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Biomedical subjects

S C Li

Publications and source records attributed to S C Li.

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

Altered expression of extracellular matrix and proteinases in Noble rat prostate gland after long-term treatment with sex steroids.

BACKGROUND: Interactions between the epithelial cells and stromal tissues, which include the epithelial basement membrane, extracellular matrix, inducible factors, and various cell types, are believed to play a significant role in prostate gland carcinogenesis. Remodeling of extracellular matrix and degradation of basement membrane are the prerequisites for tumor cell invasion, and these changes are correlated with the expression of various proteinases. METHODS: The present study examined the alterations of epithelial basement membrane, extracellular matrix, and proteinase activities in the Noble rat prostate gland after long-term treatments with androgen and estrogen (T+DES or T+E(2) for 4-12 months) by histochemistry, immunohistochemistry, electron microscopy, and gelatin-gel zymography. RESULTS: After hormonal treatments, defects of epithelial basement membranes, such as focal disruption, diffuse staining and multilayering, were observed by histochemistry and immunohistochemistry in the dysplastic and neoplastic lesions induced in the lateral (LP) and ventral prostates (VP) but not in dorsal prostate (DP). An increase in the amount of extracellular matrix components, including hyaluronan (HA), chondroitin sulfate proteoglycan (CSPG) and tenascin, in the stroma of hormone-treated LP and VP was revealed by histochemistry and immunohistochemistry. Positive immunolabeling of matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) was detected in the fibromuscular layer surrounding the adenoma and adenocarcinoma induced in LP and VP after treatments with steroids for over 9-12 months. Zymography also detected an increase in activities of proteinases of apparent MW 120, 90, 86 and 68 kDa in the hormone-treated LP and VP, and these proteinases were characterized as metalloproteinases. In addition, two serine proteinases of MW 100 and 30 kDa were identified as being overexpressed in the hormone-treated LP and VP. Compared to LP and VP, there was no significant change in the proteinase activities in the hormone-treated DP. CONCLUSIONS: The present study demonstrated that the epithelial basement membrane and stromal extracellular matrix were altered in dysplastic and neoplastic Noble rat prostates. Since HA and CSPG (or their complexes) are highly anionic molecules, their increased accumulation in the altered prostatic stroma would tend to hydrate this tissue. This would create an environment more favorable for tumor growth and invasion. These morphological changes were also correlated with the concurrent increase in gelatinolytic proteinase activities induced in these prostates. The results suggest that the remodeling of the stromal tissue might play a role in the early stage of prostate carcinogenesis as shown in the Noble rat model.

Adenocarcinoma↗
PubMed2001

Characterization of SH2D1A missense mutations identified in X-linked lymphoproliferative disease patients.

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency characterized by extreme susceptibility to Epstein-Barr virus. The XLP disease gene product SH2D1A (SAP) interacts via its SH2 domain with a motif (TIYXXV) present in the cytoplasmic tail of the cell-surface receptors CD150/SLAM, CD84, CD229/Ly-9, and CD244/2B4. Characteristically, the SH2D1A three-pronged interaction with Tyr(281) of CD150 can occur in absence of phosphorylation. Here we analyze the effect of SH2D1A protein missense mutations identified in 10 XLP families. Two sets of mutants were found: (i) mutants with a marked decreased protein half-life (e.g. Y7C, S28R, Q99P, P101L, V102G, and X129R) and (ii) mutants with structural changes that differently affect the interaction with the four receptors. In the second group, mutations that disrupt the interaction between the SH2D1A hydrophobic cleft and Val +3 of its binding motif (e.g. T68I) and mutations that interfere with the SH2D1A phosphotyrosine-binding pocket (e.g. C42W) abrogated SH2D1A binding to all four receptors. Surprisingly, a mutation in SH2D1A able to interfere with Thr -2 of the CD150 binding motif (mutant T53I) severely impaired non-phosphotyrosine interactions while preserving unaffected the binding of SH2D1A to phosphorylated CD150. Mutant T53I, however, did not bind to CD229 and CD224, suggesting that SH2D1A controls several critical signaling pathways in T and natural killer cells. Because no correlation is present between identified types of mutations and XLP patient clinical presentation, additional unidentified genetic or environmental factors must play a strong role in XLP disease manifestations.

Amino Acid Sequence↗
PubMed2001

A novel endo-beta-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAcalpha 1-->Gal from glycans specifically expressed in the gastric gland mucous cell-type mucin.

We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcalpha1-->4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-alpha-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcalpha1-->4Gal. The specificity of this enzyme as an endo-beta-galactosidase was established by analyzing the liberation of GlcNAcalpha1-->4Gal from GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAcalpha1--> 4Galbeta1-->3)GalNAc-ol by mass spectrometry. Because this novel endo-beta-galactosidase specifically releases the GlcNAcalpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcalpha1-->4Gal-releasing endo-beta-galactosidase (Endo-beta-Gal(GnGa)). Endo-beta-Gal(GnGa) was found to remove the GlcNAcalpha1-->4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4-N-acetylglucosaminyltransferase cDNA. Endo-beta-Gal(GnGa) should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcalpha1-->4Gal epitope.

Adenocarcinoma↗
PubMed2001

A unique sialidase that cleaves the Neu5Gcalpha2-->5-O(glycolyl)Neu5Gc linkage: comparison of its specificity with that of three microbial sialidases toward four sialic acid dimers.

We found that the hepatopancreas of oyster, Crassostrea virginica, contained a sialidase capable of releasing Neu5Gc from the novel polysialic acid chain (-->5-O(glycolyl)Neu5Gcalpha2-->)n more efficiently than from the conventional type of polysialic acid chains, (-->8Neu5Acalpha2-->)n, or (-->8Neu5Gcalpha2-->)n. We have partially purified this novel sialidase and compared its reactivity with that of microbial sialidases using four different sialic acid dimers, Neu5Gcalpha2-->5-O(glycolyl)Neu5Gc (Gg2), Neu5Acalpha2-->8Neu5Ac (A2), Neu5Gcalpha2-->8Neu5Gc (G2), and KDNalpha2-->8KDN (K2) as substrates. Hydrolysis was monitored by high performance anion-exchange chromatography with a CarboPac PA-100 column and pulsed amperometric detection, the method by which we can accurately quantitate both the substrate (sialiac acid dimers) and the product (sialic acid monomers). The oyster sialidase effectively hydrolyzed Gg2 and K2, whereas A2 and G2 were poor substrates. Neu5Ac2en but not KDN2en effectively inhibited the hydrolysis of Gg2 by the oyster sialidase. Likewise, the hydrolysis of K2 by the oyster sialidase was inhibited by a cognate inhibitor, KDN2en, but not by Neu5Ac2en. Using the new analytical method we found that Gg2 was hydrolyzed less efficiently than A2 but much more readily than G2 by Arthrobacter ureafaciens sialidase. This result was at variance with the previous report using the thiobarbituric acid method to detect the released free sialic acid [Kitazume, S., et al. (1994) Biochem. Biophys. Res. Commun. 205, 893-898]. In agreement with previous results, Gg2 was a poor substrate for Clostridium perfringens sialidase, while K2 was refractory to all microbial sialidases tested. Thus, the oyster sialidase is novel and distinct from microbial sialidases with regards to glycon- and linkage-specificity. This finding adds an example of the presence of diverse sialidases, in line with the diverse sialic acids and sialic acid linkages that exist in nature. The new sialidase should become useful for both structural and functional studies of sialoglycoconjugates.

Animals↗
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

Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings.

Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.

Chromatography, Affinity↗
PubMed2001

Cloning and characterization of a DEAD box RNA helicase from the viable seedlings of aged mung bean.

Seeds stored under adverse conditions will reduce the viability of germination as a result of induced aging. We have established a procedure to induce accelerated aging for studying the process of aging in mung bean (Vigna radiata) seeds at the molecular level. A full-length cDNA was isolated from acceleratedly aged mung bean seedlings. The cDNA, VrRH1 (Vigna radiata RNA helicase 1), contains an open reading frame of 2139 bp encoding a protein of 713 amino acids. VrRHI has seven highly conserved motifs including the DEAD box as in the case of other plant RNA helicases. VrRHI was sub-cloned into an expression vector pET-28b (+), over-expressed in Escherichia coli BL 21 and purified by a Ni2+-agarose column. The expressed protein showed double-stranded RNA unwinding and ATPase activities. Either ATP or dATP is required for the unwinding activity, indicating that VrRHI is an ATP/dATP-dependent RNA helicase. Northern blot analysis showed the presence of mRNAs hybridized with a full-length cDNA fragment of VrRHI (VrRH transcripts) in mung bean seeds that were imbibed for 16 to 32 h after accelerated aging treatment. The amount of these mRNAs reached a maximum in 24 h imbibed seeds after the treatment. The accumulation of VrRH transcripts was shown to lead to the appearance of 25S and 18S rRNAs in the imbibed aging mung bean seeds. The results suggest that VrRHI may play a role in the viability of mung bean seeds.

Adenosine Triphosphatases↗
PubMed2001

Evaluation of the hepatic and renal-protective effects of Ganoderma lucidum in mice.

The antioxidative effect of hot water extract of the mushroom Ganoderma lucidum on ethanol-induced free radical generation had been studied. In order to further investigate the hepatic and renal protective mechanism of Ganoderma lucidum, rates of lipid peroxidation were determined. The hot water extract of Ganoderma lucidum dose-dependently exhibited antioxidative effect on mouse liver and kidney lipid peroxidation; our results indicated that hepatic and renal homogenates have a higher malonic dialdehyde level in an ethanol administered group than in the Ganoderma lucidum treated group. It was concluded that the hepatic and renal protective mechanism of Ganoderma lucidum, might be due at least in part to its prominent superoxide scavenging effect. Ganoderma extract could protect the liver and kidney from superoxide induced hepatic and renal damages.

Animals↗
PubMed2001

Longitudinal study of blood pressure and stroke in over 37,000 People in China.

As part of a longitudinal study performed in urban China, 37,655 subjects were evaluated for stroke risk factors, including having their blood pressure measured in a standard fashion. The cohort was followed for 3.5 years during which time 427 subjects experienced incident strokes--221 ischemic, 203 hemorrhagic, and 3 undefined. Both systolic and diastolic blood pressure were significantly related to risk of stroke and stroke type. Associations were stronger for systolic than diastolic blood pressure. These results emphasize the importance of systolic blood pressure, as opposed to diastolic, as a risk factor for stroke. In this study, the risk of stroke is increased by about 25% with each 10 mm Hg increase in systolic blood pressure.

Adult↗
PubMed2000

Crystal structure of human GM2-activator protein with a novel beta-cup topology.

GM2 activator protein (GM2-AP) belongs to a small group of non- enzymatic lysosomal proteins that act as cofactors in the sequential degradation of gangliosides. It has been postulated that GM2-AP extracts single GM2 molecules from membranes and presents them in soluble form to beta-hexosaminidase A for cleavage of N-acetyl-d-galactosamine and conversion to GM3. The high affinity of GM2-AP for GM2 is based on specfic recognition of the oligosaccharide moiety as well as the ceramide lipid tail. Genetic defects in GM2-AP result in an atypical form of Tay-Sachs disease known as variant AB GM2 gangliosidosis. The 2.0 A resolution crystal structure of GM2-AP reported here reveals a previously unobserved fold whose main feature is an eight-stranded cup-shaped anti-parallel beta-pleated sheet. The striking feature of the GM2-AP structure is that it possesses an accessible central hydrophobic cavity rather than a buried hydrophobic core. The dimensions of this cavity (12 Ax14 Ax22 A) are suitable for binding 18-carbon lipid acyl chains. Flexible surface loops and a short alpha-helix decorate the mouth of the beta-cup and may control lipid entry to the cavity.

Amino Acid Sequence↗
PubMed2000

Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb.

The phosphotyrosine-binding (PTB) domain of the cell fate determinant Numb is involved in the formation of multiple protein complexes in vivo and can bind a diverse array of peptide sequences in vitro. To investigate the structural basis for the promiscuous nature of this protein module, we have determined its solution structure by NMR in a complex with a peptide containing an NMSF sequence derived from the Numb-associated kinase (Nak). The Nak peptide was found to adopt a significantly different structure from that of a GPpY sequence-containing peptide previously determined. In contrast to the helical turn adopted by the GPpY peptide, the Nak peptide forms a beta-turn at the NMSF site followed by another turn near the C-terminus. The Numb PTB domain appears to recognize peptides that differ in both primary and secondary structures by engaging various amounts of the binding surface of the protein. Our results suggest a mechanism through which a single PTB domain might interact with multiple distinct target proteins to control a complex biological process such as asymmetric cell division.

Amino Acid Sequence↗
PubMed2000

Semicontinuous production of lipase by Acinetobacter radioresistens in presence of nonwoven fabric.

The effect of a hydrophobic nonwoven fabric on the lipase production by Acinetobacter radioresistens was investigated with semicontinuous culture. The fermentation medium contained n-hexadecane as the carbon source. The nonwoven fabric was made from nylon 6 and coated with an acrylic resin. Equipping the nonwoven fabric around the baffles of a 2.5-L agitated fermentor could provide a fine dispersion of n-hexadecane, thus enhancing lipase production. The improvement on lipase yield by using the nonwoven fabric was found to be comparable to that of using an emulsifier (gum arabic). Compared with the corresponding culture in the absence of nonwoven fabric, the employment of the nonwoven fabric could significantly enhance both lipase yield and volumetric productivity.

Acinetobacter↗