature of galactosemia and suggest a hypothesis for it.

Galactokinase↗
PubMed1995

Galactose-containing glycoconjugates of the iris, the aqueous outflow passages and the cornea in capsular glaucoma. A lectin histochemical study.

BACKGROUND: The aim of the study was to compare galactose-containing glycoconjugates of the iris, the aqueous outflow passages and the cornea with exfoliation material in capsular glaucoma. METHODS: Six formalin-fixed, paraffin-embedded human eyes with capsular glaucoma and six control eyes were studied by using a panel of 11 biotinylated lectins to galactose- and N-acetylgalactosamine-containing glycoconjugates. RESULTS: The Gal (beta 1-->3) GalNAc-reactive lectins peanut agglutinin (PNA) and Bauhinia purpurea alba agglutinin (BPA) and the Gal (beta 1-->4)GlcNAc-reactive lectins Ricinus communis agglutinin (RCA-I) and Phaseolus vulgaris erythroagglutinin (PHA-E) gave the strongest label with exfoliation material. Lectin binding to the iris was variable. The binding of PNA, BPA, RCA-I, Erythrina cristagalli agglutinin (ECA), PHA-E and Glycine max agglutinin (SBA) to the subendothelial region of iris blood vessels closely resembled their binding to exfoliation material. RCA-I and PHA-E bound moderately to the aqueous outflow passages. The surface of the corneal epithelium showed positive reaction with most lectins studied, but the keratocytes reacted with RCA-I and PHA-E only. Neuraminidase pretreatment generally increased the reaction intensity. CONCLUSIONS: The findings suggest that the glycoconjugate composition of exfoliation material in the classical locations along the anterior and posterior chamber closely resembles that in the subendothelial region of iris blood vessels.

Aged↗
PubMed1980

The distribution and fate of 3H-glucose and 3H-galactose in oocytes of Oryzias latipes: Formation of the cortical alevoli.

The distribution and fate of 3H-glucose and 3H-galactose and the formation of the cortical alveoli in the oocytes of the teleost Oryzias latipes were investigated. Fish were given intraperitoneal injections of either labeled hexose, and the relative amounts of each were measured in autoradiographs prepared for light and electron microscopy. There were significant differences in the amounts of each hexose in the oocytes at all time intervals studied. During the earlier time periods, the oocyte incorporates more 3H-glucose than 3H-galactose in both the cytoplasmic and cortical alveoli compartments; however, the overall rate of formation of the cortical alevoli appears to be similar for the two tracers. Electron microscopic autoradiographs reveal the participation of cytoplasmic elements, e.g., endoplasmic reticulum and Golgi apparatus, in the formation of the cortical alveoli. The chorion does not incorporate significant amounts of either tracer, while the yolk granules contain small but significant amounts.

Animals↗
PubMed1993

Galactose-binding lectins as markers of pregnancy-related glycoproteins.

Protein extracts from pregnant mouse endometria were compared with those obtained from non-pregnant and pseudopregnant mice to detect early pregnancy-specific galactose-rich glycoproteins. Gradient gel electrophoresis combined with lectin overlay and lectin histochemistry were used to identify Ricinus communis I (RCA-I), R. communis II (RCA-II) and Cytisus scoparius (CSA) lectin binding glycoproteins. Using this approach, galactose-rich glycoproteins were identified that were maximally expressed in the estrus phase of non-pregnant endometria and also those that had peak expression in pregnancy. Lectin histochemistry revealed pregnancy related changes in three portions of mouse endometrium: endometrial glands, luminal epithelium and its basement membrane. Two major glycoproteins (RCA-I reactive 64 kDa and RCA-II reactive 35 kDa) were specifically expressed in peri-implantation endometrium on days 3 and 4 of pregnancy. The appearance of these glycoproteins during the period of the implantation window in mouse suggests that they could serve as markers of uterine receptivity to the implanting blastocyst.

Alkaline Phosphatase↗
PubMed1977

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae.

Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80s-1 and GAL80s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80s-1 GAL81-1 strain was inducible and the GAL80s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.

Carboxy-Lyases↗
PubMed1978

Effect of galactose treatment in the puffing pattern of Chironomus thummi Balbiani rings.

Galactose feeding of Chironomus thummi larvae induces the regression of Balbiani ring c (BRc) and the full expansion of BRb, both localized in the IV salivary gland chromosome. This effect coincides with that described on BR2 and BR1 of Ch. pallidivittatus and Ch. tentans. The puffing changes of BRb and BRc throughout develoment have been studied and also show identical variations as in BR1 and BR2 of Ch. pallidivittatus and Ch. tentans. The similar behaviour of BRb and BR1, and of BRc and BR2 respectively after galactose treatment and throughout development strongly suggests that these BRs play the same physiological role in the three Chironomus species, with BRb = BR1 and BRc= BR2.

Animals↗
PubMed1983

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4.

The GAL4 gene positively regulating the expression of the gene cluster GAL7-GAL10-GAL1 in the yeast Saccharomyces cerevisiae was isolated for its ability to suppress a recessive mutation in that gene. When the isolated gene was incorporated into a multi-copy plasmid, the GAL cluster genes in the host chromosome partially escaped the normal control; a yeast that harbors the plasmid bearing the GAL4 gene synthesized the galactose-metabolizing enzymes encoded by the GAL cluster genes at a low but significant level in the absence of galactose. If the GAL7 gene was amplified along with GAL4 on the multi-copy plasmid, the constitutive synthesis of Gal-1-P uridylyl transferase encoded by GAL7 was further pronounced and the enzyme activity reached the level of the fully induced wild-type yeast. Such an escape synthesis of the GAL enzymes was not detected if GAL4 or both GAL4 and GAL7 were carried by a single-copy plasmid. The results suggest that the escape synthesis of GAL enzymes observed in the GAL4-amplified yeast was a consequence of overproduction of the GAL4 protein. The GAL80 gene negatively regulating the GAL cluster genes was also isolated, and when amplified together with GAL4, no escape synthesis of the GAL enzymes was observed, suggesting that the balanced synthesis of two regulatory proteins was essential to maintain the repressed state of the GAL cluster genes.

DNA, Fungal↗
PubMed1984

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. II. The isolation and dosage effect of the regulatory gene GAL80.

The galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.

Base Sequence↗
PubMed1990

Sorbinil does not prevent galactose-induced glomerular capillary basement membrane thickening in the rat.

We investigated the role of the polyol pathway in the pathogenesis of glomerular basement membrane thickening in galactosaemic rats, an animal model that develops basement membrane lesions comparable to those of human diabetic subjects. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for nine months developed significant glomerular basement membrane thickening by comparison with rats on a control test diet (p = 0.008). However, addition of an aldose reductase inhibitor, sorbinil (250 mg/kg diet), to the galactose diet did not prevent the increase in glomerular basement membrane thickness. Furthermore, by using a quantitative electron microscopic immunogold technique, we examined biochemical alterations in the composition of glomerular basement membranes in this animal model. The labelling density (comparable to relative concentration) of collagen type IV in thickened glomerular basement membranes of galactosaemic animals was significantly increased by comparison to those of control rats (p = 0.015). However, there was no significant difference in labelling densities of laminin and heparan sulfate proteoglycan core protein of these animals. Thus, our results indicate that an increase in glomerular basement membrane thickness accompanied by an increase in the labelling density of collagen type IV occurs in the galactosaemic rats, but this thickening is not prevented by sorbinil at the dose used in this experiment. Our study raises the strong possibility that glomerular basement membrane thickening in galactosaemic rats may not be due to excessive polyol pathway activity.

Aldehyde Reductase↗
PubMed1986

Glycoconjugate with terminal alpha galactose. A property common to basal cells and a subpopulation of columnar cells of numerous epithelia in mouse and rat.

Glycoconjugates associated with the basal cell layer of various types of epithelia in the mouse and rat were examined histochemically with a battery of lectin-horseradish peroxidase (HRP) conjugates of differing sugar binding specificities. Basal cells in paraffin sections of composite tissue blocks stained with an isolectin from Griffonia simplicifolia (GSA I-B4) specific for terminal alpha-galactose residues but failed to react with the other lectins. Basal cells in epithelium lining striated and excretory ducts of salivary and lacrimal glands, tongue, esophagus, trachea, renal calyx, ureter, urinary bladder, urethra, epididymis and vas deferens stained selectively and intensely for content of a glycoconjugate with terminal alpha-galactose. This galacto-conjugate appeared associated with the plasmalemma of basal cells. Basal cells with a galactocalyx formed an intermittent to continuous layer generally increasing in prevalence distally in glandular duct systems. A minor population of pyramido-columnar cells with cytosolic GSA I-B4 reactivity occurred in striated ducts and appeared less numerous in intralobular excretory ducts and more prevalent in extraglandular ducts. In trachea and renal pelvis, the GSA I-B4 positive cell profiles ranged from low cuboidal to tall pyramidal in contour, but the latter appeared not to reach the lumen. In contrast, no GSA I-B4 positive basal cells were seen in any segment of the pancreatic or bile ducts or in the epithelium of the gastrointestinal tract. These findings suggest that the basal cells found in similar sites in different epithelia and possessing in common a unique alpha-galactoconjugate may function in a manner common to all and not simply in providing progenitor cells for epithelial renewal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

The histochemistry of galactose residues of complex carbohydrates as studied by peroxidase-labeled Ricinus communis agglutinin.

A peroxidase-labeled Ricinus communis agglutinin diaminobenzidine (PO-RCA-DAB) procedure has been utilized to determine the light microscopic localization of galactose residues of complex carbohydrates in a variety of tissues from different vertebrate species. In the same tissues, the localization of mannose and glucose residues was studied by means of a concanavalin A peroxidase diaminobenzidine (Con A-PO-DAB) procedure for comparison. The distribution pattern of galactose residues was found to be in distinct contrast with that of mannose and glucose residues in connective and epithelial tissues such as those involved in the umbilical cord, cartilage, aorta, comb, thyroid gland, submaxillary gland, duodenum, jejunum, colon and gill of various vertebrates. The results of the present study indicate that the PO-RCA-DAB procedure is useful in providing a means of dividing periodic acid-Schiff reactive neutral complex carbohydrates into subgroups in comparison with the intensity of their Con A-PO-DAB reaction in the histochemistry of complex carbohydrates.

Animals↗