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S Y Lee

Publications and source records attributed to S Y Lee.

At least 19 recordsLinked to original sources
PubMed1997

A regulatory role for TRAF1 in antigen-induced apoptosis of T cells.

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF1 were found as components of the TNFR2 signaling complex, which exerts multiple biological effects on cells such as cell proliferation, cytokine production, and cell death. In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-kappaB, but the role of TRAF1 has not been previously determined. Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8(+) T lymphocytes. Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

Animals↗
PubMed1997

Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli.

A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography. The molecular mass of RGA1 bearing a His tag was approx. 49 kDa. Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots. It exists at particularly high levels in the immature embryo after pellicle extrusion. In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants. When bound to guanosine 5'-(gamma-thio)triphosphate (GTP[S]), the RGA1 protein was partially protected from tryptic proteolysis. In the presence of GTP[S], trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size. When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion. Photoaffinity labelling with [alpha-32P]GTP and a GTP[S]-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide. Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM). The rate of guanine nucleotide binding of RGA1 (kon,GTP[S]=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2. The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP.

Affinity Labels↗
PubMed1997

Effects of pleiotrophic mutations, degUh and spoOA, on the production of foreign proteins using the heterologous secretion system of Bacillus subtilis.

To elevate the production of foreign target proteins using a heterologous protein secretion system of Bacillus subtilis, two different pleiotrophic mutations, degUh for increase of transcriptional level of target genes and spoOA for reduction of extracellular protease activity of a host strain were introduced, respectively. The productivities of three differently originated enzymes, beta-lactamase, streptokinase and human pancreatic trypsin inhibitor (hPSTI) were examined under the each mutation background. By the degUh mutation, the activities of all three enzymes secreted in the culture were increased, although the increased levels were different from 1.2-fold (streptokinase) to 1.8-fold (beta-lactamase). The lower productivity of streptokinase compared to other enzymes under the degUh background was caused by the higher susceptibility to proteolytic degradation. The increased transcriptional level of the beta-lactamase gene by degUh mutation resulted in the accumulation of unprocessed precursor protein in the cytoplasm and cytoplasmic membrane. In the case of the spoOA mutation background, the differences in the levels of the secreted target proteins were not significant and observed only after a stationary phase of the growth.

Bacillus subtilis↗
PubMed1997

Purification and characterization of an antifungal PR-5 protein from pumpkin leaves.

A 28-kDa antifungal PR-5 protein (PLTP) was purified from pumpkin leaves to homogeneity by using ammonium sulfate fractionation, a regenerated chitin column, and reversed-phase column chromatographies on butyl-Toyopearl and HPLC C18 columns. Analysis of 14 N-terminal amino acid sequences of PLTP shows 100% sequence identity to those of two PR-5 proteins, NP24 from tomatoes and AP24 from tobacco. The identical sequence also exhibited high amino acid sequence homology to that of an osmotin-like protein (OLP; 71%) from tobacco cells and thaumatin (64%), a sweet-tasting protein of Thaumatococcus danielli Bench. When the PLTP was immuno-blotted with antiserum raised against the tobacco OLP, the OLP antibody specifically cross-reacted with the PLTP, suggesting that they share several common epitopes in their tertiary structure of the proteins. The purified PLTP rapidly lyzed hyphal tips of Neurospora crassa at a concentration greater than 200 nM and significantly inhibited the fungal growth of Fusarium oxysporum in an agar-disc plate at a concentration greater than 2 microM. It also shows a synergistic effect with nikkomycin, a chitin synthase inhibitor, for the growth inhibition of Candida albicans.

Amino Acid Sequence↗
PubMed1997

Molecular characterization of a rab-related small GTP binding protein cDNA from rice (Oryza sativa L. IR-36).

To study physiological roles of plant small GTP binding proteins, we isolated a cDNA clone (ORrab2) encoding the rab-related small GTP binding protein from rice (Oryza sativa L. IR-36) by using human cDNA rab2 as a probe. The deduced amino acid sequence of the ORrab2 gene shared all the conserved regions, important for GTPase/GTP binding activities, with those of other small GTP binding proteins. ORrab2 is a 1028 bp long cDNA, encodes a 23.2 kDa protein which shows 85.2% similarity on the amino acid sequence level to the Hrab2 protein, and was used as a probe. Through Southern and Northern blot analyses, we found that ORrab2 is a single copy gene and actively expressed at the stages of cell division and elongation. We investigated GTP binding abilities by a filter assay procedure. Deletion of a binding motif, GDTGVGKS, within an ORrab2 protein showed a significant decrease of GTP binding affinity, suggesting its important role in nucleotide binding.

Amino Acid Sequence↗
PubMed1997

Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1.7 A resolution.

alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of alpha-1, 4-glucosidic linkages of starch components, glycogen, and various oligosaccharides. Thermostable alpha-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose. Thermostable alpha-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability. This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins. The three-dimensional structure of thermostable alpha-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography. The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with F0 > 2 sigma F0 between 8.0 and 1.7 A resolution, with root mean square deviations of 0.013 A from ideal bond lengths and 1.72 degrees from ideal bond angles. The final model consists of 469 amino acid residues and 294 water molecules. Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192. Like other alpha-amylases, the polypeptide chain folds into three distinct domains. The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (beta/alpha)8-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between the third beta-strand and the third alpha-helix of domain A. The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel beta-barrel. Neither calcium ion nor chloride ion is located near the active site. This study reveals the architecture of the thermostable alpha-amylase from Bacillus licheniformis. By homology with other alpha-amylases, important active site residues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (beta/alpha)8-barrel. Since many of the stabilizing and destabilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appears to be important for thermostability. The factors responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.

Amino Acid Sequence↗
PubMed1997

TRAF-interacting protein (TRIP): a novel component of the tumor necrosis factor receptor (TNFR)- and CD30-TRAF signaling complexes that inhibits TRAF2-mediated NF-kappaB activation.

Through their interaction with the TNF receptor-associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor-TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-kappaB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor-proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.

Amino Acid Sequence↗
PubMed1997

Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase.

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.

3T3 Cells↗
PubMed1997

Purification and characterization of prophenoloxidase from the hemolymph of coleopteran insect, Holotrichia diomphalia larvae.

Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named pro-PO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction, was observed in the presence of 5 mM Ca2+, with a specific limited proteolysis. The NH2-terminal sequence of generated PO was determined to be NH2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, alpha-chymotrypsin, and SDS.

Amino Acid Sequence↗
PubMed1997

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase.

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 b of recovery period after the depletion of LPL, with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.

3T3 Cells↗
PubMed1997

Molecular mass of poly[(R)-3-hydroxybutyric acid] produced in a recombinant Escherichia coli.

Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 degrees C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phb-CAB genes in Luria-Bertani media containing glucose at 10-30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB.

Acyltransferases↗
PubMed1997

The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1.

Two new members (Bsar1a and Bsar1b) of the Sar1 gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed. The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region. The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sar1 family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER). The sequences contain all the conserved motifs of the Ras superfamily (G1-G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sar1 specific. Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sar1 family and that they have a close relationship to the ARF family. The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sar1 protein. It also exhibited GTP-binding activity. Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B. campestris Sar1 family that consists of 2 to 3 genes. RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds. Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S. cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport. The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sar1 protein in S. cerevisiae and that they may perform similar biological functions.

Amino Acid Sequence↗
PubMed1997

Characterization and solubility measurements of uranium-contaminated soils to support risk assessment.

Remediation of uranium-contaminated soils is considered a high priority by the U.S. Department of Energy because these soils, if left untreated, represent a hazard to the environment and human health. Because the risk to human health is a function of the solubility of uranium in the soils, the objectives of this work are to measure the uranium solubility of two contaminated soils, before and after remedial treatment, and determine the health risk associated with these soils. Two carbonate-rich, uranium-contaminated soils from the U.S. Department of Energy Fernald Environmental Management Project facility near Cincinnati, Ohio, as well as two nearby background soils were characterized and their uranium solubility measured in a 75-d solubility experiment using acid rain, groundwater, lung serum, and stomach acid simulants. Results show that the soluble uranium levels of each soil by each simulant are greatly influenced by their contamination source term. Risk calculations and biokinetic modeling based on the solubility data show that the risks from the soil ingestion and groundwater ingestion pathways are the predominant contributors to the total carcinogenic risk, whereas the risk from the soil inhalation pathway is the smallest contributor to this risk. However, kidney toxicity was the greater health concern of the Fernald Environmental Management Project soils, primarily from undiluted ingestion of the groundwater solution following contact with the contaminated soils. Sensitivity analyses indicate that uranium solubility is a key parameter in defining kidney toxicity; therefore, without proper consideration of the solubility of radionuclides/metals in untreated and treated soils, important factors may be overlooked which may result in soil cleanup goals or limits which are not protective of human health and the environment. We recommend that characterization and measurement of target radionuclides/metals solubilities should also be required by the regulatory community to support the establishment of scientifically-sound, site-specific, soil cleanup goals or limits.

Acid Rain↗
PubMed1997

Evaluation of swallowing safety with fiberoptic endoscope: comparison with videofluoroscopic technique.

Videofluoroscopy has long been viewed as the "gold standard" of swallowing examination for the comprehensive information it provides. However, it is not very efficient and accessible in some practical situations. In this study, we tried to use a modified technique of fiberoptic endoscopic examination of swallowing (FEES) in evaluating dysphagic patients. For each examination, a spoonful of pudding and dyed water were fed in sequence three times. The pharyngeal swallowing events were observed with fiberscope panoramically and videotaped. Twenty-eight chronic dysphagic patients underwent both videofluoroscopy and FEES in 2 weeks. Comparison of the results revealed that disagreements in premature oral leakage to the pharynx, pharyngeal stasis, laryngeal penetration, aspiration, effective cough reflex, and velopharyngeal incompetence were 39.3%, 10.7%, 14.3%, 14.3%, 39.3%, and 32.1%, respectively. FEES was found to be more sensitive in detecting these risky features of swallowing, except with respect to premature leakage. Possible causes of the discrepant results are discussed, and the limitation of videofluoroscopy in practical usage is discussed. FEES is conclusively a safer, more efficient, and sensitive method than videofluoroscopy in evaluating swallowing safety.

Adult↗
PubMed1997

Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.

Attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. However, it is often difficult to ensure correct processing or secretion of the encoded peptides. Here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. We selected the c-myc gene for two main reasons: (i) its protein is known to be well conserved in evolution and to have multiple essential functions during development; and (ii) c-myc family genes have yet to be characterized in insects, thus blockage of essential genes by anti-sense transcripts from a strong virus promoter could provide a sensitive test for the existence of myc-like gene products. An appropriate fragment of the human c-myc gene was inserted downstream from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus and tested in bioassays on Spodoptera frugiperda larvae. Western blot analysis with a human c-myc antibody revealed an endogenous protein band which bound specifically to these antibodies. This band disappeared more rapidly from cells infected with the antisense c-myc recombinant virus than from those infected with c-myc-negative virus. Results of bioassays showed that the antisense construct stopped feeding as soon as the polyhedrin promoter-driven transcripts accumulated, followed shortly by death of the larvae. These results suggest that c-myc-like protein(s) exist in insects and that the antisense strategy is an effective approach to virus insecticide productions.

Animals↗
PubMed1997

A preceding airway reaction to one allergen may lead to priming of the airway responses to another allergen.

This study aimed to determine whether a preceding airway response to one allergen leads to priming of the airway responses to another allergen. Twelve asthmatic children who had positive prick tests to two allergens, Dermatophagoides pteronyssinus (D.p.) and German cockroach (CR), participated in a randomized, placebo-controlled crossover study. We performed two consecutive inhalation challenges, D.p. challenge being followed 48 h later by CR challenge. The effect of initial (D.p.) challenge on the early and late airway responses to the subsequent (CR) challenge (CR2) was examined by comparing the responses with those to CR challenge preceded by sham challenge (CR1). The geometric mean PD20 of CR allergen in the CR2 was 2.8 BU (breath unit) (range of 1 SD; 0.77-10.4), which was 12.0-fold less than that (33.7 BU, 10.8-105.2) in the CR1. The administration of a 6.1-fold less dose (8.9 BU, 2.7-28.8) in the CR2 than in the CR1 (54.5 BU, 44.1-69.3) provoked a similar degree of late-phase reactions (18.7 +/- 7.3% vs 15.8 +/- 9.6%). Our data indicated that the early- and late-phase reactions to CR challenge were augmented by the preceding reaction to D.p. This suggests that a preceding airway response to one allergen may lead to priming, with enhancement of the early and late airway responses to the subsequent challenge with another allergen.

Adolescent↗