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Biomedical subjects

K B Lee

Publications and source records attributed to K B Lee.

At least 91 records · Page 5Linked to original sources
PubMed1998

Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3'-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli.

Screening of cDNA libraries for the homologous vertebrate proteins high mobility group (HMG) 1 and 2 using DNA probes based on the coding sequences is likely to result in isolation of both HMG1 and HMG2 clones, as well as pseudogenes, which may be transcribed at low levels. However, the 3'-untranslated regions (UTRs) of HMG1 and 2 are quite distinct, and unusually conserved across species. We have used this property to select the true chicken HMG1 cDNA clone from a chicken lymphocyte cDNA library in lambdagt11, using a probe based on the 3'-UTR of rat HMG1 cDNA. The chicken HMG1 cDNA clone is very similar to all the complete HMG1 cDNA clones isolated so far. We suggest that the sequence designated chicken HMG1 in the GenBank Data Library (Accession number D14314) is, in fact, that of HMG2a [and moreover that the recently reported mouse clone (Accession number AF022465), proposed to encode a new HMG protein, HMG4, is also likely to encode an HMG2a, based on the translated amino-acid sequence and 3'-UTR]. We also report much improved expression of intact recombinant HMG1 in Escherichia coli by the use of chloramphenicol rather than ampicillin selection and conditions that limit cell growth. This should be general for all members of the HMG1 (and 2) family which may be toxic to cells (possibly because of the long acidic tail), and may also prove useful in the production of other such proteins.

3' Untranslated Regions↗
PubMed1998

Determination of free acetaldehyde in total blood for investigating the effect of aspartate on metabolism of alcohol in mice.

To explore the effect of sodium L-aspartate monohydrate (aspartate) as a NAD+ regenerating agent for acetaldehyde in alcohol metabolism, a simple HPLC method has been developed for the measurement of free acetaldehyde in total mice blood digested with alcohol and aspartate. The blood samples were collected in EDTA Vacutainer tubes, and treated with 2,4-dinitrophenylhydrazine (DNP hydrazine) reagent in total blood. Acetaldehyde DNP hydrazone was extracted from total blood and analyzed by HPLC using an Ultrasphere ODS column. The compounds were separated using acetonitrile-water (50:50, v/v) as mobile phase and detected at 356 nm. The detection limit for acetaldehyde DNP hydrazone was 0.1 ppm. A blank determination was carried out for each analysis and subtracted from the results. The amount of acetaldehyde in blood has been determined as a function of time lapse after sole alcohol administration and aspartate ingestion followed by alcohol administration, respectively. This comparative analysis demonstrates that the ingestion of aspartate before the administration of alcohol dramatically decreases the aldehyde level in blood, and aspartate may be utilized as a prospective antagonist for acceleration of ethanol metabolism and prevention of acetaldehyde toxicity.

Acetaldehyde↗
PubMed1998

A phase I trial of continuous hyperthermic peritoneal perfusion with tumor necrosis factor and cisplatin in the treatment of peritoneal carcinomatosis.

BACKGROUND: Tumor necrosis factor (TNF), hyperthermia, and cisplatin have synergistic cytotoxicity against cancer cells in vitro. This combination may be well suited to the regional treatment of peritoneal tumor spread in patients. Continuous hyperthermic peritoneal perfusion (CHPP) is a technique that allows uniform delivery of cytotoxic agents and heat to the peritoneal surface. A Phase I trial of CHPP with TNF and cisplatin was conducted to define the maximum tolerated dose (MTD) for TNF and cisplatin under moderate hyperthermia in the treatment of peritoneal carcinomatosis. METHODS: Twenty-seven patients with peritoneal carcinomatosis underwent exploratory laparotomy and tumor debulking followed by a 90-minute CHPP with cisplatin (100-350 mg/m2) and TNF (0-0.3 mg/L). Perfusion parameters included a perfusate volume of 3-9 L, a peritoneal temperature of 42-43 degrees C, and a flow rate of 1.5 L/minute. Sodium thiosulfate was administered systemically during and after the perfusion as a cisplatin binding agent. RESULTS: There was no operative or treatment-related mortality in this study. CHPP resulted in a 14-fold higher area under the concentration versus time curve (AUC) for cisplatin in the perfusate compared with plasma, and a 4854-fold higher AUC for TNF. The MTD was defined as 250 mg/m2 cisplatin plus 0.1 mg/L TNF. The dose-limiting toxicity was renal insufficiency. No other systemic toxicity was identified, and no significant regional toxicity was identified. The median time to toleration of a regular diet was 8 days (range, 5-20 days). CONCLUSIONS: The favorable regional pharmacologic profile of the combination of cisplatin and TNF suggests that these agents administered via CHPP warrant further evaluation as prophylaxis against or treatment for peritoneal carcinomatosis.

Adult↗
PubMed1998

Familial schizophrenia with pericentric inversion of chromosome 9: a case report.

We report a case with schizophrenia who had a pericentric inversion of chromosome 9 (inv. 9) and whose mother has the same illness and karyotype. Although inv. 9 has been considered to be a common chromosomal rearrangement without specific phenotype, recent evidence has shown that there are several different forms of inv. 9 with differential breakpoints. This indicates that phenotypes of inv. 9 may vary depending on the location of breakpoints. Thus our cases may be valuable material to localize breakpoints on chromosome 9, which may lead to cloning of a susceptibility gene for schizophrenia.

Adult↗
PubMed1998

Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors.

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.

Arrestin↗
PubMed1998

DNA-binding properties of the tandem HMG boxes of high-mobility-group protein 1 (HMG1).

High-mobility-group protein 1 (HMG1) is a conserved chromosomal protein with two homologous DNA-binding HMG-box domains, A and B, linked by a short basic region to an acidic carboxy-terminal tail. NMR spectroscopy on the free didomain (AB) shows that the two HMG boxes do not interact. The didomain has a higher affinity for all DNA substrates tested than single HMG-box domains and has a significantly higher ability to distort DNA by bending and supercoiling. The interaction of the didomain with DNA is stabilized by the presence of the basic region (approximately 20 residues, 9 of which are Lys) that links the second HMG box to the acidic tail in intact HMG1; this may be, at least in part, why this region also enhances supercoiling of relaxed circular DNA by the didomain and circularization of short DNA fragments (in the presence of ligase). Competition assays suggest significantly different structure-specific preferences of single and tandem HMG boxes for four-way junction and supercoiled plasmid DNA. Binding to supercoiled DNA appears to be promoted by protein oligomerization, which is pronounced for the didomains. Electron microscopy suggests that the oligomers are globular aggregates, associated with DNA looping. One box versus two (or several) is likely to be an important determinant of the properties of (non-sequence specific) HMG-box proteins.

Binding Sites↗
PubMed1998

(Ta/Si) multilayer as a wide-bandpass monochromator material.

Specular and non-specular X-ray reflectivity intensities of a (Ta/Si)(60) multilayer sample were measured to characterize its interface structure. Since the multilayer has a good reflectance at its multilayer peaks, its performance as a wide-bandpass monochromator for X-ray scattering experiments of polymers has been tested.

Journal Article↗
PubMed1998

Design and feasibility of quick EXAFS scans for a 'broomstick' double-crystal monochromator at PLS beamline.

A data-collection technique for quick extended X-ray absorption fine-structure spectroscopy (QEXAFS) was developed with a new 'broomstick' double-crystal monochromator, which has been installed for X-ray absorption fine-structure (XAFS) applications at the Pohang Light Source. The monochromator operates in a fixed-exit scan mode as the Bragg angle is varied from 8 to 80 degrees, corresponding to 2-14 keV, using an Si(111) crystal. The monochromator scan capability was investigated by analysing EXAFS data quality from step-scan and from continuous rotation of the Bragg crystal reflection angle. In our fast continuous-scan design, the electronic pulsing speed of the step motor is adjustable to avoid the monochromatic beam instability caused by serious mechanical resonance. The feasibility of QEXAFS scanning is demonstrated by a typical EXAFS scan (e.g. 1 keV range) being taken within 1 min.

Journal Article↗
PubMed1998

New N-glycans in horseradish peroxidase.

We describe here the structures of several new N-glycan oligosaccharides from horseradish peroxidase (HRP). Glycopeptides from HRP were digested with glycoamidase A (from sweet almond) to release the N-glycans. The oligosaccharides were reductively aminated with 2-aminopyridine, and separated by high-performance liquid chromatography on octadecylsilyl- and amide-silica columns for two-dimensional mapping (Tomiya et al., Anal. Biochem. 171, 73-90, 1988). The N-glycans were also analyzed by electrospray ionization mass spectrometry. Two different lots of HRP showed a considerable difference in their N-glycan composition.

Amidohydrolases↗
PubMed1998

Prolyl endopeptidase inhibitors from caryophylli flos.

Three prolyl endopeptidase inhibitors were isolated and identified as luteolin, quercetin and beta-sitosterol-3-O-beta-D-glucopyranoside with IC50 of 0.17, 0.19 and 27.5 ppm, respectively. The inhibition of two flavonoids were non-competitive with substrate. Twenty authentic flavonoids were tested in order to investigate structure-activity relationship. No significant relationship was found in them, however, catechol moiety of B-ring and 7-OH group in flavonoid skeleton were seemed to be responsible for the stronger activity.

Hydrolysis↗
PubMed1998

Isolation and identification of chondroitin sulfates from the mud snail.

Chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitinase ABC, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S) were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of delta Di-OS/delta Di-6S/delta Di-4S was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.

Adjuvants, Immunologic↗
PubMed1998

The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II and a solution-based assay for the binding of a biantennary glycopeptide.

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299-303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (lambda[ex] = 295 nm, lambda[em] = 350 nm) decay is complex and can be described by four decay times with the following values: tau1 = 7.4 nsec, alpha1 = 0.22; tau2 = 2.9 nsec, alpha2 = 0.25: tau3 = 1.0 nsec, alpha3 = 0.34, tau4 = 0.2 nsec, alpha4 = 0.18. The addition of a biantennary glycopeptide (carbohydrate sequence [see text]) to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8 x 10(5) M(-1). The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.

Carbohydrate Sequence↗
PubMed1998

Multi-drug resistance (MDR1) gene expression in de novo acute leukemia cells: correlations with CD surface markers and treatment outcome.

One important mechanism of drug resistance in acute leukemia is the overexpression of the multi-drug resistance (MDR1) gene that encodes a 170-kDa membrane protein called P-glycoprotein. To estimate the incidence and role of MDR1 gene expression in patients with acute leukemia, we investigated the expression of MDR1 by using the RT-PCR method in blast cells from 40 cases of de novo acute leukemia. We found a high frequency of MDR1 gene expression: 10 out of 20 with de novo acute myeloid leukemia (AML), 8 out of 17 with de novo acute lymphoblastic leukemia (ALL), and none of the 3 with de novo acute mixed leukemia, were MDR1 mRNA-positive. No correlation between cluster designation (CD) surface markers (CD19, CD7, CD13, CD33, CD34, CD14, HLA-DR) and MDR1 gene expression in AML was found. The complete remission rate was correlated with MDR1 gene expression. Among 40 evaluable patients examined, 17% (3 of 18) with MDR1 mRNA-positive reached complete remission versus 77% (17 of 22) with MDR1 mRNA-negative (p=0.044). These results suggest that MDR1 gene expression can be used as a prognostic factor and may be helpful in determining chemotherapeutic protocol for patients with acute leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Pancytopenic prodrome (pre-ALL) of acute lymphoblastic leukemia in adults: possible pathogenesis.

We report two cases of adult acute lymphoblastic leukemia presenting with preleukemic phase of pancytopenia with a few abnormal lymphoid cells in bone marrow aspirates. The initial diagnosis of each case was suspicious aplastic anemia and hypoplastic anemia. Both cases progressed to overt acute lymphoblastic leukemia within 1 year. We suggest that initial pancytopenic phase (pre-ALL) may precede the diagnosis of acute lymphoblastic leukemia in adults and differential diagnosis from myelodysplastic syndrome and primary aplastic anemia will be needed. We also suggest that primary bone marrow lymphoma and "primary unknown metastatic lymphoma of bone marrow" may be possible as the pathogenesis in a case like ours.

Adult↗
PubMed1997

Internalization of the m2 muscarinic acetylcholine receptor. Arrestin-independent and -dependent pathways.

Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergic receptors (mAChR) from G-proteins and sequestration of the receptors away from the cell surface. However, mutant m2 mAChRs were identified that were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs. We have tested whether these properties might stem from differences in the abilities of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor/G-protein uncoupling and internalization. We have determined that arrestin binding requires phosphorylation at a cluster of Ser/Thr residues in amino acids 307-311 in the m2 mAChR. A strong correlation was found between the ability of WT and mutant receptors to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays. However, the phosphorylation-dependent internalization of the m2 mAChR in HEK-tsA201 cells did not require arrestins and did not proceed via clathrin-mediated endocytosis. While the m2 mAChR was able to enter a clathrin- and arrestin-dependent pathway when arrestin 2 or arrestin 3 was significantly overexpressed, the preferred pathway of internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may involve arrestin-dependent and -independent events.

Animals↗
PubMed1997

Synergistic effect and possible mechanisms of tumor necrosis factor and cisplatin cytotoxicity under moderate hyperthermia against gastric cancer cells.

BACKGROUND: Peritoneal carcinomatosis is a difficult management problem, and intraperitoneal treatment approaches may provide an opportunity to intensify dose and minimize toxicity. The current experiments were conducted to characterize the cytotoxic effects of cisplatin (cDDP), tumor necrosis factor (TNF), and hyperthermia (HT) on a gastric cancer cell line in vitro under conditions achievable with intraperitoneal treatment. METHODS: Seoul National University gastric cancer cell line (SNU-5), a poorly differentiated gastric cancer cell line, was tested for sensitivity to various doses of cDDP, TNF, or combinations of the two at normothermia (37 degrees C) or HT (42.5 degrees C). The effect of TNF on cellular rates of cDDP accumulation, efflux, and cDDP-DNA adduct formation were evaluated using atomic absorbance spectrometry with Zeemen background correction. RESULTS: During a 2-h exposure to various doses of cDDP HT, we observed a supraadditive cytotoxicity of SNU-5 with 1 to 50 micrograms/ml of TNF (p2 = 0.0001). In the presence of the three-agent combination (HT, TNF, and cDDP) we observed statistically significant increases in total cellular accumulation of cisplatin (p2 = 0.016); a nonsignificant decrease in cellular efflux of drug (p2 = 0.098); and a 40% increase in persistent cisplatin DNA damage as measured by atomic absorption spectrophotometry (p2 = 0.06). These patterns were specifically not seen with the combinations of cDDP and HT, or cDDP and TNF. CONCLUSIONS: These data provide the experimental basis for the use of TNF and cDDP with HT in the treatment of gastric cancer and support the investigation of these agents in vivo in the regional treatment of peritoneal carcinomatosis.

Antineoplastic Agents↗