Biomedical subjects

K C Lee

Publications and source records attributed to K C Lee.

At least 55 records · Page 3Linked to original sources

PubMed2001

Mechanisms of histamine-induced intracellular Ca2+ release and extracellular Ca2+ entry in MG63 human osteosarcoma cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca2+ dye. Histamine increased ([Ca2+](i)) in a concentration-dependent fashion with an EC(50) value of 0.5 microM. Extracellular Ca2+ removal inhibited the ([Ca2+](i)) signals. Histamine failed to increase ([Ca2+](i)) in Ca2+-free medium after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Addition of Ca2+ induced concentration-dependent ([Ca2+](i)) increases after preincubation with histamine in Ca2+-free medium. Histamine-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The ([Ca2+](i)) increase induced by histamine in Ca2+ medium was abolished by cimetidine, but was not altered by pyrilamine, nifedipine, verapamil, and La(3+). Together, this study shows that histamine increased in ([Ca2+](i)) in osteosarcoma cells by stimulating H2 histamine receptors. The Ca2+ signal was caused by Ca2+ release from the endoplasmic reticulum in a phospholipase C-dependent manner. The Ca2+ release was accompanied by Ca(2+) influx.

Biological Transport↗

PubMed2001

Determination of growth hormone-releasing hexapeptide by reversed-phase high-performance liquid chromatography with electrochemical detection.

A novel HPLC method with electrochemical detection is described for the determination of a growth-hormone-releasing hexapeptide (GHRP-6). HPLC conditions, such as the column, mobile phase, and oxidation potential, were optimized for sensitivity and selectivity of analysis. GHRP-6 was separated on a reversed-phase CN column with 37% acetonitrile in 100 mM phosphate buffer (pH 7.0) as the mobile phase. The optimum electrochemical oxidation signal was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to two electroactive tryptophans and a histidine residue. Solid-phase extraction using octadecyl cartridges was optimized for sample cleanup of GHRP-6 from serum samples and the method was successfully applied over the concentration range of 5 to 100 ng/ml of analyte. reserved.

Chromatography, High Pressure Liquid↗

PubMed2001

Identification of the modifying sites of mono-PEGylated salmon calcitonins by capillary electrophoresis and MALDI-TOF mass spectrometry.

A capillary electrophoretic method (CE) was developed for the determination of the PEG-modification sites of three positional isomers of mono-PEG modified salmon calcitonins (mono-PEG-sCTs). Resistance to proteolytic degradation on the PEG modification sites resulted in different patterns of CE electropherograms for the tryptic digested mono-PEG-sCTs isomers, and the PEG modification sites were assigned accordingly. The PEG-modification sites were also confirmed directly by determining the molecular masses of the tryptic digested PEG-modified fragments of respective mono-PEG-sCT by the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry.

Amino Acid Sequence↗

PubMed2001

Fendiline-Induced Ca2+ movement in A10 smooth muscle cells.

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca2+ levels ([Ca2+]i) in A10 smooth muscle cells was explored by using fura-2 as a Ca2+ indicator. Fendiline at concentrations between 10-50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 20 microM. External Ca2+ removal reduced the Ca2+ signal by 75%. Addition of 3 mM Ca2+ increased [Ca2+]i in cells pretreated with fendiline in Ca2+-free medium. The 50 microM fendiline-induced [Ca2+]i increase in Ca2+-containing medium was inhibited by 10 microM of La3+, nifedipine, or verapamil. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ store partly inhibited 50 microM fendiline-induced Ca2+ release; whereas pretreatment with 50 microM fendiline abolished 1 microM thapsigargin-induced Ca2+ release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 50 microM fendiline-induced Ca2+ release. Incubation with 50 microM fendiline for 10-30 min decreased cell viability by 10-20%. Together, the findings indicate that in smooth muscle cells fendiline induced [Ca2+]i increases. Fendiline acted by activating Ca2+ influx via L-type Ca2+ channels, and by releasing internal Ca2+ in a phospholipase C-independent manner. Prolonged exposure of cells to fendiline induced cell death.

Animals↗

PubMed2001

Differential roles of spinal cholera toxin- and pertussis toxin-sensitive G proteins in nociceptive responses caused by formalin, capsaicin, and substance P in mice.

The aim of the present study is to characterize the roles of spinal cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G proteins in the regulation of various nociceptive responses. The effects of intrathecal (i.t.) pretreatments with CTX and PTX on the formalin (subcutaneous)-, capsaicin (i.t.)-, and substance P (SP; i.t.)-induced nociceptive behaviours were examined in mice. Pretreatment with CTX (i.t.; 24 h before) significantly and dose-dependently (0.05-0.5 microg) suppressed both the first and second phases of the formalin-induced nociceptive behaviour. On the other hand, pretreatment with PTX (i.t., 6 days before) at the same doses (0.05-N0.5 microg) did not affect the formalin-induced response. Capsaicin (i.t., 0.5 microg)- and SP (i.t., 0.7 microg)-induced nociceptive behaviours were attenuated by the pretreatment with CTX. In addition, SP-induced nociceptive response was also attenuated by the pretreatment with PTX. However, the capsaicin-induced nociceptive response was not influenced by PTX pretreatment. These findings suggest that, at the spinal cord level, CTX-sensitive G-proteins are involved in the formalin-, capsaicin-, and SP-induced nociceptive behavioural responses, whereas PTX-sensitive G proteins are involved in SP-induced nociceptive response.

Animals↗

PubMed2001

Sepharose-unbinding ricin E as a source for ricin A chain immunotoxin.

To evaluate the Sepharose-unbinding ricin E as a preference source material for ricin A chain (RTA) in immunotoxin studies, RTA of ricin E (RTA(E)) was characterized and compared with RTA of the Sepharose-binding ricin D (RTA(D)). RTA(E) and RTA(D) were separated into two subunits of A(1) and A(2) by capillary electrophoresis. The isoelectric points of A(1) and A(2) subunits were determined to be 7.6 and 7.4, respectively, for RTA(E), while they were 7.4 and 7.3, respectively, for RTA(D). The molecular masses of A(1) and A(2) isomers determined by the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were 31059 and 32266 Da, respectively, for RTA(E), while they were 30892 and 32179 Da, respectively, for RTA(D). There were no significant differences in the cell surface affinity and cytotoxicity between RTA(E) and RTA(D). Anti-CD4-RTA(E) immunotoxin was prepared by conjugating RTA(E) with anti-CD4 monoclonal antibody using a heterobifunctional crosslinker, 4-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio) toluene. Anti-CD4-RTA(E) immunotoxin showed comparable cytotoxic effects to anti-CD4-RTA(D) immunotoxin to antigen-positive CEM cells in vitro. It is concluded that RTA(E) from ricin E is one of different variants of RTA(D) and may be used as a preference source material of RTA in immunotoxin studies.

Animals↗

PubMed2001

The discovery of indoxacarb: oxadiazines as a new class of pyrazoline-type insecticides.

The evolution of the insecticidal pyrazoline moiety that was originally discovered in 1972 has led to the discovery of a new crop insecticide, indoxacarb, which is the first commercialized pyrazoline-type sodium-channel blocker. Both monocyclic and fused-tricyclic pyrazolines and pyridazines, as well as structurally related semicarbazones were examined prior to the discovery of analogous tricyclic oxadiazines which had similarly high activity as well as favorable environmental dissipation rates and low toxicity to non-target organisms. The eventual leading candidate, DPX-JW062, was originally obtained as a racemic molecule, but a chiral synthesis was developed which produces material that is 50% ee in the insecticidal (+)-S-enantiomer (DPX-MP062, indoxacarb).

Animals↗

PubMed2001

Fine-needle aspiration cytology of pleuropulmonary blastoma: a case report with unusual features.

Pleuropulmonary blastoma (PPB) is a rare and aggressive intrathoracic neoplasm of childhood, typically presenting as a pulmonary and/or pleural-based mass with cystic, solid, or combined features. Histologically, the tumor is well characterized with a mixture of primitive mesenchymal and variably differentiated sarcomatous components, and the cytologic features described in the three previous reports are also compatible to the histologic ones. Now, we present another case of PPB showing unusual features. A 3-yr-old boy presented with a pleural- or chest wall-based tumor. The fine-needle aspiration cytologic smears were highly cellular with poorly differentiated cells showing variable size and shape rather than those of typical blastemal cells. The histologic findings were also distinctive, exhibiting diffuse sheets of poorly differentiated cells without typical blastemal cell component. Diagnosis was confirmed by the support of immunohistochemical and ultrastructural features. The patient underwent a typical aggressive clinical course to death within 8 mo after diagnosis.

Biomarkers, Tumor↗

PubMed2001

Differential expression of p16/p21/p27 and cyclin D1/D3, and their relationships to cell proliferation, apoptosis, and tumour progression in invasive ductal carcinoma of the breast.

In order to understand the intricate relationship of cell proliferation and apoptosis in tumour development, proliferation markers (Ki-67 and c-myc), apoptosis, cell-cycle inducers cyclin D1 and D3, and cell-cycle inhibitors p16(INK4), p21(CIP1), and p27(KIP1) were evaluated in ductal breast carcinoma. The heterogeneous nature of breast tumours provides a system by which the changes in cell-cycle genes can be explored under a wide range of proliferation and apoptotic indices. To address the above issues, immunohistochemical studies were conducted in 40 pairs of tumours and adjacent normal ductal tissues. The TUNEL method was used to identify apoptotic cells. Except for p27/KIP1, the proliferation (Ki-67, c-myc) and the apoptotic indexes together with levels of p16/INK4a, p21/CIP1, cyclin D1, and cyclin D3, were clearly elevated among tumour tissues, while absent in the adjacent normal tissues. Spearman correlation analysis indicated strong associations among apoptotic index, Ki-67, c-myc, and tumour grade. In addition, p21/CIP1 and cyclin D3 were positively correlated, while p16/INK4a, p27/KIP1, and cyclin D1 were negatively correlated with tumour grade. There was clear decoupling between p21 and p27, as well as decoupling between cyclin D1 and cyclin D3, in terms of their relationship to cell proliferation and apoptosis, indicating differential roles in tumour progression.

Apoptosis↗

PubMed2001

Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.

Calcium↗

PubMed2001

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells.

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.

Anticarcinogenic Agents↗

PubMed2001

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells.

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.

Animals↗

PubMed2001

Fluoxetine-induced Ca2+ signals in Madin-Darby canine kidney cells.

The effect of fluoxetine on Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca2+]i concentration-dependently between 5 microM and 200 microM with an EC50 value of 40 microM. The response was reduced by external Ca2+ removal by 30%40%. In Ca2+-free medium pretreatment with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, abolished 100 microM fluoxetine-induced Ca2+ release. Addition of 3 mM Ca2+ to Ca2+-free medium increased [Ca2+]i when cells were pretreated with 100 microM fluoxetine. Suppression of 1,4,5-trisphosphate (IP3) formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 100 microM fluoxetine-induced Ca2+ release. Fluoxetine (5-100 microM) also increased [Ca2+]i in neutrophils, prostate cancer cells and bladder cancer cells from human and rat glioma cells.

Adenosine Triphosphate↗

PubMed2001

Fendiline, an anti-anginal drug, increases intracellular Ca2+ in PC3 human prostate cancer cells.

BACKGROUND: The effects of the anti-anginal drug fendiline on intracellular Ca2+ concentrations ([Ca2+]i) in human PC3 prostate cancer cells were examined. METHODS: [Ca2+]i was measured using the fluorescent dye fura-2. RESULTS: Fendiline (0.5-100 microM) increased [Ca2+]i in a concentration-dependent manner. Ca2+ removal partly inhibited the Ca2+ signals. In Ca2+-free medium, pretreatment with 100 microM fendiline inhibited most of the [Ca2+]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and pretreatment with thapsigargin abolished the fendiline-induced [Ca2+]i increases. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.5-200 microM fendiline in Ca2+-free medium. Pretreatment with 1 microM U73122 to block the formation of inositol-1.4.5-trisphosphate (IP3) did not alter fendiline-induced internal Ca2+ release. CONCLUSIONS: The anti-anginal drug fendiline induced internal Ca2+ release and external Ca2+ entry. Because prolonged increases in [Ca2+]i may lead to cell injury and death, the long-term effect of fendiline on the function of prostate cancer cells should be investigated.

Adenosine Triphosphate↗

PubMed2001

Application of the stretched exponential function to fluorescence lifetime imaging.

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

Animals↗

PubMed2001

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells.

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.

Animals↗

PubMed2001

Cucurbit protoplast isolation for the study of plant virus replication.

A cucurbit protoplast isolation protocol was established for the study of plant virus replication in vivo. This protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods. A combination of Cellulase RS, Macerozyme R10 and mannitol was used as digestion enzymes and osmoticum. An average of 1.7x10(7) protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-alpha. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0x10(6) protoplasts per g of fresh leaf tissue. Electroporation of 10 microg of Zucchini yellow mosaic potyvirus (ZYMV-S) RNA into the protoplasts resulted in virus replication and synthesis of coat protein (CP). SDS-PAGE and immunoblotting were used to detect the CP 48 h post-electroporation. This protocol is highly reproducible and will assist researchers who require cucurbit protoplasts to study virus replication.

Cell Separation↗

PubMed2001

Inhibitory action of water soluble fraction of Terminalia chebula on systemic and local anaphylaxis.

We investigated the effects of the water soluble fraction of Terminalia chebula (Combretaceae) (WFTC) on systemic and local anaphylaxis. WFTC administered 1h before compound 48/80 injection inhibited compound 48/80-induced anaphylactic shock 100% with doses of 0.01-1.0 g/kg. When WFTC was administered 5 or 10 min after compound 48/80 injection, the mortality also decreased in a dose-dependent manner. Passive cutaneous anaphylaxis was inhibited by 63.5+/-7.8% by oral administration of WFTC (1.0 g/kg). When WFTC was pretreated at concentrations ranging from 0.005 to 1.0 g/kg, the serum histamine levels were reduced in a dose-dependent manner. WFTC (0.01-1.0 mg/ml) also significantly inhibited histamine release from rat peritoneal mast cells (RPMC) by compound 48/80. However, WFTC (1.0 mg/ml) had a significant increasing effect on anti-dinitrophenyl IgE-induced tumor necrosis factor-alpha production from RPMC. These results indicate that WFTC may possess a strong antianaphylactic action.

Anaphylaxis↗