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Biomedical subjects

J C Lee

Publications and source records attributed to J C Lee.

At least 19 recordsLinked to original sources
PubMed1999

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.

Carbon-Oxygen Lyases↗
PubMed1999

High frequency of p16INK4A gene alterations in hepatocellular carcinoma.

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR-SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5' CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5' CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.

Carcinoma, Hepatocellular↗
PubMed1999

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli.

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.

Amino Acid Substitution↗
PubMed1999

The transcriptional program in the response of human fibroblasts to serum.

The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Blood↗
PubMed1999

Combined morphological and interphase fluorescence in situ hybridization study in multiple myeloma of Chinese patients.

To gain insight into the real incidence of the numeric chromosomal aberrations and the cell lineage involvement of the neoplastic process in multiple myeloma (MM), we examined 18 Chinese MM patients by May-Grunwald-Giemsa (MGG) staining and fluorescence in situ hybridization using three DNA centromeric probes specific for chromosomes 3, 7, and 9. In this investigation, cytogenetic abnormalities were detected in plasma cells (PCs), myeloid cells (MCs), and lymphoid cells (LCs) in all of the MM patients studied. This is the first demonstration of the cytogenetic aberration involved in the myeloid series. Furthermore, the MCs and PCs of 16 MM patients had the same aneuploidies in one or more of the chromosomes analyzed. These data suggest that the neoplastic transformation of MM may occur early in the hematopoietic development. Chromosomal aberrations involving mainly subclones and considerable cellular heterogeneity with gain of a variety of copy numbers of the same chromosome were demonstrated within PCs, which may possibly be the result of an underlying defect of PCs in the control of their number of chromosomes. Whereas PCs showed evidence suggestive of increased polyploidization, MCs and LCs, which exhibited similar chromosomal patterns as the former, rarely did. Thus, the clonal evolution from LC to PC, if that happens in MM, is characterized by chromosomal instability favoring growth of tumor cells with polysomies and polyploidies.

Adult↗
PubMed1999

A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort.

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation, typically starting in early adulthood. IBD is subdivided into two subtypes, on the basis of clinical and histologic features: Crohn disease and ulcerative colitis (UC). Previous genomewide searches identified regions harboring susceptibility loci on chromosomes 1, 3, 4, 7, 12, and 16. To expand our understanding of the genetic risk profile, we performed a 9-cM genomewide search for susceptibility loci in 268 families containing 353 affected sibling pairs. Previous linkages on chromosomes 12 and 16 were replicated, and the chromosome 4 linkage was extended in this sample. New suggestive evidence for autosomal linkages was observed on chromosomes 1, 6, 10, and 22, with LOD scores of 2.08, 2.07, 2.30, and 1.52, respectively. A maximum LOD score of 1.76 was observed on the X chromosome, for UC, which is consistent with the clinical association of IBD with Ullrich-Turner syndrome. The linkage finding on chromosome 6p is of interest, given the possible contribution of human leukocyte antigen and tumor necrosis-factor genes in IBD. This genomewide linkage scan, done with a large family cohort, has confirmed three previous IBD linkages and has provided evidence for five additional regions that may harbor IBD predisposition genes.

Cohort Studies↗
PubMed1999

Persistent Hz-1 virus infection in insect cells: evidence for insertion of viral DNA into host chromosomes and viral infection in a latent status.

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.

Animals↗
PubMed1999

Detection of anti-colon antibodies in inflammatory bowel disease using human cultured colonic cells.

BACKGROUND: Investigation of anti-colon antibodies may be simplified if a sensitive method and homogeneous source of antigen were available. AIMS: To examine the anti-colon antibody response using human colonic carcinoma cell lines as antigen. SUBJECTS: Patients with inflammatory bowel disease and other gastrointestinal disorders and healthy controls were studied. METHODS: Comparative enzyme linked immunosorbent assays (ELISAs) were performed to assess the value of whole Caco-2, HT-29, and LS-180 cells as antigen. The antigenic determinants of the immune response were characterised by western blot analysis. RESULTS: Sera demonstrated immunoreactivity against each of the cell lines, but different epitopes were recognised. Applying whole Caco-2 cells as antigen in an ELISA, the prevalence of anti-colon antibodies was significantly greater in patients with ulcerative colitis (36%) than Crohn's disease (13%), other gastrointestinal disorders (13%) and healthy controls (0) (p<0. 05). The immune response was not associated with one predominant antigen. CONCLUSIONS: Fixed whole cell ELISA is a simple and feasible method for studying the anti-colon antibody response. This response is non-specific, being directed against multiple antigens, and is likely to be an epiphenomenon of inflammatory bowel disease, more so for ulcerative colitis than Crohn's disease.

Adolescent↗
PubMed1998

Hematopoietic cells from -spectrin-deficient mice are sufficient to induce thrombotic events in hematopoietically ablated recipients.

Thrombotic events are life-threatening complications of human hemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sickle cell disease, and thalassemia. It is not clear whether these events are solely influenced by aberrant hematopoietic cells or also involve aberrant nonhematopoietic cells. Spherocytosis mutant (Spna1(sph)/Spna1(sph); for simplicity referred to as sph/sph) mice develop a severe hemolytic anemia postnatally due to deficiencies in -spectrin in erythroid and other as yet incompletely defined nonerythroid tissues. Thrombotic lesions occur in all adult sph/sph mice, thus providing a hematopoietically stressed model in which to assess putative causes of thrombus formation. To determine whether hematopoietic cells from sph/sph mice are sufficient to initiate thrombi, bone marrow from sph/sph or +/+ mice was transplanted into mice with no hemolytic anemia. One set of recipients was lethally irradiated; the other set was genetically stem cell deficient. All mice implanted with sph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histological analyses of marrow recipients showed that thrombi were present in the recipients of sph/sph marrow, but not +/+ marrow. The results indicate that the -spectrin-deficient hematopoietic cells of sph/sph mice are the primary causative agents of the thrombotic events.

Anemia, Hemolytic↗
PubMed1998

Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA.

In vitro synthesized radioactive yeast 35S precursor rRNA (35S pre-rRNA) molecules were used to determine the binding characteristics of 13 proteins from the yeast 60S ribosome subunit. L4, L17, L20 and L25 were found to bind the 35S pre-rRNA molecule in vitro in the absence of any other cellular components as determined by a modified membrane filtration assay and an agarose gel mobility shift assay. In all cases, RNA-protein complex formation was proportional to the amount of protein added to the binding reaction mixture. Binding to the pre-rRNA could be saturated yielding a molar RNA/protein ratio approaching one. Non-radioactive 35S pre-rRNA transcript competed for the binding in a dosage-dependent manner. Presence of 18S rRNA species and poly(A) did not affect their binding to the 35S RNA. However, in the presence of the 25S rRNA species, the four proteins exhibited distinct binding characteristics for the pre-rRNA molecule. L4 did not bind the 25S rRNA but interacted specifically with the 35S pre-rRNA molecule with a binding constant of 4.4x10(6)/M. L17 bound the pre-rRNA molecule preferentially (Ka=17x10(6)/M) but also bound the mature 25S rRNA species (Ka=10x10(6)/M). L20 bound both the pre-rRNA molecule and the 25S rRNA species equally well (Ka=11-12x10(6)/M). L25 also bound both the 35S pre-rRNA and the mature 25S rRNA with slightly different affinities, with Ka=3.1 vs. 2.5x10(6)M, respectively. We speculate that L4, L17, and L25 are among the early assembled ribosomal proteins but L4 may be one of the first ribosomal proteins that bind to the 35S pre-rRNA molecule during ribosome biogenesis.

Binding, Competitive↗
PubMed1998

Pyrimidinylimidazole inhibitors of CSBP/p38 kinase demonstrating decreased inhibition of hepatic cytochrome P450 enzymes.

Pyrimidine analogs of the pyrimidinylimidazole class of CSBP/p38 kinase inhibitors were prepared in an effort to reduce the potent inhibition of hepatic cytochrome P450 observed for the pyridinyl compounds. The substitution of pyrimidin-4-yl, 2-methoxypyrimidin-4-yl, or 2-methylaminopyrimidin-4-yl for pyridin-4-yl effectively dissociates CSBP/p38 kinase from P450 inhibition for this series and furthermore achieves an increase in oral activity.

Animals↗
PubMed1998

Allostery in rabbit pyruvate kinase: development of a strategy to elucidate the mechanism.

Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics. The only amino acid changes in the two isozymes, originating from alternative RNA splicing, occur at a stretch of 55 amino acids in the C domain near the subunit interface. The self-correcting distance geometry (SECODG) program DIAMOD was used to calculate a homology model of these interfacial contacts in the four helix bundle of the kidney PK dimer, based on the X-ray structure of the tetrameric rabbit muscle PK [Larsen et al. (1994) Biochemistry 33, 6301-6309]. Energy refinement with the program FANTOM, using the ECEPP/2 force field to assess packing and electrostatic interactions between the two subunits, yielded two groups of energetically favorable conformations. The primary difference in the two groups is the loop conformation of residue Pro 402, which is serine in muscle PK. In one loop conformation, the conserved Lys 421 can form an intersubunit salt bridge as observed in the muscle PK crystal structure. The other loop conformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used for generating the model. The intersubunit salt bridge leads to an intersubunit hydrogen bonding between Lys 421 of one subunit and Tyr 443 of the other. To provide direct evidence on the roles of these residues, site-directed mutagenesis of the muscle PK gene was conducted. Converting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neither the secondary nor the tetrameric structure, as measured by far UV-CD and sedimentation velocity, respectively. However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more reponsive to regulation by effectors, while the mutant Y443F was essentially equivalent to wild-type muscle PK protein except for a lower affinity to phosphoenolpyruvate. These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK.

Allosteric Regulation↗
PubMed1998

Postischemic hypothermia induced by eugenol protects hippocampal neurons from global ischemia in gerbils.

We studied whether eugenol provides neuroprotection against delayed neuronal death in the hippocampal CA1 region following a 5 min occlusion of the common carotid arteries bilaterally under either free-regulating temperature (TF) or maintained temperature (TM, 37 degrees C) conditions in gerbils. Right after occlusion of the carotid arteries, we injected eugenol intraperitoneally at concentrations of either 50, 100, or 200 mg/kg. There was significant preservation of neuronal cells in the CA1 region in the eugenol-treated groups 7 days after the ischemic insult in the TF condition, with respective survival values of 26, 43, and 68%. In the TM condition, however, significant neuroprotection was only seen with eugenol concentrations of 100 and 200 mg/kg (32% and 52%, respectively). When the rectal temperature was maintained at 38 degrees C for 30 min after occlusion of the carotid arteries, no reduction in CA1 damage was observed with any dose of eugenol. These results suggest that eugenol may provide neuroprotection against ischemic damage by its hypothermic action.

Animals↗
PubMed1998

Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.

Adenosine Triphosphate↗
PubMed1998

The negative dominant effects of T340M mutation on mammalian pyruvate kinase.

A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.

Adenosine Diphosphate↗
PubMed1998

Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL.

TRAIL is a tumor necrosis factor-related ligand that induces apoptosis upon binding to its death domain-containing receptors, DR4 and DR5. Two additional TRAIL receptors, TRID/DcR1 and DcR2, lack functional death domains and function as decoy receptors for TRAIL. We have identified a fifth TRAIL receptor, namely osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homologue that inhibits osteoclastogenesis and increases bone density in vivo. OPG-Fc binds TRAIL with an affinity of 3.0 nM, which is slightly weaker than the interaction of TRID-Fc or DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apoptosis of Jurkat cells. Conversely, TRAIL blocks the anti-osteoclastogenic activity of OPG. These data suggest potential cross-regulatory mechanisms by OPG and TRAIL.

Animals↗
PubMed1998

Specific latent membrane protein 1 gene sequences in type 1 and type 2 Epstein-Barr virus from nasopharyngeal carcinoma in Hong Kong.

We reported previously that a characteristic Epstein-Barr virus latent membrane protein 1 (EBV-LMP1) gene was associated with nasopharyngeal carcinoma (NPC) in Hong Kong. It showed a 30 bp deletion at the carboxyl terminus with specific amino acid substitution Asp at codon 335 with reference to Gly in B95-8 LMP1. This deletion variant Asp335 was present in over 90% of NPC biopsy specimens. The present study attempted to determine the whole encoding sequence of the LMP1 gene in different EBV isolates from NPC, and its relation with EBV types. We found that 92% (34/37) of primary NPC tumours harboured EBV-1 and possessed the LMP1 deletion variant, of which 86% were Asp335 and 6% were Gly335. EBV-2 was present in 8% (3/37) of tumours and all contained the retention variant of the LMP1 gene. Sequencing of the whole encoding region of the LMP1 gene revealed that the deletion variant Asp335 and deletion variant Gly335 carried similar sequences. They showed 43 common nucleotide substitutions in 41 codons with reference to B95-8. The retention variant showed 52 base changes in 46 codons compared with B95-8. The amino acid alterations in both the deletion and retention variants were mostly clustered at the transmembrane domain of the protein. Furthermore, half of the substitutions were common to both variants, suggesting a common evolutionary selection pressure. Nonetheless, the 2 LMP1 variants showed differences in nucleotide alterations and were associated with different EBV types, suggesting the presence of 2 distinct EBV strains in Hong Kong NPC.

Adolescent↗