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I Lee

Publications and source records attributed to I Lee.

At least 127 records · Page 7Linked to original sources
PubMed1997

A LEAFY co-regulator encoded by UNUSUAL FLORAL ORGANS.

BACKGROUND: . Development of petals and stamens in Arabidopsis flowers requires the function of the organ-identity gene APETALA3 (AP3), whose RNA is expressed specifically in petal and stamen primordia. AP3 expression is positively regulated by the meristem-identity gene LEAFY (LFY), which is expressed ubiquitously in young flowers. It is unknown how the transition from ubiquitous expression of LFY to region-specific expression of AP3 is made. It has previously been proposed for Antirrhinum that another gene, FIMBRIATA (FIM), mediates between the LFY and AP3 orthologs, with the three genes acting in a simple regulatory hierarchy. FIM is activated later than the LFY ortholog, and its expression is more restricted than that of the LFY ortholog. RESULTS: . We have tested whether the model proposed for Antirrhinum applies to Arabidopsis, by creating transgenic plants in which the FIM ortholog UNUSUAL FLORAL ORGANS (UFO) was expressed constitutively from the promoter of the cauliflower mosaic virus 35S gene. In 35S::UFO flowers, AP3 was expressed precociously and ectopically, confirming that UFO is an upstream regulator of AP3. However, 35S::UFO could not restore petal and stamen development in lfy mutants, indicating that UFO can only function in the presence of LFY activity. The failure of 35S::UFO to rescue lfy mutants is consistent with our observation that UFO expression levels are not markedly changed in lfy mutants. CONCLUSIONS: . We conclude that UFO is not a simple mediator between meristem- and organ-identity genes, but is likely to be a partially dispensable co-regulator that acts together with LFY. The interplay between LFY and UFO provides a paradigm for how a global regulator such as LFY activates selected target genes only in restricted regions within its expression domain.

Arabidopsis↗
PubMed1997

Interlaboratory variation in oxygen tension measurement by Eppendorf "Histograph" and comparison with hypoxic marker.

BACKGROUND AND OBJECTIVES: The median of pO2 values in tumor measured by Eppendorf "Histograph" with a needle-type electrode has been used as a prognostic indicator in cancer patients. However, it is not established that a pretreatment measured pO2 value can be used as a universal predictor of local control probability, because the variation in pO2 values, especially in hypoxic tissue, among institutes may not allow comparison of measured "absolute pO2 values." The purpose of this study was to examine the variation in oxygen tension measurement by Eppendorf "Histograph" among six laboratories using a single batch of mice and tumors and the same detailed protocol. These results were also compared to the immunohistochemical staining of 2-nitromidazole adducts. METHODS: C3H mice bearing FSaII murine fibrosarcoma subcutaneously were shipped to all laboratories, and the oxygen status in tumors and in normal subcutis was examined using Eppendorf "Histograph" and immunohistochemical hypoxic marker. RESULTS: All laboratories showed that the FSaII tumor was hypoxic with at least 77% of measured points under 10 mmHg in pO2 and with a median pO2 value less than that of normal subcutis. These results were further confirmed immunohistochemically. These findings are interpreted as evidence that the pO2 values measured by Eppendorf "Histograph" can be useful. However, the median values of tumor pO2 varied from 1.5 mmHg to 5.6 mmHg among the laboratories, and pO2 of normal subcutis also varied from 28 mmHg to 38 mmHg. There were also significant differences in hypoxic fraction, defined as the fraction under a given oxygen partial pressure (i.e., under 2.5, 5, or 10 mmHg), among institutes. CONCLUSIONS: Caution needs to be exercised in using the absolute, median, or distribution of pO2 values measured by the Eppendorf "Histograph" to compare the data between laboratories or to predict the radiation response in an individual subject.

Animals↗
PubMed1997

Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin.

Molecular dynamics simulations in solution are performed for a rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus (RdPf) and one from the mesophilic organism Desulfovibrio vulgaris (RdDv). The two proteins are simulated at four temperatures: 300 K, 373 K, 473 K (two sets), and 500 K; the various simulations extended from 200 ps to 1,020 ps. At room temperature, the two proteins are stable, remain close to the crystal structure, and exhibit similar dynamic behavior; the RMS residue fluctuations are slightly smaller in the hyperthermophilic protein. An analysis of the average energy contributions in the two proteins is made; the results suggest that the intraprotein energy stabilizes RdPf relative to RdDv. At 373 K, the mesophilic protein unfolds rapidly (it begins to unfold at 300 ps), whereas the hyperthermophilic does not unfold over the simulation of 600 ps. This is in accord with the expected stability of the two proteins. At 473 K, where both proteins are expected to be unstable, unfolding behavior is observed within 200 ps and the mesophilic protein unfolds faster than the hyperthermophilic one. At 500 K, both proteins unfold; the hyperthermophilic protein does so faster than the mesophilic protein. The unfolding behavior for the two proteins is found to be very similar. Although the exact order of events differs from one trajectory to another, both proteins unfold first by opening of the loop region to expose the hydrophobic core. This is followed by unzipping of the beta-sheet. The results obtained in the simulation are discussed in terms of the factors involved in flexibility and thermostability.

Amino Acid Sequence↗
PubMed1997

LEAFY expression and flower initiation in Arabidopsis.

During the initial vegetative phase, the Arabidopsis shoot meristem produces leaves with associated lateral shoots at its flanks, while the later reproductive phase is characterized by the formation of flowers. The LEAFY gene is an important element of the transition from the vegetative to the reproductive phase, as LEAFY is both necessary and sufficient for the initiation of individual flowers. We have analyzed in detail the expression of LEAFY during the plant life cycle, and found that LEAFY is extensively expressed during the vegetative phase. In long days, Arabidopsis plants flower soon after germination, and this is paralleled by rapid upregulation of LEAFY. In short days, Arabidopsis plants flower several weeks later than in long days, but LEAFY expression increases gradually before flowering commences. Application of the plant hormone gibberellin, which hastens flowering in short days, enhances the gradual change in LEAFY expression observed in short days. Changes in LEAFY expression before the transition to flowering suggest that the time point of this transition is at least partly controlled by the levels of LEAFY activity that are prevalent at a given time of the life cycle. This assumption is borne out by the finding that increasing the copy number of endogenous LEAFY reduces the number of leaves produced before the first flower is formed. Thus, LEAFY combines properties of flowering-time and flower-meristem-identity genes, indicating that LEAFY is a direct link between the global process of floral induction and the regional events associated with the initiation of individual flowers.

Arabidopsis↗
PubMed1996

Pharmacological characterization of inositol-1,4,5,-trisphosphate binding to membranes from retina and retinal cultures.

Light and excitatory amino acids (EAA) stimulate the phosphoinositide cycle in the vertebrate retina. The regulation of Ca2+ release from intracellular stores by inositol-1,4, 5-trisphosphate (IP3) involves an interaction of this compound with specific receptors. By means of [3H]IP3-specific binding, we studied the kinetic and pharmacological properties of IP3 receptors in the chick retina as well as in primary cultures of neurons and glia from this tissue. The equilibrium time for the binding reaction was 15 min and was optimal at alkaline pH (8.3). IP3 receptor displayed high affinity (K(B) approximately 40 nM) and selectivity for D-IP3, compared to D-IP4 > L-IP3 > D-IP2 > D-IP1. These characteristics were the same in subcellular fractions from outer (P1) and thinner (P2) plexiform layers, binding sites being more abundant in P2 (2.65 pmol/mg protein). IP3 receptors were present in both neuronal and glial cultures, but were concentrated in neuronal cultures. Binding was not affected by ryanodine, or caffeine, related to calcium-induced calcium release (CICR) channels, nor by the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin, while heparin affectively inhibited IP3 binding. GSSG and thimerosal increased the affinity of [3H]IP3 binding from IC50 approximately 80 nM to IC50 approximately 40 nM; this effect was reversed by DTT. Binding in zero Ca2+ was decreased by low concentrations of Ca2+ (350 nM). These results suggest that actions of IP3 in the retina are regulated by physiological changes in intracellular pH and Ca2+ concentrations, as well as by the oxidation state of the receptor. Additionally, the presence of IP3 receptors in Müller glia opens the possibility of IP3 participation in nonsynaptic signalling through Ca2+ waves in glial cells.

Analysis of Variance↗
PubMed1996

Oxygenation in tumors by modified hemoglobins.

The effect of systemic injection of modified hemoglobin (Hb) prepared from bovine, human, or mouse Hb on tumor oxygenation was investigated. Hb was modified by (1) diisothiocyanatobenzenesulfonate (DIBS) to yield cross-linking within a tetramer; (2) glycolaldehyde (Glyal) to yield cross-linking between and within tetramers; (3) carboxymethylation (Cm) to change oxygen affinity; or (4) poly(ethylene glycol) (PEG) to yield attachment between tetramers. HGL9 (human glioma) in nude mice and FSaII (mice fibrosarcoma) in C3H mice were used as tumor models. Dose and time dependency were detected in the oxygenation effect by bovine-PEG-Hb. Internal cross-linkage prolonged the half-life in the circulation, and thus showed a significant effect. Compared to bovine-CmHb, bovine-DIBS-Hb and bovine-DIBS-CmHb were more effective. Decreasing the oxygen affinity by Cm significantly enhanced tumor oxygenation. Human-DIBS-CmHb was more effective than human-DIBS-Hb. These effects were caused by oxygen carrying capacity of modified Hbs as well as hemodynamic factors, and the injection seemed to reduce both perfusion-limited (acute) and diffusion-limited (chronic) hypoxia.

Animals↗
PubMed1996

Comparison of acute aquatic effects of the oil dispersant Corexit 9500 with those of other Corexit series dispersants.

The acute aquatic toxicity of a new Corexit series dispersant, Corexit 9500, was evaluated and compared with that of others in the series using early life stages of two common nearshore marine organisms: the red abalone (Haliotis rufescens) and a kelp forest mysid (Holmesimysis costata). Spiked-concentration testing was performed under closed, flowthrough conditions, with dispersant concentrations measured in real time using UV spectrophotometry. Median-effect concentrations ranged from 12.8 to 19.7 initial ppm for Haliotis and from 158.0 to 245.4 initial ppm for Holmesimysis. The difference in sensitivity of the two types of tests was consistent with patterns seen with other oil dispersants. Also, these data indicate Corexit 9500 to be of similar toxicity to Corexit 9527 and 9554. Corexit 9500 represents a reformulation of a long-time industry "standard," Corexit 9527, to allow use on higher viscosity oils and emulsions. The present data suggest that acute aquatic toxicity concerns surrounding the use of this newer dispersant should not be significantly different from those associated with the use of Corexit 9527.

Animals↗
PubMed1996

A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones.

This study addresses two important technical problems: how to perform targeted alterations such as site-directed mutagenesis and deletions in large fragments of DNA and how to construct full-length genes from two partly overlapping bacterial artificial chromosome (BAC) plasmids. Given the size and the lack of convenient unique restriction sites in these large-insert bacterial clones, these are nontrivial tasks. Here we describe a simple and efficient protocol based on RecA-assisted restriction endonuclease (RARE) cleavage, a method that enables sequence-specific cleavage of genomic DNA. The same protocol has been used with minor modifications to introduce site-specific mutations into an apolipoprotein-B 90-kb P1 clone, to generate deletions in a 160-kb BAC, and to generate a 160-kb BAC containing the complete 92-kb gene for low-density lipoprotein-related protein-1 (LRP-1) from two smaller overlapping BACs ("BAC marriage").

Apolipoproteins↗
PubMed1995

A chemiluminescent immunoassay for the identification of functional single-chain antibodies.

Random mutagenesis of a template single-chain Fv (scFv) antibody generates a mutant library with members of varying ligand affinities; however, only a small population of these mutants exhibit strong (Kd approximately 1 nM) or improved affinities. We have therefore designed a chemiluminescent sandwich assay which can identify strong ligand-binding mutants expressed by Escherichia coli. A model system involving scFv 43C9 and selected mutants with varying hapten affinities was used to demonstrate the application of this methodology. Both the plaque and colony lifts of these scFvs were screened for hapten-binding activities with the chemiluminescent assay, and only strong binders were detected.

Antibodies, Bacterial↗
PubMed1995

Isolation and characterization of the chicken trypsinogen gene family.

Based on genomic Southern hybridizations and cDNA sequence analyses, the chicken trypsinogen gene family can be divided into two multi-member subfamilies, a six-member trypsinogen I subfamily which encodes the cationic trypsin isoenzymes and a three-member trypsinogen II subfamily which encodes the anionic trypsin isoenzymes. The chicken cDNA and genomic clones containing these two subfamilies were isolated and characterized by DNA sequence analysis. The results indicated that the chicken trypsinogen genes encoded a signal peptide of 15 to 16 amino acid residues, an activation peptide of 9 to 10 residues and a trypsin of 223 amino acid residues. The chicken trypsinogens contain all the common catalytic and structural features for trypsins, including the catalytic triad His, Asp and Ser and the six disulphide bonds. The trypsinogen I and II subfamilies share approximately 70% sequence identity at the nucleotide and amino acid level. The sequence comparison among chicken trypsinogen subfamily members and trypsin sequences from other species suggested that the chicken trypsinogen genes may have evolved in coincidental or concerted fashion.

Amino Acid Sequence↗
PubMed1995

Molecular electronics of a single photosystem I reaction center: studies with scanning tunneling microscopy and spectroscopy.

Thylakoids and photosystem I (PSI) reaction centers were imaged by scanning tunneling microscopy. The thylakoids were isolated from spinach chloroplasts, and PSI reaction centers were extracted from thylakoid membranes. Because thylakoids are relatively thick nonconductors, they were sputter-coated with Pd/Au before imaging. PSI photosynthetic centers and chemically platinized PSI were investigated without sputter-coating. They were mounted on flat gold substrates that had been treated with mercaptoacetic acid to help bind the proteins. With tunneling spectroscopy, the PSI centers displayed a semiconductor-like response with a band gap of 1.8 eV. Lightly platinized (platinized for 1 hr) centers displayed diode-like conduction that resulted in dramatic contrast changes between images taken with opposite bias voltages. The electronic properties of this system were stable under long-term storage.

Journal Article↗
PubMed1995

Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

Animals↗
PubMed1995

Identification and characterization of a human cDNA homologous to yeast SKI2.

A monoclonal antibody, 170A1, which recognizes a nucleolar peptide of molecular weight 90,000, was raised. The protein was conserved among various vertebrates. To characterize the antigen, we screened a human fetal liver expression library using the monoclonal antibody as a probe. Molecular analyses of immunopositive clones suggested the presence of a novel cDNA. It appeared to be a single-copy gene and encoded about 4- and 5-kb mRNAs. The gene appeared to be expressed in every cell tested so far. Its deduced amino acid sequence revealed an overall homology to recently described yeast SKI2. The SKI2 gene of Saccharomyces cerevisiae encoded a nucleolar protein that is involved in the antiviral system. We report here the partial human cDNA sequence and the localization of the corresponding gene on chromosome 6p21.

Amino Acid Sequence↗
PubMed1995

Acute toxicity of the oil dispersant corexit 9554 to marine organisms.

The acute toxicity of a surfactant-based oil dispersant, Corexit 9554, to the early life stages of four marine species was identified using a closed, flow-through exposure system. Standardized, spiked exposures were used in order to impart a measure of reality to the data. The species were taxonomically diverse: a mollusc, the red abalone (Haliotis rufescens); a fish, the topsmelt (Atherinops affinis); a crustacean, the kelp forest mysid (Holmesimysis costata); and a macroalga, the giant kelp (Macrocystis pyrifera). Results indicated the Haliotis test to be most sensitive, followed by the Macrocystis test, and the Atherinops test, with the Holmesimysis test being least sensitive. Median-effect concentration estimates for the four species ranged from 8.0 to 184.3 initial ppm, a > 20-fold difference. Differences in sensitivity of the four tests were likely the result of both internal (morphological, physiological, etc.) and external (life stage, endpoint, etc.) factors.

Animals↗