Biomedical subjects

K B Lee

Publications and source records attributed to K B Lee.

At least 127 records · Page 7Linked to original sources

PubMed1994

Transfer of modified sialic acids by Trypanosoma cruzi trans-sialidase for attachment of functional groups to oligosaccharide.

Donor specificity of Trypanosoma cruzi trans-sialidase (TcTs) has been investigated with modified 2-[4-methylumbelliferone]-alpha- ketoside of N-acetyl-D-neuraminic acid (4MU-NANA) as donor and lactose as acceptor. 4MU-NANA was treated with periodate under mild conditions to generate an aldehyde on the exocyclic side chain. The oxidized 4MU-NANA was derivatized with various primary amines by reductive amination to yield potential donors. High-performance anion-exchange chromatography equipped with pulsed amperometric detector was used to assay the transglycosylation activity of TcTs. Several modified 4MU-NANA derivatives served as substrates by TcTs and they may be utilized to make valuable intermediates, including those for fluorescence energy transfer measurement or photoaffinity labeling experiment.

Amination↗

PubMed1994

Health care innovation: progress report and focus on biotechnology.

Funding for biomedical research has shifted from government to the private sector. One reason is rapid expansion in the number and strength of U.S. biotechnology companies, which collectively spend more than $6 billion a year on biomedical research. Most of these companies are not yet profitable and therefore depend on flows of capital from private investors, Wall Street, and large pharmaceutical company collaborations. Investment in the new drugs, devices, and vaccines in this pipeline is sensitive to signals emanating from the debate on health care reform, suggesting that new federal policy will have a major impact on steering the type of innovation to emerge in the future.

Biotechnology↗

PubMed1994

Chest wall implantation of carcinoma after fine needle aspiration biopsy.

Implantation of malignant cells along the needle aspiration tract in patients with lung cancer is a rare but potential complication following percutaneous fine needle aspiration biopsy. Dissemination of cancer cells by aspiration biopsy can change resectable, potentially curable lung cancer to unresectable cancer. We report a 55 year male patient who underwent completion pneumonectomy due to squamous cell carcinoma and one cycle of chemotherapy. He developed outgrowing chest wall tumor at the site of needle aspiration biopsy performed prior to completion pneumonectomy and was pathologically diagnosed as metastatic squamous cell carcinoma. The lesion was successfully treated by radical full-thickness resection of the chest wall and reconstruction with latissimus dorsi musculocutaneous island flap.

Biopsy, Needle↗

PubMed1994

Modulation of cytochrome P-450 induction by long-term food restriction in male rats.

The influence of food restriction (FR) on the induction of liver microsomal cytochrome P-450 (P-450) was examined in rats, FR by 40% for 6 weeks caused increase in the levels of P-450, cytochrome b5 (b5), NADPH P-450 reductase, NADH b5 reductase, P-450 dependent testosterone 6 beta-, 16 alpha- and 16 beta-hydroxylases (P < 0.05). Treatment of 3-methylcholanthrene (MC) to food restricted rats (FR rats) induced more P-450, but phenobarbital (PB) induced it less. MC-inducible P-450 from FR rats did not increase its specific ethoxycoumarin O-deethylase activity but it increased testosterone 2 alpha-hydroxylase activity, and the maximum absorption of its CO complex was shifted from 448 nm to 450 nm. PB-inducible P-450 showed less increase in the activities of its specific benzphetamine N-demethylase and it rather significantly decreased testosterone 16 alpha-hydroxylase activity (P < 0.05). These results indicate that the drugs did not induce their unique types of P-450 in FR rats. Such alterations in P-450 induction in FR rats seem to be closely related to the level of P-450 and its related components enhanced by FR, and it could result in modulation of mixed function oxidase system.

Animals↗

PubMed1993

Interpretation of hyperfine shift patterns in ferricytochromes b5 in terms of angular position of the heme: a sensitive probe for peripheral heme protein interactions.

The 1H-NMR hyperfine shift pattern of the heme in a variety of low-spin ferricytochromes b5 has been analyzed in terms of the angular position of the prosthetic group within a structurally and magnetically-conserved protein matrix. A simple model is presented in which the changes in the spread of the predominantly contact shifted methyl and predominantly dipolar shifted meso-H signals of the heme, as well as shift trends for individual signals, provide sensitive indicators of the orientation of the heme relative to the orbital hole (singly-occupied d orbital), which in turn is related to the rhombic magnetic axes. The invariance of the axial His and non-coordinated residue hyperfine shifts show that it is the heme within a relatively rigid protein matrix, rather than the magnetic coordinate system, which is displaced angularly about the heme normal in order to accommodate variations in the polypeptide, orientation of the heme about the alpha,gamma-meso axis, and the length of heme carboxylate chains. Native heme shows increased counterclockwise rotation about the heme normal in the order rat-->beef-->chicken ferricytochrome b5, which is attributed largely to increased bulk of a variable sequence hydrophobic cluster consisting of residues 23, 25 and 32. The two alternate heme orientations about the alpha,gamma-meso axis are shown to also differ by rotation about the heme normal. A semiquantitative estimate of the degree of angular accommodation based on the spread of the meso-H rhombic dipolar shifts indicate rotations of 2-10 degrees. Possible functional consequences of such angular accommodation in relation to the role of these proteins in electron transfer are discussed.

Animals↗

PubMed1993

Cisplatin sensitivity/resistance in UV repair-deficient Chinese hamster ovary cells of complementation groups 1 and 3.

We assessed the possible role of the human repair genes, ERCC1 and ERCC3, in resistance to cisplatin-induced cytotoxicity. The UV repair-deficient Chinese hamster ovary (CHO) 43:3B [designated ERCC1(-)] cell line and its paired subline 83-J5, which is stably transfected with the human DNA excision repair gene ERCC1 [designated ERCC1(+)], were used in this study. UV repair-deficient CHO 27-1 cells [designated ERCC3(-)] and its paired subline designated 'ERCC3(+)', which is stably transfected with the human DNA excision repair gene ERCC3, were also used. In each pair of cell lines, we assessed cisplatin cytotoxicity, cellular drug accumulation and platinum-DNA adduct repair after 1 h drug exposures. Drug accumulation and DNA repair were assessed by atomic absorption spectrometry with Zeeman background correction. ERCC1(+) cells (IC50 = 4.0 microM) were 5-fold more resistant to cisplatin than ERCC1(-) cells (IC50 = 0.75 microM). ERCC1(+) cells repaired 25% of DNA lesions in cellular DNA within a 6 h time period following an IC50 drug exposure and repaired 48% over 24 h. No DNA repair was observed in ERCC1(-) cells during the same time periods. Both cell lines showed similar patterns of drug accumulation. For ERCC3(-) cells (IC50 = 54 microM) and ERCC3(+) cells (IC50 = 49 microM), the profiles of cisplatin sensitivity and cellular drug accumulation were similar. When treated with 50 microM cisplatin, these cells showed similar patterns of drug accumulation, and were equally efficient at forming and repairing lesions in cellular DNA. These data show that in UV repair-deficient CHO cells, ERCC1 confers resistance to cisplatin and confers the ability to remove platinum from cellular DNA. In contrast, ERCC3 does not influence cisplatin drug sensitivity or adduct repair capability. This suggests that ERCC1 may be a determinant of cisplatin resistance, whereas ERCC3 is probably not.

Animals↗

PubMed1993

Taxol effect on cisplatin sensitivity and cisplatin cellular accumulation in human ovarian cancer cells.

Taxol and cisplatin are the two most effective agents discovered to date for treating advanced-stage cancer of the ovary. Learning how best to combine these agents is the focus of preclinical and clinical studies conducted at a number of institutions. Taxol's effect on cellular sensitivity to cisplatin was studied in paired cisplatin-sensitive A2780 and cisplatin-resistant A2780/CP70 human ovarian cancer cell lines. Cisplatin growth curves were generated under conditions of specific sequencing with Taxol, and IC50s (concentrations at which growth is inhibited to 50% of control) for cisplatin were obtained and compared. Taxol was used at an IC10 dose in all experiments. Taxol treatments were for 24 hours and cisplatin treatments were for 1 hour in all experiments. Dimethyl sulfoxide (DMSO) was the diluent for all Taxol stock solutions. Separately, the effects of Taxol and DMSO on cisplatin cellular accumulation were measured. End points reported include measures of cytotoxicity and Taxol effects on cisplatin cellular accumulation. Using a microculture tetrazolium assay, cisplatin growth curves were obtained under the influence of Taxol, at a Taxol dose of 3 nM for both cell lines. DMSO alone had no effect on tumor cell growth. In A2780 cells, the influence of Taxol on cisplatin cytotoxicity was modest, whereas cisplatin-induced cell kill was augmented 1.5-fold when cisplatin was given immediately after Taxol. In A2780/CP70 cells, Taxol augmented cisplatin-induced cell kill by 30-fold when cisplatin was given immediately after Taxol; 75-fold when cisplatin was given 24 hours after completion of Taxol; and 19-fold when cisplatin was given 48 hours after completion of Taxol.(ABSTRACT TRUNCATED AT 250 WORDS)

Cisplatin↗

PubMed1992

An electrophoresis-based assay for glycosyltransferase activity.

Polyacrylamide gel electrophoresis (PAGE) and capillary zone electrophoresis (CZE) were used to measure the activity of glycosyltransferases. Acceptor molecules were prepared by reductive amination of the monopotassium 7-amino-1,3-naphthalenedisulfonic acid (AGA) Schiff base with sugars. The resulting sugar conjugates were purified by gradient PAGE and recovered using semidry electrotransfer into a positively charged nylon membrane. The beta(1----4)galactosyltransferase was shown, by PAGE analysis, to transfer a beta-galactosyl residue to the AGA conjugate of beta-D-GlcNAc-(1----4)-beta-D-GlcNAc-(1----4)-D-GlcNAc (compound 4). Similarly, alpha(1----2)fucosyltransferase isolated from porcine submaxillary glands was shown to transfer fucose from GDP-fucose to the AGA conjugate of beta-D-Gal-(1----4)-beta-D-GlcNAc-(1----6)-D-Gal (compound 5). This conjugate (compound 5) was also an acceptor for the alpha(1----3/4)fucosyltransferase partially purified from human milk. The latter reaction was followed by both gradient PAGE and CZE, having sensitivities of 200 pmol and 80 fmol, respectively.

2-Naphthylamine↗

PubMed1992

Lectin affinity electrophoresis for the separation of fluorescently labeled sugar derivatives.

Lectin affinity electrophoresis was applied to the separation of charged, fluorescent conjugates of disaccharides. Four fluorescent conjugates were prepared by reductive amination of alpha-D-Man-(1----3)-D-Man, alpha-D-Gal-(1----4)-D-Glc, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc in the presence of 7-amino-1,3-naphthalenedisulfonic acid. These charged fluorescent-disaccharide conjugates all have identical molecular weight and in the absence of conconavalin A lectin failed to separate either by agarose or by polyacrylamide gel electrophoresis. In the presence of either free or immobilized concanavalin A, agarose gel electrophoresis and polyacrylamide gel electrophoresis could separate the fluorescent conjugate of alpha-D-Man-(1----3)-D-Man from that of alpha-D-Gal-(1----4)-D-Gal, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc.

2-Naphthylamine↗

PubMed1992

Point mutation at codon 12 of the c-Ha-ras gene in human gastric cancers.

The molecular mechanisms of the carcinogenic process of gastric cancer have not been fully understood yet. In order to know whether c-Ha-ras gene is being involved in the process of gastric carcinogenesis, 8 gastric cancer cell lines, 8 cases of gastric cancer and same number of adjacent dysplasia were analyzed for the presence of mutation at codon 12, 13 and 61 of the c-Ha-ras gene by using polymerase chain reaction (PCR) and mutant-specific oligonucleotide hybridization. Point mutations at codon 12 of the c-Ha-ras gene were found in 2 out of 8 gastric cancer and dysplasia samples in one case, but we found no mutation at codon 13 or 61 of the c-Ha-ras gene. These results suggest that the frequency of mutation of the c-Ha-ras gene detected by sensitive PCR technique is low indeed, however it would be notable that such a genetic change has been detected in the dysplastic lesion of the gastric cancer patient.

Base Sequence↗

PubMed1992

The value of immunohistochemical detection of P-glycoprotein in breast cancer before and after induction chemotherapy.

We have studied the patterns of P-glycoprotein expression before and after 3 cycles of induction chemotherapy (5-fluorouracil, adriamycin and cyclophosphamide) using immunohistochemically stained paraffin-embedded specimen of 28 patients with locally advanced breast cancer. The frequency of P-glycoprotein expression in untreated breast cancer turned out to be very low: only one out of 28 untreated, biopsy specimen at the time of diagnosis was positive. The frequency of P-glycoprotein expression was markedly increased from 9.1% before chemotherapy to 63.6% after induction chemotherapy (p = 0.006). After 3 cycles of induction chemotherapy, 25 patients had obtained clinical response to chemotherapy (4, CR; 21, PR). Eleven out of 25 tumors (44%) showing clinical response and all three tumors (100%) with minimal response have expressed P-glycoprotein. One out of 6 patients (16.7%) with microscopic residual tumor seen in mastectomy specimen expressed P-glycoprotein, whereas 13 of 22 patients (59.1%) with gross residual tumor showed the presence of P-glycoprotein (p = 0.08). The frequency of intrinsic P-glycoprotein expression in untreated breast cancer was quite low, but approximately half of the patients do acquire P-glycoprotein expression during the cycles of induction chemotherapy. Therefore, the results suggest that the immunohistochemical detection of P-glycoprotein on residual tumor cells after induction chemotherapy can predict acquired drug resistance in breast cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

A new method for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates.

A new method is described for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates. The reducing ends of various mono-, di-, tri-, and tetra-saccharides were conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (a fluorescent and negatively charged compound) by reductive amination using sodium cyanoborohydride. The sugar conjugates were purified by preparative gradient polyacrylamide gel electrophoresis followed by a newly developed technique involving their semi-dry transfer to positively charged nylon membranes and elution with sodium chloride. The structures of a monosaccharide- and trisaccharide-conjugate were established by f.a.b.-m.s. and 2D n.m.r. Seven linear oligosaccharide-fluorescent conjugates were treated sequentially with exoglycosidases and with endoglycosidases. Analysis of the products by gel electrophoresis provided sequence information. These methods may be useful for sequencing oligosaccharides that are chemically or enzymically (endoglycosidase) released from glycoproteins, glycolipids, and proteoglycans.

Carbohydrate Sequence↗

PubMed1991

1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation.

The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination. The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.

Cytochrome b Group↗

PubMed1991

1H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b5.

1H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b5 reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl)8-nporphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains at interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b5 is judged much less polar and less sterically accommodating than that of myoglobin. The propionate occupational preference was greatest as the native 7-propionate site, but also possible at the nonnative crystallographic 5-methyl or 8-methyl positions. Only for a propionate at the crystallographic 8-methyl position was a significant perturbation of the native molecular/electronic structure observed, and this was attributed to an alternative propionate-protein hydrogen bond at the crystallographic 8-methyl position. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links at either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.

Animals↗

PubMed1991

Capillary zone electrophoresis for the quantitation of oligosaccharides formed through the action of chitinase.

Capillary zone electrophoresis with fluorescence detection was used to analyze the products formed by chitinase acting on N-acetylchitooligosaccharide-fluorescent conjugates. Six oligosaccharides of the structure [N-acetylglucosamine(1----4)]n (where n = 1-6) were conjugated to 7-amino-1,3-naphthalene disulfonic acid by reductive amination. Each oligosaccharide-fluorescent conjugate was purified by preparative gradient polyacrylamide gel electrophoresis, semi-dry electrotransfer to a positively-charged nylon membrane and recovered by washing the membrane with salt solution. The products formed by treating each oligosaccharide-fluorescent conjugate with chitinase were analyzed by capillary zone electrophoresis. The chitinase treatment hexasaccharide-fluorescent conjugate was also examined kinetically to study the action pattern of this enzyme.

Amination↗

PubMed1991

Expression of prognostic factors (EGFR, ER) by immunohistochemical staining method in male breast cancer.

Twelve male patients with operable breast cancer were evaluated for the expression of prognostic factors by immunohistochemical staining assay. Seven patients were stage I & II, and five patients were stage III. Axillary lymph node positivity was 42%. Nine patients were nuclear grade I, three were nuclear grade II, and none were nuclear grade III. The expression rate of EGFR (epidermal growth factor receptor), ER (estrogen receptor) were 8.3%, 70.0% respectively. This limited data suggest better tumor behavior in male than in female breast cancer. Adjuvant treatment should be considered in male breast cancer just as in females, based on axillary lymph node and ER states.

Breast Neoplasms↗

PubMed1991

A case of intrapericardial extralobar pulmonary sequestration--first case in Korea.

Extralobar pulmonary sequestration, known as accessory lung, is a rare congenital anomaly and intrapericardial lung sequestration is extremely rare. To the best of our knowledge, only four reported cases were intrapericardial. We report a case with intrapericardial extralobar pulmonary sequestration confirmed by operation and morphologic basis.

Adult↗

PubMed1990

1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5.

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme pi system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes b5 is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗