Biomedical subjects
Publications and source records attributed to C Y Lee.
The chromosomal abnormality t(1;19) is an infrequent finding in adult hematopoietic malignancies. This is only the second report of t(1;19) in association with myelodysplastic syndrome in which there was an apparent excellent response to oral cyclosporin A and a very indolent clinical course.
By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.
Eugenia jambos L. (Myrtaceae) is an antipyretic and anti-inflammatory herb of Asian folk medicine. A 70% acetone extract exerted the strongest cytotoxic effects on human leukemia cells (HL-60) from a preliminary screening of 15 plants. The cytotoxic principles were separated by bio-assay-guided fractionation to HL-60 cells; two hydrolyzable tannins (1-O-galloyl castalagin and casuarinin) were isolated from the 70% acetone extract. All significantly inhibited human promyelocytic leukemia cell line HL-60 and showed less cytotoxicity to human adenocarcinoma cell line SK-HEP-1 and normal cell lines of human lymphocytes and Chang liver cells. Thus, these compounds were exhibited the dose-dependent manner in HL-60 cells and the IC(50) were 10.8 and 12.5 microM, respectively. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G(2)/M phase, and a concomitant increase of cell population at G(1) phase. The apoptosis induced by these two compounds was also demonstrated by DNA fragmentation assay and microscopic observation. These results suggest that the cytotoxic mechanism of both antitumor principle constituents might be the induction of apoptosis in HL-60 cells.
Hyperthermia is a potential anti-cancer regimen but the mode of action is far from clear. Based on the flow cytometric analysis with FITC-annexin V and propidium iodide, apoptosis was found to be the major form of cell death after the treatment with hyperthermia (43 degrees C, 3 h) and/or recombinant murine tumour necrosis factor-alpha (TNF-alpha, 50 ng/ml) in L929 cells. Since mitochondria are thought to play a key role in apoptosis, experiments were done to assess their role in the hyperthermia-mediated apoptosis. Our results indicate that hyperthermia was able to depolarize the mitochondrial membrane potential (delta psi m) and release cytochrome c to the cytoplasm, in a way very similar to the action of TNF-alpha. With the use of cyclosporin A to inhibit the delta psi m dissipation, the cytotoxicity mediated by hyperthermia or TNF-alpha was suppressed. Taken together, our results indicate that hyperthermia and TNF-alpha can induce apoptosis in L929 cells and the mitochondrial dysfunction plays a key role in the cell death process.
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Cardiac hypertrophy is an adaptive response to chronic hemodynamic overload. We employed a whole-genome approach using expressed sequence tags (ESTs) to characterize gene transcription and identify new genes overexpressed in cardiac hypertrophy. Analysis of general transcription patterns revealed a proportional increase in transcripts related to cell/organism defense and a decrease in transcripts related to cell structure and motility in hypertrophic hearts compared to normal hearts. Detailed comparison of individual gene expression identified 64 genes potentially overexpressed in hypertrophy, of 232 candidate genes derived from a set of 77,692 cardiac ESTs, including 47,856 ESTs generated in our laboratory. Of these, 29 were good candidates (P < 0.0002) and 35 were weaker candidates (P < 0.005). RT-PCR of a number of these candidate genes demonstrated correspondence of EST-based predictions of gene expression with in vitro levels. Consistent with an organ under various stresses, up to one-half of the good candidates predicted to exhibit differential expression were genes potentially involved in stress response. Analyses of general transcription patterns and of single-gene expression levels were also suggestive of increased protein synthesis in the hypertrophic myocardium. Overall, these results depict a scenario compatible with current understanding of cardiac hypertrophy. However, the identification of several genes not previously known to exhibit increased expression in cardiac hypertrophy (e.g., prostaglandin D synthases; CD59 antigen) also suggests a number of new avenues for further investigation. These data demonstrate the utility of genome-based resources for investigating questions of cardiovascular biology and medicine.
The present study investigated the site of action of 5-hydroxytryptamine (5-HT) and pharmacologically characterized the receptors involved in regulating blood glucose levels in the crayfish, Procambarus clarkii. Injection of 5-HT into intact animals increased glucose levels in a dose-dependent manner. In contrast, 5-HT failed to elicit a hyperglycemic response in eyestalk-ablated animals. Effects of several 5-HT receptor agonists and antagonists were examined. 5-CT, oxymetazoline (both 5-HT(1) receptor agonists) and alpha-methyl-5-HT (a 5-HT(2) receptor agonist), but not 1-phenylbiguanide, m-CPBG (both 5-HT(3) receptor agonists), or RS 67333 (a 5-HT(4) receptor agonist), induced hyperglycemic responses in a dose-dependent manner. In addition, 8-OH-DPAT (a 5-HT(1A) receptor agonist), L-694,247 (a 5-HT(1B/1D) receptor agonist), and DOI (a 5-HT(2A) receptor agonist) were effective in significantly increasing the glucose levels, whereas both BW 723C86 (a 5-HT(2B) receptor agonist) and m-CPP (a 5-HT(2C) receptor agonist) were ineffective. Finally, ketanserin (a 5-HT(2A) receptor antagonist), but not p-MPPF (a 5-HT(1A) receptor antagonist), GR 55562 (a 5-HT(1B/1D) receptor antagonist), SB 206553 (a 5-HT(2B/2C) receptor antagonist), or tropisetron (a 5-HT(3) receptor antagonist), was able to block 5-HT-induced hyperglycemia. The combined results support the hypothesis that 5-HT exerts its hyperglycemic effect by enhancing the release of hyperglycemic factor(s) from the eyestalks, and suggest that 5 HT-induced hyperglycemia is mediated by 5-HT(1)- and 5-HT(2)-like receptors.
The presence, characteristics and localization of nitric oxide synthase (NOS) in the eyestalk ganglia of the crayfish (Procambarus clarkii) were investigated. Biochemical assay indicated that NOS activity was significantly inhibited by N(G)-nitro-L-arginine (a NOS inhibitor), fluphenazine-N-2-chloroethane (a calmodulin inhibitor) as well as by omitting beta-NADPH or Ca2+ from the incubations. Immunohistochemical studies localized NOS immunoreactivity (IR) to the first chiasma and the sinus gland. Finally, a NOS-immunoreactive protein (138 kDa) was detected by Western blot analysis. These data demonstrate for the first time the existence in crustacean eyestalk ganglia of NOS with biochemical characteristics similar to mammalian constitutive NOS. The localization of NOS-IR suggests that NO may be involved in regulating visual processes and neuroendocrine function in crustaceans.
We have previously reported an association of 14-3-3epsilon isoform with calmodulin. Using the voltage-clamp technique, the present study investigated the potential role of 14-3-3 in modulating the Ca(2+)-activated Cl(-) channel (CaCC) endogenously expressed in Xenopus oocytes. Injection of 14-3-3epsilon antisense oligodeoxynucleotides resulted in potentiation of the ionomycin-induced Cl(-) current, while 14-3-3 peptide and calmodulin inhibitor, W13, suppressed the antisense-potentiated current. The data suggest that 14-3-3epsilon plays an inhibitory role in modulating the CaCC by interacting with the calmodulin-dependent pathway. The potential role of 14-3-3epsilon in other tissues and its therapeutic potential for cystic fibrosis are discussed.
We transplanted rat livers cryopreserved at high subzero temperatures with a protocol that mimics freezing in freeze-tolerant animals. The results of nine transplants show that: (a) every single transplanted liver produced bile, which suggests that the cryopreserved livers retained some physiological function; (b) eight of the animals survived between 2 and 4 h with loss of microvascular integrity which suggests that transplantation failure is related to the circulation and tests of bile production are not indicative of transplantation success; and (c) one animal survived for 5 days with an intact circulation which might be due to an unidentified technical variation or could indicate that when the circulation recovers animals with transplanted livers survive.
A method for demonstrating whether a gene of Staphylococcus aureus is essential for growth in a rich medium is described. We have used this method to determine whether the murE gene, which encodes the UDP-N-acetylmuramyl tripeptide synthetase required for peptidoglycan synthesis, is essential for growth in S. aureus. In this study, strain CYL368 was constructed from S. aureus RN4220 by placing the murE gene in the chromosome under the control of the spac promoter (a hybrid promoter of the Escherichia coli lac operator and the Bacillus subtilis SPO1 phage promoter). To regulate the murE gene in CYL368, the E. coli lacI gene was expressed from the B. licheniformis penicillinase gene (pcn) promoter in plasmid pMJ8426. Strain CYL368(pMJ8426) grew normally in the presence of isopropyl-beta-d-thiogalactopyranoside but could not grow in the absence of the inducer. These results indicate that the murE gene expressed from the spac promoter in CYL368(pMJ8426) is needed for bacterial growth. We concluded that murE is an essential gene of S. aureus.
Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. Here we show that the Drosophila E93 gene determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. We propose that the steroid induction of E93 determines a programmed cell death response during development.
This research was conducted to understand the effects of heat processing and storage on flavanols and sensory qualities of green tea extract. Fresh tea leaves were processed into steamed and roasted green teas by commercial methods and then extracted with hot water (80 degrees C) at 1:160 ratio (tea leaves/water by weight). Green tea extracts were heat processed at 121 degrees C for 1 min and then stored at 50 degrees C to accelerate chemical reactions. Changes in flavanol composition and sensory qualities of green tea extracts during processing and storage were measured. Eight major flavanols (catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, catechin gallate, epigallocatechin gallate, and gallocatechin gallate) were identified in the processed tea extract. Among them, epigallocatechin gallate and epigallocatechin appeared to play the key role in the changes of sensory qualities of processed green tea beverage. The steamed tea leaves produced a more desirable quality of processed green tea beverage than the roasted ones.
Oxalic acid has a strong antibrowning activity. The inhibitory pattern on catechol-PPO model system appeared to be competitive, with a K(i) value of 2.0 mM. When the PPO was incubated with oxalic acid, the activity was not recovered via dialysis, but the inactivated enzyme partially recovered its activity when cupric ion was added. Comparing the relative antibrowning effectiveness of oxalic acid with other common antibrowning agents, oxalic acid with I(50) value of 1.1 mM is as effective as kojic acid and more potent than cysteine and glutathione.
Biotechnology research is developing into genomic analyses that involve the simultaneous monitoring of thousands of genes. The development of various bioinformatics resources that provide efficient access to information is necessary. We have used single-pass sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs). These ESTs data has been widely used to study gene expression in a variety of heart libraries [1, 21]. Data annotation on our recent finding allows us to construct the profiles of genes in the energy metabolizing pathways (glycolysis and glycogen metabolism) that are expressed in heart cDNA libraries. In silico studies of genes of energy metabolism yields data that are consistent with results derived from conventional metabolic experiments. The change in gene profiles describing the metabolism of diseased hearts is also presented here.
Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypodiploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by alpha-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.
Programmed cell death plays an important role in maintaining homeostasis during animal development, and has been conserved in animals as different as nematodes and humans. Recent studies of Drosophila have provided valuable information toward our understanding of genetic regulation of death. Different signals trigger the novel death regulators rpr, hid, and grim, that utilize the evolutionarily conserved iap and ark genes to modulate caspase function. Subsequent removal of dying cells also appears to be accomplished by conserved mechanisms. The similarity between Drosophila and human in cell death signaling pathways illustrate the promise of fruit flies as a model system to elucidate the mechanisms underlying regulation of programmed cell death.
The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.