Biomedical subjects

Y C Lee

Publications and source records attributed to Y C Lee.

At least 19 recordsLinked to original sources

PubMed1992

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.

Acetylglucosamine↗

PubMed1992

Ligand binding characteristics of the major mistletoe lectin.

Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.

Binding Sites↗

PubMed1992

Multivalent ligand binding by serum mannose-binding protein.

The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.

Acute-Phase Proteins↗

PubMed1992

Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.

Amidohydrolases↗

PubMed1992

Alterations in proglucagon processing and inhibition of proglucagon gene expression in transgenic mice which contain a chimeric proglucagon-SV40 T antigen gene.

The proglucagon gene is expressed in A cells of the pancreas and L cells of the large and small intestine. Transgenic mice expressing SV40 large T antigen under the control of proglucagon regulatory sequences develop neuroendocrine carcinoma of the large intestine. To determine the consequences of coexpression of SV40 large T antigen and proglucagon in different cell types, the levels of proglucagon mRNA transcripts and proglucagon-derived peptides were determined in tumor-bearing transgenic mice and in age-matched paired controls. Plasma levels of proglucagon-derived peptides (glicentin, oxyntomodulin, and glucagon, as determined by high pressure liquid chromatography and radioimmunoassay) were markedly elevated in association with tumor growth (p < 0.001). Northern blot analysis demonstrated that the increased concentration of proglucagon-derived peptides was associated with significant inhibition of the endogenous proglucagon gene in pancreas, and to a lesser extent, small intestine. Concomitantly, the concentrations of proglucagon-derived peptides fell to 1-10% of control values in pancreas (p < 0.001) and to 62% of control values in small intestine (p < 0.001). Analysis of proglucagon-derived peptides in mice of different ages demonstrated that tumor growth was associated with a switch in the post-translational processing of proglucagon. Compared with normal mouse intestine, tumors contained increased proportions of glucagon and glucagon-like peptide-1(7-37) relative to glicentin, oxyntomodulin, and glucagon-like peptide-1(1-37). The results of these studies provide evidence for humorally-mediated tissue-specific inhibition of proglucagon gene expression.

Animals↗

PubMed1992

Ultrasonography and ultrasonographically guided fine-needle aspiration biopsy of impalpable cervical lymph nodes in patients with non-small cell lung cancer.

BACKGROUND: Neck ultrasonography (US) and ultrasonographically guided fine-needle aspiration (UGFNA) cytologic examination were used in the staging work-up of 51 consecutive patients with non-small cell lung cancer (NSCLC) with clinically impalpable cervical lymphadenopathy. METHODS: All patients had chest radiography, fiberoptic bronchoscopic examination, computed tomography (CT) scans of the chest and abdomen, bone scan, and US of the abdomen and the neck. RESULTS: Six patients (12%) were found to have cervical lymph nodes by neck US. The cytologic examination of the lymph node aspirates obtained from UGFNA confirmed the metastasis. The clinical stage thus was changed from Stage IIIa to Stage IIIb in one patient. In the other five patients, UGFNA of cervical lymph nodes prevented more invasive diagnostic procedures. CONCLUSIONS: This preliminary study shows that neck US and UGFNA of lymph nodes may be helpful in the pretreatment evaluation of patients with NSCLC with impalpable cervical lymph nodes. However, additional study is needed to confirm this result.

Adenocarcinoma↗

PubMed1992

Purification and identification of the heat-stable factor required for pregnenolone-binding protein activity. Evidence that the factor is adenosine 3',5'-diphosphate.

This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.

Adenosine Diphosphate↗

PubMed1992

Comparison of N-glycosides of fetuins from different species and human alpha 2-HS-glycoprotein.

Complex type N-glycosides of commercial bovine fetuin preparations from pooled fetal calf serum have been shown to contain comparable amounts of Gal4,4,4TRI (see structure A below) and Gal4,4,3TRI (structure B) as major asialo-structures. To investigate whether there is a clear genetic specificity for synthesis of these oligosaccharides, N-glycosides from two preparations of bovine fetuin, each from a single calf, were examined. Both of these structures were present in each calf, and there was only a subtle quantitative difference in the ratio of these two structures between the calves. Thus, a specific galactosyltransferase, presumably required for the biosynthesis of the Gal4,4,3TRI structure, may exist in both of these individual calves. Comparison of fetuin N-glycosides was also extended to sheep, pig, and human alpha 2-HS-glycoprotein, the human counterpart of bovine fetuin, using high-pH anion-exchange chromatography of the reducing oligosaccharides as well as HPLC of their pyridinylamino derivatives. The N-glycosides of ovine fetuin also have both Gal4,4,4TRI and Gal4,4,3TRI structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin is of a fucosyl biantennary complex type structure (structure C below) and human alpha 2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated biantennary structure (structure D). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases. [formula: see text]

Amidohydrolases↗

PubMed1992

Glucagon gene 5'-flanking sequences direct expression of simian virus 40 large T antigen to the intestine, producing carcinoma of the large bowel in transgenic mice.

Glucagon and the glucagon-like peptides play important roles in the regulation of glucose homeostasis. Previous studies have demonstrated that approximately 1300 base pairs of rat glucagon gene 5'-flanking sequences direct transgene expression to the pancreas and brain, but not to the intestine, of transgenic mice. These observations suggested that different tissue-specific enhancer elements mediate activation of glucagon gene transcription in the pancreas and intestine. We have now generated mice that express SV40 large T antigen under the control of approximately 2000 base pairs of glucagon gene 5'-flanking sequences. Transgene expression was observed in the brain and pancreas in association with the development of pancreatic endocrine tumors. In contrast to the mice described previously, we also detected transgene expression throughout the gastrointestinal tract in endocrine cells of the stomach and small and large intestine. Focal areas of enteroendocrine cell hyperplasia in the large bowel invariably progressed to invasive and metastasizing plurihormonal endocrine carcinoma, which was clinically and pathologically evident by 4 weeks of age. In contrast, transgene expression in the small bowel and stomach was not associated with progression to either hyperplasia or carcinoma. The results of these studies provide functional evidence for the existence of an upstream cis-acting regulatory domain that directs glucagon gene transcription to the endocrine cells of the intestine in transgenic mice.

Animals↗

PubMed1992

Ultrasonographically guided biopsy of thoracic tumors. A comparison of large-bore cutting biopsy with fine-needle aspiration.

A prospective study to compare the safety and diagnostic accuracy of ultrasonographically guided transthoracic large-bore cutting biopsy histologic examination with fine-needle aspiration cytologic examination was conducted in 149 patients with thoracic tumors (29 mediastinal tumors and 120 pulmonary masses). The authors found that large-bore cutting biopsy under ultrasonographic guidance could be as safe as fine-needle aspiration, whereas diagnostic accuracy was significantly higher (97% versus 59% in malignant tumors, respectively, P less than 0.05; 85% versus 33% in benign lesions, respectively, P less than 0.05). The size, depth, and location of lesions did not influence the results of transthoracic needle aspiration or cutting biopsy. In 77 patients with primary lung cancer, fine-needle aspiration cytologic examination, although achieving 88% positive cytologic results, identified the histologic cell type accurately in only 70%, whereas Tru-Cut (Top Surgical, Tokyo, Japan) biopsy was 97% accurate in confirmative histologic diagnosis. Fourteen patients had discordant cytologic and histologic diagnoses, and the cases of 3 (3.9%) were between small cell lung cancer and non-small cell lung cancer. The diagnostic accuracy of Tru-Cut biopsy also was significantly higher than that of fine-needle aspiration in metastatic cancers (90% versus 33%, respectively) and mediastinal tumors (100% versus 46%, respectively). The authors conclude that transthoracic cutting biopsy under ultrasonographic guidance is safe and has a higher diagnostic accuracy as compared with fine-needle aspiration. This technique is particularly useful for benign lesions or tumors with pleomorphic morphologic characteristics, such as lymphomas and thymomas.

Adult↗

PubMed1992

A non-covalent NH2-terminal pro-region aids the production of active aqualysin I (a thermophilic protease) without the COOH-terminal pro-sequence in Escherichia coli.

The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production. Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential. A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence. In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted.

Base Sequence↗

PubMed1992

Deletion studies to reveal the basis for size discrepancy in proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), an essential component for DNA replication in eukaryotes, is a highly conserved nonhistone nuclear protein of 261 amino acids. The molecular weight of mammalian PCNA, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), differs notably from that predicted by the cDNA sequences, that is, 36,000 in comparison with 29,261 and 28,748 for human and rat PCNA, respectively. To investigate if this discrepancy is due to posttranslational modifications, we studied the PCNA protein synthesized by an in vitro transcription/translation system as well as the protein overproduced in bacteria. We found that both PCNA protein samples were indistinguishable from the authentic protein from the protein mobility in SDS-PAGE. The finding indicates that the size discrepancy is not due to the posttranslational modifications. Hence, the size discrepancy may be due to the protein sequence per se, namely a sequence-related anomaly in SDS-PAGE. Results from the analyses of a series of PCNA derivatives with various lengths of C- or N-terminal deletion indicate that the putative sequence is in the region of residues 128-150.

Amino Acid Sequence↗

PubMed1992

Structure-cardiovascular activity relationships of parathyroid hormone.

The effects of parathyroid hormone (PTH) analogues on isolated rat tail arteries and frog arteries and heart were examined. Synthetic bovine PTH fragment 1 to 34 [bPTH-(1-34)], porcine PTH-(1-34), [Ala1] pPTH-(1-34), and [Nle8] pPTH-(1-34) were tested for vasorelaxant activity on helical strips of the rat tail artery and the frog iliac and femoral artery, preconstricted with arginine vasopressin or arginine vasotocin in the rat and the frog, respectively. For cardiac activity, PTH analogues were administered to isolated frog atria, and atrial rate and atrial tension (AT) were measured. The N-terminal amino acid alanine appears to be pivotal for vasorelaxant activity of PTH molecules on the rat tail artery strips. Data from the frog preparations supported the finding in the rat, although the iliac and femoral arterial preparations differed in their responses. The N-terminal serine in pPTH-(1-34), compared with the other PTH analogues, appeared to be responsible for different cardiac responses, being negatively chronotropic and without effect on AT.

Animals↗

PubMed1992

Quantitative carbohydrate analyses of the tectorial and otoconial membranes of the guinea pig.

Carbohydrate composition of the tectorial membrane (TM) and the otoconial membrane (OM) of the guinea pig was analyzed after hydrolysis, using high-performance anion-exchange chromatography and pulsed amperometric detection. Both of the tissues were highly glycosylated; the carbohydrate content being 24-42% of protein. GlcN, Gal, Glc and Man were found to be the major component sugars of TM, whereas little GalN was found. Fuc and NANA were also present, but NGNA was not detectable. After digestion with thermolysin for solubilization, OM was separated into two fractions: insoluble mineral particles of the otoconia (OM-ppt) and a soluble fraction from the gelatinous layer (OM-sup). These two fractions showed distinct carbohydrate composition from each other. Further analyses using glycosidases revealed that TM contained asialyl and monosialyl but little di-, tri- and tetrasialyl N-glycosides, and OM-sup did not seem to be susceptible to endo-beta-galactosidase, which is known to cleave some N-acetyl-polylactosamine and keratan sulfate. Based on these analyses, it can be suggested that most of the carbohydrates in TM are likely to be asialyl and monosialyl N-glycosides. N-Glycosides may be predominant in the otoconia as well, and a polymer structure consisting of GlcN(Ac) and Gal other than N-acetyl-polylactosamine may exist in the gelatinous layer of OM. O-Glycosylation of the usual type appeared to be minor in all the fractions.

Acetylation↗

PubMed1992

Cardiac activity of calcitonin gene-related peptide in amphibian species (Rana tigrina, Triturus sp).

1. Calcitonin gene-related peptide (CGRP) produced dose-related positive chronotropic and inotropic responses in isolated atria of a newt, Triturus sp. and a frog, Rana tigrina. It was apparent that CGRP was stimulatory on the heart of the amphibian species. 2. In the frog, these dose-related responses were attenuated in the presence of a CGRP antagonist (hCGRP 3 x 10(-8) M). This was indicative of the presence of cardiac CGRP receptors. 3. In the presence of beta-adrenergic (propranolol 10(-6) M) and calcium channel (verapamil 10(-8) M, but not nifedipine 10(-8) M) blocker, the basal AR in the frog were decreased. AR induced by CGRP were increased however. 4. The maximal AR and AT responses of the isolated atria to CGRP were not affected by the presence of propranolol. 5. In the presence of calcium channel blockers (verapamil and nifedipine), the maximal AT response was attenuated. These changes suggest the cardiac effects of CGRP are related to calcium mobilization.

Animals↗

PubMed1992

Cardiac activity of some peptide hormones in the frog, Rana tigrina.

1. The cardiac responses of isolated frog (Rana tigrina) atria to peptide hormones were studied. 2. Calcitonin gene-related peptide (CGRP), arginine vasotocin (AVT), bovine parathyroid hormone fragment (bPTH-(1-34)) and oxytocin (OXY) produced dose-related positive chronotropic and inotropic responses; atrial natriuretic peptide (ANP) was negative chronotropic and inotropic; cholecystokinin (CCK), vasoactive intestinal peptide (VIP) were without effects. 3. The dose-related responses under bPTH-(1-34) stimulation but not CGRP or AVT were attenuated in the presence of ANP (300 ng/ml, approximately 0.98 x 10(-7) M). As expected ANP decreased the basal AR and AT responses of the isolated atria and the inhibitory effects were dose-dependent. 4. As shown previously, propranolol blocked the atrial tension stimulated by bPTH (1-34) but did not alter the cardiac responses to CGRP and AVT. 5. In the presence of beta-adrenergic blocker (propranolol 10(-7) M) or ANP (10(-7) M), the AR and AT changes under ISO stimulation in the frog were also decreased. 6. These cardiac changes suggest the cardiac inhibitory effects of ANP are related to beta-adrenoceptor activity and ANP might be a beta antagonist.

Animals↗

PubMed1992

Biochemistry of carbohydrate-protein interaction.

Recognition of glycoconjugates is an important event in biological systems, and is frequently in the form of carbohydrate-protein interactions. To thoroughly understand these interactions, well-defined carbohydrate ligands must be available. Naturally derived glycoconjugates can be highly purified, and their structures (including conformational structures) can be elucidated to provide such ligands. This requires highly effective methods of separation, such as various forms of high-performance liquid chromatography. Alternatively, structurally well-defined glycoconjugates can be synthesized for this purpose. These include conjugates of carbohydrate derivatives to proteins, lipids, and nonbiological carriers and polymers. The efficacy of these conjugates is amply demonstrated in the studies of carbohydrate-binding proteins from animals. Hepatic carbohydrate receptors, requiring calcium for binding, recognize only the terminal sugar residues. Although different sugar specificities are manifested by different species, there is some commonality in the requirement of the substituents of the sugar rings. Clustering of the target sugars in proper geometric arrangement greatly enhances the binding by these proteins. Some other animal carbohydrate-binding proteins, however, may require penultimate sugars for optimal binding.

Animals↗

PubMed1992

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.

Candida albicans↗