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J Lee

Publications and source records attributed to J Lee.

At least 19 recordsLinked to original sources
PubMed1992

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R. Significance of amino acid conservation within the Int family and the yeast sub-family.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg308, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed "step-arrest" phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

DNA Mutational Analysis↗
PubMed1992

Fluorescence study of the ligand stereospecificity for binding to lumazine protein.

6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0). D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM. All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay. Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.

Apoproteins↗
PubMed1992

The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor.

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.

Amino Acid Sequence↗
PubMed1992

Construction of lambda gt103, a derivative of phage lambda gt10 that has unique EcoRI, NotI, SacI and SpeI sites and retains positive selection for recombinants.

A phage vector, lambda gt103, that has unique EcoRI, NotI, SacI and SpeI sites within the imm434 cI repressor gene, was constructed by PCR-aided site-directed mutagenesis of lambda gt10 [Huynh et al., DNA Cloning Techniques: A Practical Approach, 1985, pp. 49-78]. This vector allows directional cloning and retains positive selection for recombinants on Escherichia coli C600hfl strains (since only phages with disrupted cI genes plate on this host). Libraries made with this phage vector can be efficiently screened for clones in which a part of the insert is homologous to probe DNAs derived from a plasmid-based library, without cross-hybridization.

Amino Acid Sequence↗
PubMed1992

Comparison of long-term dietary recall between cancer cases and noncases.

A quantitative history of current dietary intake based on 83 food items was administered by interview to a representative sample of 4,809 subjects in Hawaii in 1977-1979. In 1983-1987, this history was readministered to the 131 original respondents who had subsequently developed cancer, as well as 413 randomly selected subjects who remained cancer free. A surrogate was interviewed when the original subject was unavailable. The repeat interview elicited information about diet at the time of the original interview. The authors found that recall values for macronutrients were consistently higher than original levels for both cases and noncases, which may be due in part to a modification in the administration of the repeat questionnaire. Although there were no marked differences overall between cases and noncases in the ability to recall past diet, differences between the two groups were seen in certain subsets of the sample. In the subgroup with the longest recall interval (8-10 years), cases were not able to recall their diets as well as noncases. Also, the difference between original and recall values was larger for cases with colorectal cancer and all cases diagnosed with distant stage disease, compared with noncases. This was not true for cases of breast and prostate cancer and those with localized or regional disease. The following variables were found to have no significant effect on recall for cases or noncases: sex, age, education, and type of respondent (surrogate or subject). Of the five major ethnic groups included in the study, Japanese had the best recall of their past diets, while respondents reporting a diet change between the interviews had poorer recall than did those who reported no change. These results suggest that differential misclassification in dietary case-control studies may pose a significant problem in certain instances, but that these studies can yield meaningful results with certain constraints on the study population.

Aged↗
PubMed1992

Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.

Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.

Affinity Labels↗
PubMed1992

Molecular cloning and characterization of catechol 2,3-dioxygenases from biphenyl/polychlorinated biphenyls-degrading bacteria.

Catechol 2,3-dioxygenases were cloned from Alcaligenes sp. KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putida KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.

Alcaligenes↗
PubMed1992

Characterization of two high affinity human interleukin-8 receptors.

Interleukin 8 (IL-8) and melanocyte growth-stimulatory activity/gro (MGSA) are structurally related proinflammatory cytokines that are chemoattractants and activators of neutrophils. Recently, cDNA clones encoding a high affinity IL-8 receptor (IL-8R-A) and a "low affinity" IL-8 receptor (IL-8R-B) have been isolated from human cDNA libraries. These two receptors have 77% amino acid identity and are members of the G protein-coupled superfamily of receptors with seven transmembrane domains. We have expressed these two receptors in mammalian cells and find that in this system both receptors bind IL-8 with high affinity (Kd approximately 2 nM). The receptor affinities differ for MGSA, however. IL-8R-A binds MGSA with low affinity (Kd approximately 450 nM); IL-8R-B binds MGSA with high affinity (Kd approximately 2 nM). The transfected cells respond to ligand binding with a transient increase in the intracellular Ca2+ concentration. A Ca2+ response is found for IL-8R-A following the binding of IL-8; no response is found for MGSA. A Ca2+ response for IL-8R-B follows the binding of both ligands. Blot hybridization with oligonucleotide probes specific for the two receptors shows that mRNA for both receptors is present in human neutrophils. Analysis of IL-8 and MGSA binding data on neutrophils as well as Ca2+ response and desensitization data shows that the presence of these two IL-8 receptors on the cell surface can account for the profile of these two ligands on neutrophils.

Amino Acid Sequence↗
PubMed1992

Identification of heregulin, a specific activator of p185erbB2.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Amino Acid Sequence↗
PubMed1992

DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands.

Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.

Amino Acid Sequence↗
PubMed1992

Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase.

Single crystals of the non-fluorescent flavoprotein (NFP) purified from Photobacterium leiognathi strain S1 have been grown from ammonium sulphate solutions using the hanging drop vapour diffusion technique. The crystals grow as thin (0.06 mm) plates and belong to the orthorhombic space group C222(1): a = 57.06(3) A, b = 92.41(6) A, c = 99.52(6) A. There is one NFP monomer per asymmetric unit and crystals diffract to 2.2 A spacings on film. A complete native data set to 2.5 A resolution has been collected on a San Diego Multiwire Detector system at the University of Alberta and a heavy-atom derivative search is presently in progress.

Bacterial Proteins↗
PubMed1992

Characterization of catechol 2,3-dioxygenases.

Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.

Alcaligenes↗
PubMed1992

Characterization of complementary DNA clones encoding the rabbit IL-8 receptor.

IL-8 is a proinflammatory cytokine that functions as a chemoattractant for neutrophils. Recently, cDNA clones encoding the human neutrophil IL-8R were isolated by an expression cloning strategy. The amino acid sequence of the human IL-8R was sufficiently similar to a published sequence for an isoform of the rabbit FMLP receptor that we considered the possibility that the rabbit sequence might bind IL-8 as well. In order to establish its ligand specificity, we have isolated and characterized cDNA clones encoding the rabbit receptor. These cDNA clones, when expressed in mammalian cells, confer high affinity IL-8 binding (Kd = 3.6 nM), lack detectable binding of FMLP, and produce a transient increase in the intracellular Ca2+ concentration in response to IL-8 but not to FMLP. These data demonstrate that the reported rabbit FMLP receptor is the rabbit IL-8R, not an isoform of the FMLP receptor. In addition, the amino acid sequence of the rabbit IL-8R encoded by these cDNA clones differs at 23 amino acids (of 355) from that previously published.

Amino Acid Sequence↗
PubMed1992

Synthesis and evaluation of radioiodinated 2-(2(RS)-aminopropyl)-5- iodothiophenes as brain imaging agents.

Methods have been developed for the preparation of 2-(2(RS)-aminopropyl)-5-iodothiophenes. The syntheses and physical properties of 2-(2(RS)-aminopropyl)-5-iodothiophene and N-isopropyl-2-(2(RS)-aminopropyl)-5-iodothiophene are described. The radioiodinated agents are of interest because of the high expected uptake and prolonged brain retention that may result from binding to high-capacity, relatively nonspecific amine binding sites. Radioiodine was introduced into the 5-position of 2-(2(RS)-aminopropyl)-5-iodothiophene and N-isopropyl-2-(2(RS)-aminopropyl)-5-iodothiophene by radioiodination of the corresponding 5-boronic acid or 5-(trimethylstannyl) derivatives. Tissue distribution studies in rats with 2-(2(RS)-aminopropyl)-5-[125I]iodothiophene showed high brain uptake (5 min, 2.77% dose/g; 30 min, 2.51% dose/g) and good brain/blood (B/B) ratios (5 min, 6/1; 30 min 3.8/1. A comparison of the brain uptake of the N-isopropyl derivative with the 2(RS)-aminopropyl analogue demonstrated higher initial brain uptake and brain to blood ratios (5 min, 3.2% dose/g; 10.3/1) but more rapid washout (30 min, 1.37% dose; 2.8/1). These data suggest that radiolabeled 2-(2(RS)-aminopropyl)-5-iodothiophenes are potentially useful agents for cerebral perfusion imaging by single-photon-emission computerized tomography (SPECT).

Animals↗
PubMed1992

Measurements of tumor blood flow using intraperitoneal deuterium and 2H-NMR spectroscopy.

Using i.p. delivered deuterated water, and 2H NMR spectroscopy, it is possible to non-invasively, serially, and quantitatively measure blood flow through tissue volumes as small as 0.05 ml. Measurements made with this technique show that changes in total tumor blood flow reflect local changes measured superficially by laser Doppler flowmetry, and that tumors of identical histology and size have heterogeneous blood flow rates. Finally, we have shown that tumors growing in an irradiated normal tissue have a lower blood flow rate which is manifest when tumors grow beyond 0.1 ml.

Animals↗
PubMed1992

Risk factors for breast cancer by age and menopausal status: a case-control study in Singapore.

A case-control study was conducted among Singapore Chinese women, comprised of 200 histologically confirmed cases of breast cancer and 420 hospital controls. Subjects were interviewed on family history of breast cancer, social and demographic characteristics, reproductive history, and diet one year prior to interview. Differences in risk factors were observed according to menopausal status. In the premenopausal group, the most consistently significant nondietary effect was an increased risk with late age at first birth. In postmenopausal women, the most consistent nondietary effects were increased risks with nulliparity, tall stature, high educational status, and a family history of breast cancer. In premenopausal women, the strongest dietary effects were low risks with high intakes of polyunsaturated fatty acids (PUFA), beta-carotene, soya protein as a proportion of all protein, and a high risk with high red-meat intake. No dietary effects were observed in postmenopausal women. Examination of effects by 10-year age groups suggested that the differences in the effects of age at first birth, nulliparity, height, education, beta-carotene intake, and PUFA intake between premenopausal and postmenopausal women were at least partly attributable to age-related differences in the baseline distributions of these variables. The variation in the effects of red meat and soya protein appeared to be attributable mainly to menopausal status itself, which is consistent with the hypothesis that these factors operate on risk by way of hormonal mechanisms.

Adult↗