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Biomedical subjects

J Lee

Publications and source records attributed to J Lee.

At least 613 records · Page 34Linked to original sources
PubMed1999

2-Deoxy-D-glucose protects hippocampal neurons against excitotoxic and oxidative injury: evidence for the involvement of stress proteins.

Food restriction can extend life span in rodents and was recently reported to increase the resistance of neurons in the brain to excitotoxic and metabolic insults. In principle, administration to ad libitum fed rodents of an agent that reduces glucose availability to cells should mimick certain aspects of food restriction. We now report that administration of 2-deoxy-D-glucose (2DG), a non-metabolizable analog of glucose, to adult rats results in a highly significant reduction in seizure-induced spatial memory deficits and hippocampal neuron loss. Pretreatment of rat hippocampal cell cultures with 2DG decreases the vulnerability of neurons to excitotoxic (glutamate) and oxidative (Fe2+) insults. The protective action of 2DG is associated with decreased levels of cellular oxidative stress and enhanced calcium homeostasis. 2DG treatment increased levels of the stress-responsive proteins GRP78 and HSP70 in hippocampal neurons, without affecting levels of Bcl-2 or GRP75, suggesting that mild reductions in glucose availability can increase neuronal resistance to oxidative and metabolic insults by a mechanism involving induction of stress proteins. Our findings establish cell culture and in vivo models of "chemical food restriction" which may prove useful in elucidating mechanisms of neuroprotection and in developing preventive approaches for neurodegenerative disorders that involve oxidative stress and excitotoxicity.

Animals↗
PubMed1999

RIP3, a novel apoptosis-inducing kinase.

RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-kappaB pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-kappaB activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.

Amino Acid Sequence↗
PubMed1999

Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast.

Yap1 and Skn7 are two yeast transcriptional regulators that co-operate to activate thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to redox stress signals. Although they are both important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 has a negative effect upon this response. The respective roles of Yap1 and Skn7 in the induction of defense genes by H2O2 were analyzed by two-dimensional gel electrophoresis. Yap1 controls a large oxidative stress response regulon of at least 32 proteins. Fifteen of these proteins also require the presence of Skn7 for their induction by H2O2. Although about half of the Yap1 target genes do not contain a consensus Yap1 recognition motif, the control of one such gene, TSA1, involves the binding of Yap1 and Skn7 to its promoter in vitro. The co-operative control of the oxidative stress response by Yap1 and Skn7 delineates two gene subsets. Remarkably, these two gene subsets separate antioxidant scavenging enzymes from the metabolic pathways regenerating the main cellular reducing power, glutathione and NADPH. Such a specialization may explain, at least in part, the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance.

DNA, Fungal↗
PubMed1999

Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines.

A large naive human single-chain (sc) Fv phage library was used to search for tumor-associated antigens by panning with a lung adenocarcinoma cell line, 1264, and counter-selecting with a nontumor bronchial epithelial cell line, BEAS-2B. After three rounds of subtractive panning, 239 of 673 clones analyzed bound selectively to 1264 tumor cells in a phage ELISA. Diversity analysis of these tumor-selective clones by BstNI fingerprinting and nucleotide sequencing revealed 14 distinct scFv fragments. Four clones bound selectively to 1264 over BEAS-2B cells when analyzed by a more discriminating flow cytometric assay using scFv. Moreover, these clones showed only limited cross-reactivity to several primary human cell lines. One clone, LU30, also cross-reacted strongly with the lung adenocarcinoma line, A549. The LU30 antigen was identified as decay-accelerating factor (CD55) by expression cloning from a 1264 cDNA library. The mean number of decay-accelerating factor molecules on the surface of 1264 and BEAS cells used for panning and counter-selection was estimated as 75,000 +/- 5,000 and 13,000 +/- 10,000, respectively. Thus, phage library panning combined with expression cloning permits identification of antibodies and their cognate antigens for proteins that are differentially expressed on the surface of distinct cell populations.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1999

Methionyl adenylate analogues as inhibitors of methionyl-tRNA synthetase.

Four stable analogues of methionyl adenylate (3-6) were designed as inhibitors of methionyl-tRNA synthetase and synthesized from 2',3'-isopropylideneadenosine. They strongly inhibited aminoacylation activity of methionyl-tRNA synthetases isolated from Escherichia coli, Mycobacterium tuberculosis, Saccharomyces cerevisiae and human. Among the microorganisms tested, however, these chemicals showed the growth inhibition effect only on E. coli.

Adenosine Monophosphate↗
PubMed1999

Protein structure prediction by global optimization of a potential energy function.

An approach based exclusively on finding the global minimum of an appropriate potential energy function has been used to predict the unknown structures of five globular proteins with sizes ranging from 89 to 140 amino acid residues. Comparison of the computed lowest-energy structures of two of them (HDEA and MarA) with the crystal structures, released by the Protein Data Bank after the predictions were made, shows that large fragments (61 residues) of both proteins were predicted with rms deviations of 4.2 and 6.0 A for the Calpha atoms, for HDEA and MarA, respectively. This represents 80% and 53% of the observed structures of HDEA and MarA, respectively. Similar rms deviations were obtained for approximately 60-residue fragments of the other three proteins. These results constitute an important step toward the prediction of protein structure based solely on global optimization of a potential energy function for a given amino acid sequence.

Bacterial Proteins↗
PubMed1999

Differential impact of T cell repertoire diversity in diabetes-prone or -resistant IL-10 transgenic mice.

Expression of IL-10 transgene (tg) in pancreatic beta cells failed to induce autoimmune insulitis and diabetes in (BALB/c x NOD)F1 mice. However, IL-10-expressing tg littermates from backcrosses (N2 and N3) with NOD mice became diabetic at 5 to 10 weeks of age in an MHC-dependent manner. In this study, we tested the possibility that enhancement in frequency of islet antigen (Ag)-specific T cells overrides the protective effects of a diabetes-resistant genetic background and promotes diabetes in IL-10 tg (BALB/c x NOD)F1 mice. For this test, we introduced the IL-10 transgene into tg BDC2.5 mice expressing the islet Ag-specific Vbeta4 T cell repertoire by breeding Ins-IL-10+/BALB/c mice with BDC2.5 mice. The progeny (Ins-IL-10+/BALB/c x BDC2.5+)F1 mice doubly tg for IL-10 and Vbeta4 (BDC2.5) T cell repertoire, developed diabetes at 10 to 18 weeks of age with a much more aggressive T cell infiltrate in the pancreatic islets than in single tg mice. Surprisingly, these diabetic mice were free from acute pancreatitis but had apoptotic beta cells in the islet infiltrate. Conversely, mice tg for Vbeta4 (BDC2.5) T cell repertoire but not IL-10 had no diabetes and no apoptotic beta cells in the islet infiltrate. Therefore, an increase in the frequency of islet-specific T cells apparently overcomes the protection from diabetes by a resistant genetic background. Interestingly, N2 backcross mice doubly tg for Vbeta4 (BDC2.5) T cell repertoire and IL-10, compared to N2 backcross mice tg for IL-10 only, eventually became diabetic but with a delayed onset and reduced incidence of disease. These findings demonstrate that, along with IL-10, an increase in frequency of islet antigen-specific T cells (a) overrides the protective effect of genetic resistance to autoimmune diabetes in F1 mice and (b) delays the onset of an otherwise accelerated diabetes in (Ins-IL-10+/NOD)N2 backcross mice.

Adoptive Transfer↗
PubMed1999

Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC.

The bacterial enhancer-binding protein nitrogen regulatory protein C (NtrC) activates transcription by sigma54-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms of the protein carrying single cysteine residues and derivatized them with a sulfhydryl-specific nitroxide reagent for electron paramagnetic resonance studies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phosphorylation. In only one case, derivatized C86 in alpha-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, derivatized NtrCD86C (NtrCD86C*) activated transcription normally. Additional experiments indicated that the spectral change observed upon phosphorylation of NtrCD86C* was due to interdomain interactions rather than a conformational change within the N-terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrCD86C* is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that alpha-helix 4 of the receiver domain probably plays an important role in communication with the remainder of the protein.

Bacterial Proteins↗
PubMed1999

Potent and orally bioavailable noncysteine-containing inhibitors of protein farnesyltransferase.

Potent and orally bioavailable nonthiol-containing inhibitors of protein farnesyltransferase are described. Oral bioavailability was achieved by replacement of the pyridyl ether moiety of 1 with a 2-substituted furan ether to give 4. Potency was regained with 2,5-disubstituted furan ethers while maintaining the bioavailability inherent in 4. p-Chlorophenylfuran ether 24 is 0.7 nM in vitro (FTase) and is 32% bioavailable in the mouse, 30% bioavailable in rats, and 21% bioavailable in dogs.

Alkyl and Aryl Transferases↗
PubMed1999

mE10, a novel caspase recruitment domain-containing proapoptotic molecule.

Apoptotic signaling is mediated by homophilic interactions between conserved domains present in components of the death pathway. The death domain, death effector domain, and caspase recruitment domain (CARD) are examples of such interaction motifs. We have identified a novel mammalian CARD-containing adaptor molecule termed mE10 (mammalian E10). The N-terminal CARD of mE10 exhibits significant homology (47% identity and 64% similarity) to the CARD of a gene from Equine Herpesvirus type 2. The C-terminal region is unique. Overexpression of mE10 in MCF-7 human breast carcinoma cells induces apoptosis. Mutational analysis indicates that CARD-mediated mE10 oligomerization is essential for killing activity. The C terminus of mE10 bound to the zymogen form of caspase-9 and promoted its processing to the active dimeric species. Taken together, these data suggest a model where autoproteolytic activation of pro-caspase-9 is mediated by mE10-induced oligomerization.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Ibd1p, a possible spindle pole body associated protein, regulates nuclear division and bud separation in Saccharomyces cerevisiae.

The proper spatial and temporal coordination of mitosis and cytokinesis is essential for maintaining genomic integrity. We describe the identification and characterization of the Saccharomyces cerevisiae IBD1 gene, which encodes a novel protein that regulates the proper nuclear division and bud separation. IBD1 was identified by the limited homology to byr4, a dosage-dependent regulator of cytokinesis in Schizosaccharomyces pombe. IBD1 is not an essential gene, and the knock-out cells show no growth defects except for the reduced mating efficiency [1]. However, upon ectopic expression from an inducible promoter, IBD1 is lethal to the cell and leads to abnormal nuclear division and bud separation. In detail, approximately 90% of the IBD1 overexpressing cells arrest at large bud stages with dividing or divided nuclei. In some IBD1 overexpressing cells, spindle elongation and chromosome separation occur within the mother cell, leading to anucleated and binucleate daughter cells. The anucleated cell can not bud, but the binucleate cell proceeds through another cell cycle(s) to produce a cell with multiple nuclei and multiple buds. Observations of the F-actin and chitin rings in the IBD1 overexpressing cells reveal that these cells lose the polarity for bud site selection and growth or attain the hyper-polarity for growth. Consistent with the phenotypes, the IBD1 overexpressing cells contain a broad range of DNA content, from 2 to 4 N or more. A functional Ibd1p-GFP fusion protein localizes to a single dot at the nuclear DNA boundary in the divided nuclei or to double dots in dividing nuclei, suggesting its localization on the spindle pole body (SPB). The cross-species expressions of IBD1 in S. pombe and byr4 in S. cerevisiae cause defects in shape, implicating the presence of a conserved mechanism for the control of cytokinesis in eukaryotes. We propose that Ibd1p is an SPB associated protein that links proper nuclear division to cytokinesis and bud separation.

Cell Cycle Proteins↗
PubMed1999

Cyclic AMP induces inhibition of cyclin A expression and growth arrest in human hepatoma cells.

Classical cytotoxic therapy has been minimally useful in the treatment of hepatocellular carcinoma. In an effort to develop a new approach to the treatment of this neoplasm, we have investigated the signal transduction pathways regulating the growth of human hepatoma cells. In the data reported here, cyclic AMP (cAMP), a negative growth regulator for many cells of epithelial origin, induced G1 synchronization and apoptosis in the HepG2 human hepatoma cell line. The effects of cAMP on the components of the G1/S transition were analyzed. There was no detectable effect of two different cAMP analogs, 8-bromo cAMP or dibutyryl cAMP on the level of the D-type cyclins, cyclin E, cyclin-dependent kinase 2, cyclin-dependent kinase 4, p53, or the cyclin-dependent kinase inhibitors p21 or p27. In contrast, the cAMP analogs induced a dramatic downregulation of cyclin A protein, cyclin A messenger RNA, and cyclin A-dependent kinase activity. Cyclin A-dependent kinase has been shown to be required for the G1-S transition. Furthermore, cyclin A deregulation has been implicated in the pathogenesis of hepatocellular carcinoma. The data reported here suggest a novel signal transduction-based approach to hepatoma therapy.

Apoptosis↗
PubMed1999

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata).

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.

Amino Acid Sequence↗
PubMed1999

Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Acetylcysteine↗
PubMed1999

Potent inhibitors of protein farnesyltransferase: heteroarenes as cysteine replacements.

Synthesis and biological evaluation of heteroarenes as reduced cysteine replacements are described. Of the heteroaryl groups examined with respect to FT inhibitor FTI-276 (1), pyridyl was the replacement found to be most effective. Substitutions at C4 of the pyridyl moiety did not affect the in vitro activity. Compound 9a was found to have moderate in vivo bioavailability.

3T3 Cells↗
PubMed1999

Energy-based de novo protein folding by conformational space annealing and an off-lattice united-residue force field: application to the 10-55 fragment of staphylococcal protein A and to apo calbindin D9K.

The conformational space annealing (CSA) method for global optimization has been applied to the 10-55 fragment of the B-domain of staphylococcal protein A (protein A) and to a 75-residue protein, apo calbindin D9K (PDB ID code), by using the UNRES off-lattice united-residue force field. Although the potential was not calibrated with these two proteins, the native-like structures were found among the low-energy conformations, without the use of threading or secondary-structure predictions. This is because the CSA method can find many distinct families of low-energy conformations. Starting from random conformations, the CSA method found that there are two families of low-energy conformations for each of the two proteins, the native-like fold and its mirror image. The CSA method converged to the same low-energy folds in all cases studied, as opposed to other optimization methods. It appears that the CSA method with the UNRES force field, which is based on the thermodynamic hypothesis, can be used in prediction of protein structures in real time.

Amino Acid Substitution↗
PubMed1999

A new antioxidant with alkyl hydroperoxide defense properties in yeast.

To isolate new antioxidant genes, we have searched for activities that would rescue the tert-butyl hydroperoxide (t-BOOH)-hypersensitive phenotype of a Saccharomyces cerevisiae strain deleted for the gene encoding the oxidative stress response regulator Skn7. We report the characterization of AHP1, which encodes a 19-kDa protein similar to the AhpC/TSA protein family within a small region encompassing Cys-62 of Ahp1p and the highly conserved N-terminal catalytic AhpC/TSA cysteine. Ahp1p contains a peroxisomal sorting signal, suggesting a peroxisomal localization. AHP1 exerts strong antioxidant protective functions, as demonstrated both by gene overexpression and deletion analyses, and is inducible by peroxides in an Yap1- and Skn7-dependent manner. Similar to yeast Tsa1p, Ahp1p forms a disulfide-linked homodimer upon oxidation and in vivo requires the presence of the thioredoxin system but not of glutathione to perform its antioxidant protective function. Furthermore, in contrast to Tsa1p, which is specific for H2O2, Ahp1p is specific for organic peroxides. Therefore, with respect to substrate specificity, Ahp1p differs from Tsa1p and is similar to prokaryotic alkyl hydroperoxide reductase AhpC. These data suggest that Ahp1p is a yeast orthologue of prokaryotic AhpC and justifies its name of yeast alkyl hydroperoxide reductase.

Amino Acid Sequence↗