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Biomedical subjects

T T Lee

Publications and source records attributed to T T Lee.

At least 73 records · Page 4Linked to original sources
PubMed1992

Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection.

Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A0, and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A0 reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A0 for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage.

Adult↗
PubMed1992

Sn-chlorin e6 antibacterial immunoconjugates. An in vitro and in vivo analysis.

Monoclonal antibody-Sn-chlorin e6 immunoconjugates were prepared by the site-selective covalent modification of the monoclonal oligosaccharide moiety. By carefully controlling the reaction conditions and introducing triethanolamine groups as axial ligands of the Sn moiety, conjugates with in vivo biodistribution properties similar to underivatized IgG were prepared. By varying the reaction conditions, conjugates were reproducibly prepared with a range of photosensitizer to mAb molar ratios from 1.6 to 10. Based on a competitive inhibition radioimmunoassay, conjugates prepared by this method showed selectivity and binding affinity comparable to the unmodified antibody. The immunoconjugates had only slightly lower singlet oxygen yields than that observed with the Sn-chlorin e6 precursor indicating that negligible aggregation or structural modification of the chromophores occurred during the synthesis process. In vitro cell killing experiments demonstrated that all conjugates possessed significant cytotoxic activity. Biodistribution studies in mice showed that conjugates prepared with axial ligands had significant serum retention 24 h after injection while conjugates prepared without the triethanolamine ligand were much more rapidly cleared. In vivo specificity was demonstrated using rats infected with Fisher immunotype I P. aeruginosa at a site in the left posterior thigh muscle. Target to background ratios exceeded 60 at 120 h after conjugate injection of the specific immunoconjugate, compared to a ratio of only 6 for a non-specific mouse IgG conjugate. Biodistribution patterns at 120 h post injection indicate that the conjugates were both biologically active and structurally intact.

Animals↗
PubMed1992

High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis.

A highly sensitive laser-induced (LIF) detection scheme for native, tryptophan- or tyrosine-containing proteins in capillary electrophoresis (CE) has been demonstrated. The 275.4 nm line from an argon-ion laser is used to excite native protein fluorescence. A limit of detection (LOD) (S/N = 2) of 1 x 10(-10) M for conalbumin represents a 140-fold improvement over earlier reports. With stacking at injection, the LOD is 3 x 10(-12) M. Linear dynamic ranges of at least 5 and 4 orders of magnitude for, respectively, tryptophan and bovine serum albumin are found. The practical performance and blueprint of an easily constructed, rugged, compact and user-friendly LIF detector for CE are shown.

Electrophoresis↗
PubMed1991

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing.

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.

Amino Acid Sequence↗
PubMed1991

Cytotoxic principles of Securinega virosa: virosecurinine and viroallosecurinine and related derivatives.

Virosecurinine (1) and viroallosecurinine (2) were isolated as two cytotoxic alkaloids from the leaves of Securinega virosa. A comparison of the cytotoxicity of 1 and several of its derivatives indicates that an alpha,beta- and a gamma,delta-unsaturated lactone located in a strained ring system, such as rings -B, -C, and -D of 1, is structurally required for significant cytotoxicity.

Alkaloids↗
PubMed1988

The effect of molecular weight/lipophilicity on clearance of organic compounds from lungs.

The objective of this study was to test the hypothesis that lipophilicity (as measured by the octanol/water partition coefficient, P) and/or molecular weight are determining factors in the rate of clearance of organic compounds from the lung. Previous work in our laboratory has shown that organic-soluble compounds such as pyrene, benzo[a]pyrene, 1-nitropyrene, 2-aminoanthracene, phenanthridone, dibenzo[c,g]carbazole, 1,3-dichloropropene, and methyl bromide, all of which have a log P less than 6.1, clear the lung rapidly (t 1/2 less than 12 hr). In the present study, organic compounds (mainly anthraquinone dyes) having a wider range of log P's (1.95-8.65) were instilled into rat lungs and the percentage of the compound retained in the lungs at 24 hr was determined. A positive correlation between the log of the theoretical P and the percentage of the compound retained in lungs at 24 hr was found. The lipophilicity of the series of compounds studied was highly dependent on the molecular weight, so that there was also a positive correlation between the molecular weight of the compounds and the percentage of the compound retained in the lung at 24 hr. To help understand the relative importance of lipophilicity and molecular weight in determining lung retention, an additional compound with a high molecular weight but containing a polar functional group [1,5-di(2-sulfo-p-toluidino)anthraquinone] was studied. The results indicated that the lipophilicity was the more important factor in whether the material was retained in the lung. On the basis of the results of this study, organic-soluble compounds with molecular weights less than 300 Da can be expected to clear the lungs rapidly. Nonpolar, organic-soluble compounds with a molecular weight greater than 300 Da can be expected to clear the lungs more slowly.

Animals↗
PubMed1988

Tripling of serum progesterone (P4) and embryonic morphological features in conception and nonconception cycles, after human in vitro fertilization.

A series of 26 in vitro fertilization (IVF) cycles was studied for changes of serum progesterone levels before and after injection of human chorionic gonadotropin (hCG) by a comparison of the number of follicles punctured, number of oocytes recovered, morphological features of oocytes, fertilization rate, cleavage rate, and quality of embryos before their transfer. Subjects were further divided into three groups according to their pregnancy outcome. Five pregnancies were generated and reached the stage of clinical confirmation of live baby delivery or live fetus by ultrasound scanning of the uterine contents. Six patient cycles that had a prolonged luteal phase and beta-hCG levels greater than 40 MIU/ml were described as biochemical pregnancies. Fifteen patient cycles showed no sign of pregnancy. The significant finding of this study is related to the serum progesterone level increase from 20 to 34 hr after hCG injection. The nonpregnancy group yielded a 1.2-fold increase compared with the biochemical pregnancy and pregnancy groups, which had a 3-fold increase. The usage of this multiple effect of progesterone may aid in the prediction of the "high-quality embryo" that might result from a given treatment cycle, indicate the completion of luteinization, the proper timing of hCG to induce final oocyte maturation, and the necessary length of preincubation, and aid in the prediction as to whether or not a pregnancy is likely to result.

Chorionic Gonadotropin↗
PubMed1986

Internally referenced ImmunoConcentration assays.

We have developed a method for incorporating an internal reference zone in addition to the test zone in a single immunoConcentration device. We have illustrated the method by constructing an internally referenced assay for human choriogonadotropin (HCG) in serum. The internally referenced assay, which can be performed in about 5 min, provides a measured overall precision (CV) of approximately 15% at a concentration, in serum, of approximately 50 int. units/L. The test zone consists of an anti-HCG antibody adsorbed onto latex microspheres and entrapped within a porous support. The reference zone contains antibody against alkaline phosphatase (EC 3.1.3.1), which has also been adsorbed onto latex microspheres and trapped within the support. The use of an internal reference moderates the effects of variations in assay conditions on the interpretation of assay results.

Alkaline Phosphatase↗
PubMed1984

Resistance of a hemorrhagic proteinase from timber rattlesnake venom to proteolytic degradation.

The proteolytic activity of hemorrhagic proteinase IV isolated from timber rattlesnake (Crotalus horridus horridus) venom was resistant to inactivation by trypsin, pronase and the proteolytic IIt fraction isolated from timber rattlesnake venom. SDS-polyacrylamide gel electrophoresis of the hemorrhagin incubated alone and with the three proteinases revealed that the addition of trypsin or the IIt fraction caused little apparent degradation of the hemorrhagin, whether or not the samples were reduced prior to electrophoresis. SDS electrophoresis of the hemorrhagin after incubation with pronase revealed a single band of 28,000 apparent molecular weight (as compared to 52,000 for the original hemorrhagin) if the samples were not reduced prior to electrophoresis, and a single band of 17,000 if reduced. If the hemorrhagin was reduced and alkylated, it was much more susceptible to hydrolysis by all three proteinases.

Animals↗
PubMed1983

Effect of glyphosate on ethylene production in tobacco callus.

Glyphosate (N-phosphonomethylglycine) caused a significant decrease or a slight increase in ethylene production in tobacco callus (Nicotiana tabacum L.) depending on the concentration of indole-3-acetic acid (IAA) present in the medium. IAA stimulated ethylene production, but a pretreatment with glyphosate greatly reduced the IAA-induced ethylene production. Inasmuch as glyphosate treatment promoted the metabolism of IAA, the decrease in ethylene production induced by glyphosate is attributed to the rapid loss of free IAA in the treated tissue.

Journal Article↗
PubMed1982

The effect of protease inhibitors on the polyclonal B cell activator from the serum of patients with rheumatoid arthritis.

It has been reported that polyclonal B cell stimulation results in formation of autoantibodies and immune complexes. We have previously reported that a polyclonal B cell activator (PBA) associated with alpha 2-macroglobulin (alpha 2M) is present in the serum of patients with rheumatoid arthritis and related diseases. Here we studied the possibility that patient alpha 2M (Pt-alpha 2M) carries a trypsin-like protease responsible for the PBA activity. This activity was determined by the Ig-turnover assay developed in our laboratories. The small molecular weight protease inhibitors, aprotinin (Trasylol, Bayer) and phenylmethylsulfonylfluoride (PMSF), and the large molecular weight soybean trypsin inhibitor (SBTI) were used. These inhibitors did not affect the PBA activity of dextran sulfate of LPS. However, as expected, trypsin had a PBA-activity which was blocked by all of the above mentioned inhibitors. A trypsin-normal alpha 2M complex (Tr-N alpha 2M) and PBA activity which was inhibited by PMSF or aprotinin but not by SBTI. The PBA associated with Pt-alpha 2M was also inhibited by PMSF or aprotinin but not by SBTI. Moreover, the Tr-N alpha 2M complex and the Pt-alpha 2M, but not that from normal donors, had esterase activity for p-toluenesulfonyl-L-argininemethyl ester. These data suggest a similarity between the Pt-alpha 2M and Tr-N alpha 2M complex. Thus, we concluded that the esterolytic activity is sufficient for PBA activity, that Pt-alpha 2M has esterolytic activity and that this PBA activity can be blocked by small molecular weight protease inhibitors.

Animals↗
PubMed1980

Transfer RNA-Peroxidase Interaction: INHIBITION OF INDOLE-3-ACETIC ACID OXIDATION.

Transfer RNA from wheat germ, yeast, and Escherichia coli inhibited the indoleacetic acid (IAA)-induced spectral change in horseradish peroxidase (EC 1.11.1.7) and the decarboxylation of IAA. The inhibition was limited to a delay after which the increase in A(427) and the decarboxylation of IAA resumed at the same rate as in the control; the duration of the inhibition was dependent on, but not proportional to, the concentration of tRNA. Alkaline hydrolysis destroyed the inhibitory activity of tRNA. The inhibition was completely abolished when the tRNA was added 30 seconds after IAA. Thus, the tRNA appears not to react with the enzyme intermediates formed during the reaction with IAA. The inhibition by tRNA was rapidly reversed by H(2)O(2) or additional IAA, but not by 2,4-dichlorophenol. Results suggest that the tRNA interferes with the initial reaction between IAA and the heme moiety of free peroxidase, thus preventing the formation of highly active enzyme intermediates essential for IAA degradation.

Journal Article↗
PubMed1979

Photoaffinity inactivation of the enkephalin receptor.

An arylazide enkephalin derivative, [D-Ala2,Met5]enkephalin-Tyr-N-(2-nitro-4-azidophenyl) ethylenediamine (ETN), has been synthesized. In the dark, it inhibited the binding of [3H]enkephalinamide to enkephalin receptor-rich NG-108 cell membranes with an I50 = 2.2 X 10(-8) M or KI = 7 X 10(-9) M, assuming competitive inhibition. Photolysis of membranes in the presence of ETN caused irreversible inactivation of the enkephalin receptor, but inactivation was prevented by the addition of enkephalin, the half-effective concentration being 3 x 10(-9) M. ETN appears to be an effective photoaffinity label for the enkephalin receptor.

Affinity Labels↗
PubMed1977

Role of Phenolic Inhibitors in Peroxidase-mediated Degradation of Indole-3-acetic Acid.

7-Hydroxy-2,3-dihydrobenzofuran derivatives, metabolites of a carbamate insecticide carbofuran, and five other phenolic inhibitors of indoleacetic acid (IAA) oxidase interfered with IAA-induced spectral change in the Soret band of horseradish peroxidase (HRP). The onset of IAA degradation required transformed HRP intermediates. The inhibitors, when added before IAA, protected HRP from reacting with IAA, thus preventing formation of highly reactive enzyme intermediates, and consequently, IAA degradation. When added after IAA, the inhibitors quickly reversed the IAA-induced spectral change of HRP and inhibited further IAA degradation.The phenolic inhibitors differed in stability and reactivity. 7-Hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, 3,7-dihydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, catechol, protocatechuic acid, caffeic acid, ferulic acid, and scopoletin belonged to one group which produced only a temporary inhibition to IAA-induced spectral change of HRP and IAA degradation since the inhibitors were metabolized in the reaction. The length of the lag was dependent on the IAA, inhibitor, and enzyme concentrations. 3-Keto-7-hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran and 3-keto-carbofuran belonged to the other group which produced a persistent inhibition.Degradation of IAA required both the heme group and apoprotein of HRP. Reconstituted enzyme from bovine hemin and apoprotein or HRP after unfolding by urea or guanidine treatment were inhibited by the inhibitors in a way similar to the native HRP. The inhibition was reversible by higher concentrations of IAA, but the plot of 1/v versus 1/s and 1/v versus i were curvilinear, reflecting the complex nature of a competitive inhibition.

Journal Article↗
PubMed1976

Inhibition of uridine-cytidine kinase by 5-azacytidine 5'-triphosphate.

5-Azacytidine 5'-triphosphate (5-aza-CTP) inhibited the phosphorylation of uridine, cytidine, and 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. The inhibition appeared to be competitive with respect to the adenosine 5'-triphosphate and noncompetitive with respect to nucleoside substrates. 5-aza-CTP was a potent inhibitor of 5-aza-C phosphorylation but a weak inhibitor of uridine and cytidine phosphorylation. These results suggest that the feedback inhibition of uridine-cytidine kinase by 5-aza-CTP may limit the amount of intracellular nucleotide analog formed in drug-treated cells.

Azacitidine↗