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Y T Li

Publications and source records attributed to Y T Li.

At least 37 records · Page 2Linked to original sources
PubMed2001

KDN-containing glycoprotein from loach skin mucus.

It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.

Amino Acids↗
PubMed2001

Interaction of GM2 activator protein with glycosphingolipids.

GM2 activator protein is a protein cofactor that stimulates the hydrolysis of the GalNAc and the NeuAc in GM2 by beta-hexosaminidase A and sialidase, respectively. To understand the mechanism of action of GM2 activator, the interaction of this protein with GM2 and/or beta-hexosaminidase A has been the subject of interest since the purified GM2 activator became available. Numerous techniques including ultracentrifugation, isoelectric a focusing, polyacrylamide gel electrophoresis, gel filtration, thin layer chromatogram overlay, and fluorescence dequenching assay have been used to investigate the binding and the affinity of GM2 activator to various glycosphingolipids. It has been generally accepted that GM2 activator must have a very weak binding with the enzyme, because they can be easily separated from each other by gel filtration. Therefore, the interaction of GM2 and GM2 activator has been the focus for most of he study. Although preferential association of GM2 activator with GM2 was detected by some methods, GM2 activator was found also to bind other glycosphingolipids. Isolation of the specific complex that consists of only GM2 activator and GM2 from incubation mixture containing the activator protein and mixed glycosphingolipids has not been successfully carried out. Ultracentrifugation and gel-filtration are the mildest methods for the isolation of the complexes. However, these methods do not separate the complexes formed by specific interaction from that formed by non-specific association. In fluorescence dequenching assay, the attempt to isolate the complex of R18 lipid probe with GM2 activator was also not successful. Since GM2 activator and glycosphingolipids contain hydrophobic domains in their molecules, the non-specific hydrophobic interactions between the two components can greatly interfere with the isolation of true functional complexes. Among the reported methods, thin layer chromatography overlay and the assay based on the inhibition of fluorescence dequenching by various glycosphingolipids are more informative than the others on the binding between GM2 activator and the carbohydrate head groups of glycosphingolipids.

Binding Sites↗
PubMed2001

Rectal administration of misoprostol for the management of retained placenta--a preliminary report.

BACKGROUND: Retained placenta is one of the serious complications of childbirth, and misoprostol is known to be a potent uterotonic agent. Therefore, we proposed that rectal misoprostol also may facilitate placental separation in women with retained placenta by its ability to increase uterine contractility. METHODS: The placenta was diagnosed as retained if it was not expelled within 40 minutes after vaginal birth. Then, 800 microg of misoprostol was inserted rectally and the patient observed thereafter. RESULTS: A total of 18 parturients who had retention of the placenta were studied; all the placentas were spontaneously expelled within 35 minutes. The side effects involved included nausea 17%, vomiting 11%, diarrhea 22%, shivering 33%, and pelvic cramping pain 44%. All these discomforts resolved within 24 hours. CONCLUSIONS: Our study demonstrated that misoprostol per rectum is a safe and effective technique and may be a useful alternative to manual removal of retained placentas.

Abortifacient Agents, Nonsteroidal↗
PubMed2000

A novel approach for delivery of enzyme drugs: preliminary demonstration of feasibility and utility in vitro.

A novel heparin/protamine-based approach for delivery of enzyme drugs without associated toxic effects has been proposed. This approach would allow an enzyme drug to be administered in an inactive (i.e. pro-drug) form and then released at the target site in an active form using protamine as the triggering agent. The pro-drug and the triggered release features of this approach would permit the enzyme drug to act specifically and only on its target substrates while sparing normal substrates, thereby alleviating unwanted toxic effects. The in vitro feasibility of the approach has been successfully demonstrated using trypsin as the model protease drug. In addition, the utility of the approach has also been demonstrated by applying the system in delivering streptokinase, one of the most widely used clinical drugs in thrombolytic therapy. This approach may open up the possibility of developing a wide range of new catalytic drugs that are initially thought to be impossible for therapeutic use due to their potent toxic effects.

Azure Stains↗
PubMed2000

A novel heparin/protamine-based pro-drug type delivery system for protease drugs.

Previously we proposed a heparin/protamine-based system for delivery of protease drugs such as tissue-specific plasminogen activator (tPA). To demonstrate the feasibility of this approach as well as its pro-drug and triggered release features, positively charged peptides [(Arg)(7)Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using the crosslinking agent N-succinimidyl-3-(2-pyridyldithio)- propionate. This cation-modified tPA showed much stronger heparin affinity than the parent tPA. The complex formed by mtPA and heparin was stable in human plasma, and the activity of mtPA in such a complex was inhibited by the appended heparin. Similarly, the activity of mtPA could also be inhibited by a heparin-antifibrin IgG conjugate in which heparin was linked, via endpoint attachment, to the sugar moieties in the F(c) region of anti-fibrin IgG. Aside from this pro-drug feature exhibited by the binding of the macromolecule heparin to mtPA, results from chromogenic and in vitro clot lysis assay demonstrated that the heparin-induced inhibition of the mtPA activity could be easily reversed by the addition of an adequate amount of protamine. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA. In addition to the chemical conjugation method, modified tPA could also be produced by the recombinant DNA method. The expressed modified tPA (EmtPA) thus prepared retained the full catalytic activity of the parent tPA, and this activity could also be inhibited by heparin, and the heparin-induced inhibition could be reversed following the addition of protamine.

Drug Carriers↗
PubMed2000

Biomedical application of immobilized enzymes.

Reports on chemical immobilization of proteins and enzymes first appeared in the 1960s. Since then, immobilized proteins and enzymes have been widely used in the processing of variety of products and increasingly used in the field of medicine. Here, we present a review of recent developments in immobilized enzyme use in medicine. Generally speaking, the use of immobilized enzyme in medicine can be divided into two major categories: biosensors and bioreactors. A brief overview of the evolution of the biosensor and bioreactor technology, of currently existing applications of immobilized enzymes, of problems that researchers encountered, and of possible future developments will be presented.

Animals↗
PubMed2000

Asymptomatic uterine incarceration at term gestation: a case report.

Uterine incarceration is a rare event complicating pregnancy. Herein, we present a unique case of an asymptomatic term pregnancy with uterine incarceration; this patient had had an anterior myomectomy 3 years earlier. She had an asymptomatic antenatal course and incidental identification of uterine incarceration during cesarean section. We also address the importance of early recognition and appropriate management regarding this circumstance.

Adult↗
PubMed2000

Synthesis and characterization of positively charged tPA as a prodrug using heparin/protamine-based drug delivery system.

Positively charged peptides [(Arg)7 Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7 Cys (termed modified tPA, mtPA) was 3900 IU/microg as compared to 5800 IU/microg of the parent tPA. Both activation of plasminogen with mtPA (Km= 2.7 mM(-1)) and tPA (Km= 1.1 mM(-1)) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)7 Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and alpha2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.

Antibodies↗
PubMed1999

2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)- and N-acetylneuraminic acid-cleaving sialidase (KDN-sialidase) and KDN-cleaving hydrolase (KDNase) from the hepatopancreas of oyster, Crassostrea virginica.

KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.

Amino Acid Sequence↗
PubMed1999

Catabolism of asialo-GM2 in man and mouse. Specificity of human/mouse chimeric GM2 activator proteins.

Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires beta-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66-72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn(106)-Tyr(114)) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients.

Amino Acid Sequence↗
PubMed1999

Structural basis for the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation.

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.

Acetylgalactosamine↗
PubMed1999

The 1.8 A structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism.

Intramolecular trans-sialidase from leech (Macrobdella decora) is the first member of the sialidase superfamily found to exhibit strict specificity towards the cleavage of terminal Neu5Acalpha2-->3Gal linkage in sialoglycoconjugates. Its release of 2,7-anhydro-Neu5Ac instead of Neu5Ac indicates that it catalyzes an intramolecular trans-sialosyl reaction. Crystal structures of its complexes with an inactive substrate analogue 2-propenyl-Neu5Ac, and with the product 2,7-anhydro-Neu5Ac, have been determined to 1.8 A resolution. The boat conformation of the pyranose observed in the complexes supports the proposed enzymatic mechanism that O7 of an axial 6-glycerol group attacks the positively charged C2 of the intermediate. A generalized mechanism is proposed for the sialidase superfamily.

Amino Acid Sequence↗
PubMed1999

Naturally occurring GM2 gangliosidosis in two Muntjak deer with pathological and biochemical features of human classical Tay-Sachs disease (type B GM2 gangliosidosis).

Two juvenile sibling male Muntjak deer (Muntiacus muntjak) with histories of depression, ataxia, circling and visual deficits were studied. Cerebrospinal fluid analyses revealed vacuolated macrophages that contained long parallel needle-like intracytoplasmic inclusions. Light microscopically, nerve cell bodies throughout the brain, ganglion cells within the retina and neurons in the myenteric plexuses were variably swollen and had pale granular to finely vacuolated eosinophilic cytoplasm. Neuronal cytoplasm stained specifically with sudan black and Luxolfast blue stains. Within the brain there were occasional axonal spheroids, foci of astrogliosis and scattered microglial cells with abundant pale foamy cytoplasm. Electron microscopy of the brain and retina revealed numerous neurons and ganglion cells, respectively, with multiple membrane-bound structures that contained compact electron-dense membranous whorls and fewer parallel membranous stacks. Thin layer chromatography of total lipid extracts of the cerebral cortex of both cases revealed massive accumulation of G(M2) ganglioside. Crude kidney extracts of the two affected deer were able to hydrolyze 4-methylumbelliferyl beta-GlcNAc, but not 4-methylumbelliferyl beta-GlcNAc-6-sulfate, indicating the defect of beta-hexosaminidase A. Cellogel electrophoresis of the kidney extracts also revealed the deficiency of beta-hexosaminidase A in the two deer. It is concluded that these two deer had the biochemical lesion identical to that of human type B G(M2) gangliosidosis (classical Tay-Sachs disease).

Animals↗
PubMed1999

Laparoscopic loop ligatures for bladder repair during laparoscopic surgery.

In this paper, we report a case where a bladder perforation occurred during a laparoscopically assisted vaginal hysterectomy and was repaired by laparoscopic loop ligatures. This is the first case report of using the laparoscopic loop ligatures to close the bladder perforations. The loop ligature is an easy and quick procedure, which can be performed by most surgeons who take the time to learn the endoscopic suturing techniques.

Female↗
PubMed1999

The structural basis for the susceptibility of gangliosides to enzymatic degradation.

The conformational properties of GM2, GalNacbeta-4(Neu5Acalpha-3) Galbeta-4Glcbeta-1Cer have been compared to those of 6'-GM2, in which the linkage between the GalNAc and Gal was altered from GalNacbeta-4Galbeta- to GalNacbeta-6Galbeta-, and to those of GD1a, Neu5Acalpha-3Galbeta-3GalNAcbeta-4(Neu5Acalpha-3 )Galbeta-4Glcbeta-1Cer, and GalNAc-GD1a. Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6'-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions. The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Acalpha-3Galbeta-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two 'core' GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.

Carbohydrate Sequence↗