Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 46
Filter
Add more filters










Publication year range
1.
Carbohydr Res ; 364: 22-7, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-23147042

ABSTRACT

We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.


Subject(s)
N-Acetylglucosaminyltransferases/antagonists & inhibitors , Uridine Diphosphate Galactose/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate/chemical synthesis , Animals , CHO Cells , Cricetinae , Diphosphates/chemistry , Enzyme Activation , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Galactosephosphates/chemistry , Magnetic Resonance Spectroscopy , N-Acetylglucosaminyltransferases/chemistry , Recombinant Proteins/chemistry , Solvents/chemistry , Tetrazoles/chemistry , Time Factors , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate Galactose/chemistry , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/pharmacology
3.
Am J Physiol Gastrointest Liver Physiol ; 296(4): G923-30, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19164486

ABSTRACT

P2Y receptors have been reported to modulate gastrointestinal functions. The newest family member is the nucleotide-sugar receptor P2Y14. P2ry14 mRNA was detected throughout the rat gut, with the highest level being in the forestomach. We investigated the role of the receptor in stomach motility using cognate agonists and knockout (KO) mice. In rat isolated forestomach, 100 microM UDP-glucose and 100 muM UDP-galactose both increased the baseline muscle tension (BMT) by 6.2+/-0.6 and 1.6+/-0.6 mN (P<0.05, n=3-4), respectively, and the amplitude of contractions during electrical field stimulation (EFS) by 3.7+/-1.7 and 4.3+/-2.5 mN (P<0.05, n=3-4), respectively. In forestomach from wild-type (WT) mice, 100 microM UDP-glucose increased the BMT by 1.0+/-0.1 mN (P<0.05, n=6) but this effect was lost in the KO mice (change of -0.1+/-0.1 mN, n=6). The 100 microM UDP-glucose also increased the contraction amplitude during EFS in this tissue from the WT animals (0.9+/-0.4 mN, P < 0.05, n=6) but not from the KO mice (0.0+/-0.2 mN, n=6). In vivo, UDP-glucose at 2,000 mg/kg ip reduced gastric emptying in rats by 49.7% (P<0.05, n=4-6) and in WT and KO mice by 56.1 and 66.2%, respectively (P<0.05, n=7-10) vs. saline-treated control animals. There was no significant difference in gastric emptying between WT and KO animals receiving either saline or d-glucose. These results demonstrate a novel function of the P2Y14 receptor associated with contractility in the rodent stomach that does not lead to altered gastric emptying after receptor deletion and an ability of UDP-glucose to delay gastric emptying without involving the P2Y14 receptor.


Subject(s)
Gastric Emptying/drug effects , Receptors, Purinergic P2/metabolism , Uridine Diphosphate Glucose/pharmacology , Animals , Dose-Response Relationship, Drug , Gene Expression Regulation/physiology , Lac Operon/genetics , Lac Operon/physiology , Mice , Mice, Knockout , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y , Uridine Diphosphate Galactose/pharmacology
4.
Org Lett ; 11(1): 193-6, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19067595

ABSTRACT

UDP-galactopyranose mutase (UGM or Glf), which catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose, is implicated in the viability and virulence of multiple pathogenic microorganisms. Here we report the synthesis of high-affinity ligands for UGM homologues from Klebsiella pneumoniae and Mycobacterium tuberculosis. The potency of these compounds stems from their ability to access both the substrate binding pocket and an adjacent site.


Subject(s)
Enzyme Inhibitors/pharmacology , Galactose/analogs & derivatives , Intramolecular Transferases/antagonists & inhibitors , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate/analogs & derivatives , Catalysis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Galactose/chemical synthesis , Galactose/chemistry , Galactose/pharmacology , Klebsiella pneumoniae/enzymology , Ligands , Mycobacterium tuberculosis/enzymology , Uridine Diphosphate/chemical synthesis , Uridine Diphosphate/chemistry , Uridine Diphosphate/pharmacology , Uridine Diphosphate Galactose/chemical synthesis , Uridine Diphosphate Galactose/chemistry
5.
Reprod Biol Endocrinol ; 6: 51, 2008 Nov 14.
Article in English | MEDLINE | ID: mdl-19014565

ABSTRACT

BACKGROUND: In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, since the Galactosyltransferase and N-Acetylglucosamine residues have been localized on equine gametes. METHODS: We masked the Galactosyltransferase and N-Acetylglucosamine residues before the co-incubation of gametes. Galactosyltransferase was masked either with an anti-Galactosyltransferase antibody or with the enzyme substrate, UDP Galactose. N-Acetylglucosamine residues were masked either with a purified Galactosyltransferase or with an anti-ZPC antibody. RESULTS AND DISCUSSION: The number of spermatozoa bound to the zona pellucida did not decrease after the masking of Galactosyltransferase or N-Acetylglucosamine. So, these two molecules may not be necessary in the mechanism of in vitro sperm-zona pellucida interaction in the horse. CONCLUSION: The involvement of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding may have been lost during evolution in some ungulates, such as porcine and equine species.


Subject(s)
Acetylglucosamine/physiology , Biological Evolution , Fertilization/physiology , Horses/genetics , Horses/physiology , N-Acetyllactosamine Synthase/physiology , Acetylglucosamine/chemistry , Acetylglucosamine/immunology , Acetylglucosamine/metabolism , Animals , Antibodies/pharmacology , Cells, Cultured , Female , Freezing , Male , N-Acetyllactosamine Synthase/antagonists & inhibitors , N-Acetyllactosamine Synthase/immunology , Semen Preservation , Sperm-Ovum Interactions/drug effects , Sperm-Ovum Interactions/immunology , Spermatozoa/drug effects , Spermatozoa/immunology , Uridine Diphosphate Galactose/pharmacology , Zona Pellucida/immunology , Zona Pellucida/metabolism
6.
Blood ; 111(6): 3249-56, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-18096766

ABSTRACT

Cold storage of platelets for transfusion is desirable to extend platelet storage times and to prevent bacterial growth. However, the rapid clearance of cold-stored platelets prevents their use. A novel method for preventing the rapid clearance of cold-stored platelets has previously been developed in a murine model. Cold storage induces the clustering and recognition of exposed beta-N-acetylglucosamine (betaGlcNAc) on platelet surfaces. Glycosylation of betaGlcNAc residues with uridine 5'-diphosphogalactose (UDP-galactose) results in the normal survival of short-term (2 h) 0 degrees C-stored murine platelets. Based on this finding, we developed a similar glycosylation process by adding UDP-galactose to human apheresis platelets. A phase 1 clinical trial was conducted transfusing radiolabeled autologous apheresis platelets stored for 48 hours at 4 degrees C with or without pretreatment with UDP-galactose. In contrast to the murine study, galactosylation of human platelets did not prevent the accelerated platelet clearance routinely observed after 4 degrees C storage. We next developed a murine model of platelet storage for 48 hours at 4 degrees C and showed that UDP-galactose treatment of murine platelets also did not prevent their rapid clearance, in agreement with the human platelet study. We conclude that different mechanisms of clearance may exist for short- and long-term cold-stored platelets.


Subject(s)
Blood Platelets/cytology , Blood Platelets/metabolism , Blood Preservation/methods , Cold Temperature , Galactose/metabolism , Animals , Blood Component Removal , Blood Platelets/drug effects , Cell Survival/drug effects , Female , Galactose/pharmacology , Humans , Male , Mice , Mice, Inbred C57BL , Platelet Transfusion , Time Factors , Uridine Diphosphate Galactose/pharmacology
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(2): 408-11, 2007 Apr.
Article in Chinese | MEDLINE | ID: mdl-17493358

ABSTRACT

The study was purposed to develop a novel cryopreserved agent (CPA) for platelets, to investigate the morphology of cryopreserved platelets in different CPA and the CD62P expression on membrane of platelets after stimulating by thrombin, as well as to compare the effect of adding UDP-Gal on preserved efficiency of preservation solutions. A novel cryopreserved agent consisting of 2% DMSO, thrombosol and UDP-Gal was developed on basis of using higher concentration of DMSO. The morphology of chilled platelets was observed by transmission electron microscope and compared with fresh platelets. The expression of CD62P on the membrane of platelets was detected at 0, l, 3 months. The results indicated that the significant effect of cryopreservation on morphology of platelets was found according to percentages of round, dendritic and irregular shapes of cryopreserved platelets. The protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal were better than that of 5% DMSO. Compared with fresh platelets, the expression of CD62P on platelet membrane decreased obviously after cryopreservation, but not observed difference at preservation for 1 month and 3 months, as well as among 3 kinds of different CPA. It is concluded that the protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal on morphology of platelets are similar, but better than that 5% DMSO. The reaction of cryopreserved platelets to thrombin decreases, while the significant difference is not found among these 3 kinds of CPA. The addition of UDP-Gal to cryopreserved agents not show the protective effect on platelets.


Subject(s)
Blood Platelets , Blood Preservation/methods , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Humans , P-Selectin/biosynthesis , P-Selectin/genetics , Uridine Diphosphate Galactose/pharmacology
8.
Eur J Pharmacol ; 543(1-3): 166-73, 2006 Aug 14.
Article in English | MEDLINE | ID: mdl-16820147

ABSTRACT

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y(14) receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in human neutrophils. UDP-glucose (IC(50)=1 microM) induced a small but significant inhibition (circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 microM) had no significant effect, whereas 100 microM UDP-N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 microM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y(14) receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y(14) receptor expressed in human neutrophils.


Subject(s)
Neutrophils/metabolism , Receptors, Purinergic P2/metabolism , Uridine Diphosphate Sugars/pharmacology , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Humans , In Vitro Techniques , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/chemistry , Neutrophils/drug effects , Pancreatic Elastase/metabolism , Phosphorylation , RNA, Messenger/analysis , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate Glucose/pharmacology , Uridine Diphosphate Glucuronic Acid/pharmacology
9.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 14(6): 1244-7, 2006 Dec.
Article in Chinese | MEDLINE | ID: mdl-17204203

ABSTRACT

This study was aimed to investigate the method to cold-store platelets with uridine diphosphate galactose (UDP-Gal). Rabbit heart blood was prepared for concentrated platelet suspension to which UDP-Gal was added, and then stored for ten days in 4 degrees C refrigerator. Thereafter, platelet count, mean platelet volume (MPV), platelet distributing width (PDW), platelet aggregation function, platelet activity to urge coagulation including PF3aT and APCT and apoptosis were determined. Meanwhile, survival time in vivo was tested after cold-stored rabbit platelets labeled with Cr51 were transfused into rabbits. The results showed that there was not significant difference for Plt count, MPV, PDW, PF3aT and APCT between UDP-Gal cold-stored platelet group and fresh platelet group (P > 0.05). On the contrary, platelet count decreased significantly, MPV, PDW jumped and PF3aT and APCT went down in cold control group as compared with fresh platelet group (P < 0.01). Apoptosis increased in UDP-Gal cold-stored platelet group as compared with fresh platelet group (P < 0.05), but was significantly lower than that in cold control group (P < 0.01). Although PagT (inducing reagent: C-PG) decreased, it could still be above 50% of fresh platelet. Survival time in rabbit in vivo was close between UDP-Gal cold-stored platelet group and fresh platelet group (P < 0.05). Survival rate in seventy-two hours after transfusion in the fresh platelet group, UDP-Gal cold-stored platelet group and cold control group was 57.5% +/- 7.2%, 50.3% +/- 6.3% and 0.1% +/- 0.5% respectively. It is concluded that the UDP-Gal can well protect cold-stored rabbit platelets and prolong the survival time of cold-stored platelets in vivo.


Subject(s)
Blood Platelets , Blood Preservation/methods , Cryopreservation/methods , Platelet Aggregation/drug effects , Uridine Diphosphate Galactose/pharmacology , Animals , Cellular Senescence/drug effects , Rabbits , Time Factors
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 13(6): 1113-6, 2005 Dec.
Article in Chinese | MEDLINE | ID: mdl-16403293

ABSTRACT

To study the effects of glycosylation on survival of cold-storage human platelets by using rabbit model. (51)Cr-labeling platelets were used to detect the platelet storage survival. The human platelets (2.0 x 10(12)/L) treated with 5 g/L uridine diphosphate galactose (UDP-Gal) were stored in 4 degrees C refrigeratory up to 10 days. The survival of human platelets in rabbits whose reticuloendothelial system was inhibited by the administration of ethyl palmitate was monitored in blood drawn at various times after the platelet transfusion. The results showed that the survival rate of platelets was significantly increased in cold-storage human platelets by UDP-Gal treatment. The survival rates of platelets at 2 hours after transfusion into rabbits in groups of fresh platelets group, UDP-Gal + cold platelets group and cold platelets group were (68.9 +/- 8.5)%, (65.4 +/- 8.0)% and (5.0 +/- 2.6)%, respectively. Compared with cold platelets group, significant differences were seen among all groups (P < 0.01). UDP-Gal + cold platelets group had no significant differences compared with fresh platelets group (P > 0.05). It is concluded that UDG-Gal can provide the protective effect on cold-storage human platelets and prolong the survival time of refrigerated human platelets in rabbit model.


Subject(s)
Blood Platelets/cytology , Blood Preservation , Cryopreservation/methods , Platelet Transfusion , Animals , Blood Platelets/metabolism , Cell Survival/drug effects , Glycosylation/drug effects , Humans , Models, Animal , Rabbits , Uridine Diphosphate Galactose/pharmacology
11.
Biol Reprod ; 71(6): 1822-7, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15286041

ABSTRACT

The precise cellular mechanism of primordial germ cell (PGC) migration remains unknown. Cell surface galactosyltransferase (GalTase) is known to play unique roles in the process of locomotion of many migratory cells. With an objective to seek evidence for possible involvement of GalTase in the migratory process of PGC, we evaluated germ cell migration in the rat following experimental modulation of embryonic GalTase activity. Pregnant rats were laparotomized under anesthesia on Day 10 of pregnancy. While embryos of one uterine horn received lysozyme (100 microg/fetus), those of the other received alpha-lactalbumin (LA; 100 microg/fetus), N-acetylglucosamine (GlcNAc; 250 nmole/fetus), uridine 5'-monophosphate (UMP; 2.5 micromole/fetus), uridine diphosphate-galactose (UDP-gal; 250 nmole/fetus), or a combination of 250 nmole of UDP-gal and 2.5 micromole of UMP/fetus. Between gestation Days 12 and 14, embryos were dissected out and processed for histochemical localization of PGC on the basis of binding of Dolichos biflorus agglutinin on the surface glycoconjugate of the germ cells. The number of PGC in each embryo was counted. There was a daywise increase in the number of PGC in all groups. As compared with lysozyme-exposed controls, the numbers of PGCs at the day-specific sites on all days of examination were significantly lower in the LA- as well as GlcNAc-exposed groups. UMP or UDP-gal individually exerted little or no influence, while the total PGC count rose significantly over the respective control values under simultaneous exposure to UMP and UDP-gal. The present findings suggest a likely catalytic role of GalTase in the process of germ cell migration.


Subject(s)
Cell Movement/physiology , Galactosyltransferases/physiology , Germ Cells/physiology , Gonads/embryology , Acetylglucosamine/pharmacology , Animals , Cell Count , Cell Movement/drug effects , Drug Combinations , Embryo, Mammalian/cytology , Female , Galactosyltransferases/drug effects , Gestational Age , Lactalbumin/pharmacology , Pregnancy , Rats , Rats, Sprague-Dawley , Uridine Diphosphate Galactose/pharmacology , Uridine Monophosphate/pharmacology
12.
Protein Expr Purif ; 31(1): 79-87, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12963344

ABSTRACT

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.


Subject(s)
Galactosyltransferases/chemistry , Plant Proteins/chemistry , Plants/enzymology , Protein Folding , Recombinant Proteins/chemistry , Chromatography, Gel , Chromatography, Ion Exchange , Chromatography, Thin Layer/methods , Cross-Linking Reagents/chemistry , Diglycerides/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/metabolism , Ethylmaleimide/pharmacology , Galactolipids/analysis , Galactolipids/pharmacology , Galactosyltransferases/biosynthesis , Galactosyltransferases/isolation & purification , Gene Deletion , Gene Expression , Genetic Vectors/genetics , Glycolipids/analysis , Hydroxyapatites/chemistry , Inclusion Bodies/chemistry , Kinetics , Liposomes/metabolism , Maleimides/chemistry , Plant Proteins/biosynthesis , Plants/genetics , Plants/metabolism , Protein Denaturation , Recombinant Proteins/biosynthesis , Recombinant Proteins/drug effects , Spinacia oleracea/chemistry , Temperature , Urea/chemistry , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate Galactose/pharmacology
13.
Gynecol Oncol ; 90(1): 100-8, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12821349

ABSTRACT

OBJECTIVE: Poor presentation of tumor-associated antigens (TAA) to the immune system remains a major obstacle to effective anti-tumor vaccine therapy. The aim of this study is to demonstrate the feasibility of producing a novel autologous tumor vaccine from ovarian carcinoma that is expected to have increased immunogenicity. The strategy is based on the ability of the anti-Gal IgG antibody (a natural antibody comprising 1% of IgG in humans) to target tumor membranes expressing alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) to antigen-presenting cells (APC). STUDY DESIGN: Freshly obtained ovarian carcinoma tumors are homogenized, washed, and incubated with a mixture of neuraminidase, recombinant alpha1,3 galactosyltransferase (ralpha1,3GT) and uridine diphosphate galactose (UDP-Gal) to synthesize alpha-gal epitopes on carbohydrate chains of glycoproteins of these membranes. Subsequently, the processed membranes are analyzed for expression of alpha-gal epitopes and for the binding of anti-Gal. RESULTS: Incubation of 3 g of ovarian carcinoma membranes, from five different patients, at 100 mg/ml, mixed together with ralpha1,3GT (50 microg/ml), neuraminidase (1 mU/ml), and UDP-Gal (2 mM), resulted in the effective synthesis of alpha-gal epitopes to the extent of approximately 2 x 10(11) epitopes/mg of tumor membranes. As a result of this de novo expression of alpha-gal epitopes, the tumor membranes readily bound purified anti-Gal antibody, as well as anti-Gal in autologous serum. CONCLUSIONS: The method described in this study is very effective in the synthesis of many alpha-gal epitopes on tumor membranes obtained from ovarian carcinoma. These novel epitopes readily bind the naturally occurring anti-Gal antibody. This technique of opsonization of alpha-gal-modified autologous tumor membranes carrying TAA is expected to increase effective uptake of the vaccine by APC, which is key to successful anti-tumor vaccination.


Subject(s)
Cancer Vaccines/immunology , Ovarian Neoplasms/immunology , Trisaccharides/biosynthesis , Trisaccharides/immunology , Antigen-Presenting Cells/immunology , Carbohydrate Sequence , Cell Membrane/immunology , Cell Membrane/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes/biosynthesis , Epitopes/immunology , Female , Galactosyltransferases/metabolism , Galactosyltransferases/pharmacology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Membrane Glycoproteins/immunology , Molecular Sequence Data , Neuraminidase/metabolism , Neuraminidase/pharmacology , Ovarian Neoplasms/metabolism , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate Galactose/pharmacology
14.
J Org Chem ; 65(1): 24-9, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10813891

ABSTRACT

For the enzymatic transfer of galactose, N-acetylglucosamine, and N-acetylgalactosamine, UDP-Gal (1), UDP-GlcNAc (2), and UDP-GalNAc (3) are employed, and UDP serves as a feedback inhibitor. In this paper the synthesis of the novel UDP-sugar analogues 4, 5, and 6 as potential transferase inhibitors is described. Compounds 4-6 feature C-glycosidic hydroxymethylene linkages between the sugar and nucleoside moieties in contrast to the anomeric oxygens in the natural derivatives 1-3.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Transferases/antagonists & inhibitors , Uridine Diphosphate Galactose/chemical synthesis , Uridine Diphosphate N-Acetylgalactosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Molecular Mimicry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uridine Diphosphate Galactose/chemistry , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate N-Acetylgalactosamine/chemistry , Uridine Diphosphate N-Acetylgalactosamine/pharmacology , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/pharmacology
15.
Chem Biol ; 4(9): 653-7, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9331404

ABSTRACT

BACKGROUND: Combinatorial chemistry has become an important method for identifying effective ligand-receptor binding, new catalysts and enzyme inhibitors. In order to distinguish the most active component of a library or to obtain structure-activity relationships of compounds in a library, an efficient quantitative assay is crucial. Electrospray mass spectrometry has become an indispensable tool for qualitatively screening combinatorial libraries and its use for quantitative analysis has recently been demonstrated. RESULTS: This paper describes the use of quantitative electrospray mass spectrometry for screening libraries of inhibitors of enzymatic reactions, specifically the enzymatic glycosylation by beta-1,4-galactosyltransferase, which catalyzes the transfer of galactose from uridine-5'-diphosphogalactose to the 4-position of N-acetylglucosamine beta OBn (Bn: benzene) to form N-acetyllactosamine beta OBn. Our mass spectrometric screening approach showed that both nucleoside diphosphates and triphosphates inhibited galactosyltransferase while none of the nucleoside monophosphates, including uridine-5'-monophosphate, showed any inhibition. Additional libraries were generated in which the concentrations of the inhibitors were varied and, using mass spectrometry, uridine-5'-diphosphate-2-deoxy-2-fluorogalactose was identified as the best inhibitor. CONCLUSIONS: This report introduces quantitative electrospray mass spectrometry as a rapid, sensitive and accurate quantitative assaying tool for inhibitor libraries that does not require a chromophore or radiolabeling. A viable alternative to existing analytical techniques is thus provided. The new technique will greatly facilitate the discovery of novel inhibitors against galactosyltransferase, an enzyme for which there are few potent inhibitors.


Subject(s)
Enzyme Inhibitors/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Enzyme Inhibitors/pharmacology , N-Acetyllactosamine Synthase/antagonists & inhibitors , N-Acetyllactosamine Synthase/chemistry , Uridine Diphosphate Galactose/analogs & derivatives , Uridine Diphosphate Galactose/chemistry , Uridine Diphosphate Galactose/pharmacology
16.
J Biol Chem ; 271(49): 31446-51, 1996 Dec 06.
Article in English | MEDLINE | ID: mdl-8940156

ABSTRACT

The kinetic properties of UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) purified to homogeneity from lactating bovine mammary gland have been investigated. GlcNAc-phosphotransferase transferred GlcNAc 1-phosphate from UDP-GlcNAc to the synthetic acceptor alpha-methylmannoside, generating GlcNAc-1-phospho-6-mannose alpha-methyl, the structure of which was confirmed by mass spectroscopy. GlcNAc-phosphotransferase was active between pH 5.7 and 9.3, with optimal activity between pH 6.6 and 7.5. Activity was strictly dependent on Mg2+ or Mn2+. The Km for Mn2+ was 185 microM. The Km for UDP-GlcNAc was 30 microM, and that for alpha-methylmannoside was 63 mM. The enzyme was competitively inhibited by UDP-Glc, with a Ki of 733 microM. The 166-kDa subunit was identified as the catalytic subunit by photoaffinity labeling with azido-[beta-32P]UDP-Glc. Purified GlcNAc-phosphotransferase utilizes the lysosomal enzyme uteroferrin approximately 163-fold more effectively than the non-lysosomal glycoprotein ribonuclease B. Antibodies to GlcNAc-phosphotransferase blocked the transfer to cathepsin D, but not to alpha-methylmannoside, suggesting that protein-protein interactions are required for the efficient utilization of glycoprotein acceptors. These results indicate that the purified bovine GlcNAc-phosphotransferase retains the specificity for lysosomal enzymes as acceptors previously observed with crude preparations.


Subject(s)
Transferases (Other Substituted Phosphate Groups)/chemistry , Acid Phosphatase , Animals , Calcium/metabolism , Cathepsin D/antagonists & inhibitors , Cattle , Hydrogen-Ion Concentration , Isoenzymes , Kinetics , Magnesium/metabolism , Manganese/metabolism , Metalloproteins/metabolism , Methylmannosides/pharmacology , Phosphorylation , Rabbits , Ribonucleases/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tartrate-Resistant Acid Phosphatase , Transferases (Other Substituted Phosphate Groups)/metabolism , Uridine Diphosphate Galactose/pharmacology
17.
Bioorg Med Chem ; 4(11): 1939-48, 1996 Nov.
Article in English | MEDLINE | ID: mdl-9007278

ABSTRACT

Four mono-O-methylated and one mono-O-acetylated UDP-D-Gal analogues and UDP-L-Fuc were synthesized. 2-O-Methyl-D-galactose residue was enzymatically transferred to give 2'-O-methyllactosaminide in high yield. UDP-Fuc and UDP-Man showed potent inhibitory activities against beta-1,4-galactosyltransferase. Structural requirement and steric allowance for the ground and transition states of the enzyme reaction were discussed.


Subject(s)
Fucose/pharmacology , Galactosyltransferases/metabolism , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate Sugars/pharmacology , Galactosyltransferases/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Uridine Diphosphate Galactose/pharmacology
18.
J Cell Sci ; 107 ( Pt 6): 1713-24, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7962212

ABSTRACT

Cell growth and differentiation are influenced by intercellular contact, suggesting that cell adhesion molecules may be instrumental in triggering these events. F9 embryonal carcinoma cells are an ideal system in which to examine the function of cell adhesion molecules in growth and differentiation, since the relevant cell adhesion molecules and differentiation markers are well defined. Intercellular adhesion in F9 cells is mediated by uvomorulin, or E-cadherin, and cell surface beta-(1,4)-galactosyltransferase. Since previous studies suggested that neither F9 cell growth nor differentiation is directly dependent on uvomorulin function, in this study we examined whether cell surface galactosyltransferase plays any role in F9 cell growth or differentiation. A variety of galactosyltransferase perturbants, including anti-galactosyltransferase antibodies, UDPgalactose, and the substrate modifier protein alpha-lactalbumin, inhibited the growth of F9 cells, whereas control reagents did not. To examine this in more detail, we analyzed the effects of perturbing surface galactosyltransferase on progression through the F9 cell cycle. Anti-galactosyltransferase IgG treatment inhibited ornithine decarboxylase activity and lengthened the F9 cell cycle during G1 and G2, the latter mimicking the effects of retinoic acid, a reagent known to prolong the F9 cell cycle and induce differentiation. In contrast, anti-uvomorulin antibodies had no effect on F9 cell growth, ornithine decarboxylase activity, or progression through the cell cycle. Furthermore, perturbation of surface galactosyltransferase adhesions in F9 cell aggregates induced precocious F9 cell differentiation, as assayed by increased laminin synthesis, whereas control reagents had no effect. Thus, perturbing surface galactosyltransferase adhesions in F9 cells both decreases growth and stimulates synthesis of laminin. These results imply that interactions between surface galactosyltransferase and its oligosaccharide ligand during cell adhesion may affect the normal growth-regulatory and differentiation-inducing signals, as is seen, in part, during treatment with retinoic acid.


Subject(s)
Cell Adhesion Molecules/physiology , Galactosyltransferases/physiology , Laminin/biosynthesis , Membrane Proteins/physiology , Neoplasm Proteins/physiology , Teratocarcinoma/pathology , Animals , Cell Adhesion , Cell Cycle , Cell Division , Culture Media, Serum-Free , Galactosyltransferases/antagonists & inhibitors , Galactosyltransferases/genetics , Membrane Proteins/antagonists & inhibitors , Mice , Neoplasm Proteins/antagonists & inhibitors , Ornithine Decarboxylase/metabolism , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Teratocarcinoma/metabolism , Transcription, Genetic , Tumor Cells, Cultured/drug effects , Uridine Diphosphate Galactose/analogs & derivatives , Uridine Diphosphate Galactose/pharmacology
19.
Biochem Biophys Res Commun ; 193(2): 585-90, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-8512559

ABSTRACT

The effects of nucleotides, nucleotide sugars and nucleotide dialdehydes on the activity and kinetics of cytidine 5'-monophospho-N-acetylneuraminic acid:lactosylceramide (alpha 2-->3) sialyltransferase (SAT-1) in microsomes derived from embryonic chick brain were investigated. Although under physiological conditions this enzyme utilizes a CMP-sugar as substrate, it was found that UDP-dialdehyde was an effective inhibitor of SAT-1 activity. CMP-dialdehyde was only slightly more efficient at inhibiting SAT-1 activity. Similar findings were found for the inhibitory effects of UDP versus CMP. In addition, two UDP-sugars (UDP-Gal and UDP-GalNAc) were also slightly inhibitory. Kinetic analyses demonstrate that both UDP- and CMP-dialdehydes are competitive inhibitors of SAT-1 activity. The data suggests that the substrate specificity of microsomal SAT-1 resides more in the sugar moiety, rather than in the nucleotide portion of the substrate.


Subject(s)
Brain/enzymology , Cytidine Monophosphate/analogs & derivatives , Microsomes/enzymology , Ribonucleotides/pharmacology , Sialyltransferases/antagonists & inhibitors , Uridine Diphosphate Sugars/pharmacology , Uridine Diphosphate/analogs & derivatives , Animals , Chick Embryo , Cytidine Monophosphate/pharmacology , Kinetics , Uridine Diphosphate/pharmacology , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate N-Acetylgalactosamine/pharmacology
20.
Dev Biol ; 147(2): 440-4, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1916017

ABSTRACT

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.


Subject(s)
Acrosome/physiology , Galactosyltransferases/physiology , Sperm Head/enzymology , Acetylglucosamine/pharmacology , Animals , Calcimycin/pharmacology , Female , Galactosyltransferases/immunology , Immunoglobulin Fab Fragments , In Vitro Techniques , Lactalbumin/pharmacology , Male , Mice , Sperm-Ovum Interactions/drug effects , Sperm-Ovum Interactions/physiology , Trisaccharides/pharmacology , Uridine Diphosphate Galactose/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...