Biomedical subjects
Publications and source records attributed to W K Liu.
We report the first demonstration to our knowledge of an ultrabroad emission laser using InGaAs/GaAs quantum dots by cycled monolayer deposition. The device exhibits a lasing wavelength coverage of approximately 40 nm at an approximately 1160 nm center wavelength at room temperature. The broadband signature results from the superposition of quantized lasing states from highly inhomogeneous dots.
The present study has demonstrated a differential cytotoxicity of stellettin A (1) between human leukemia HL-60 cells (IC50 0.4 microg/mL) and human prostate cancer LNCaP cells (IC50 120 microg/mL). Treatment of cells with 1 revealed the activation of NADPH oxidase, the dramatic generation of reactive oxygen species, and the dissipation of mitochondrial membrane potentials, with HL-60 cells being more sensitive than LNCaP cells by an order of magnitude. Immunoblotting analysis further demonstrated a stronger upregulation of the apoptosis marker proteins, FasL and caspase-3, in HL-60 cells, and pretreatment of cells with antisense oligonucleotide for caspase-3 abolished apoptosis. All available evidence suggests that 1 induces oxidative cell death through a FasL-caspase-3-apoptotic pathway.
Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRIamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)RIamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)RIamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)RIamide peptides. Immunocytochemistry showed the localization of -FVRIamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRIamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens.
A novel beta-carboline alkaloid, tangutorine (benz[f]indolo[2,3-a]quinolizidine) was isolated from the leaves of Nitraria tangutorum L. [Duan JA, Williams ID, Che CT, Zhou RH, Zhao RH, Tangutorine: a novel beta-carboline alkaloid from Nitraria tangutorum. Tetrahedron Lett 1999;40:2593-6], and its unique structural characters led us to initiate a study of its potential anti-proliferation activity. The in vitro treatment with low doses of tangutorine slightly stimulated the proliferation of human colon cancer HT29 cells until at concentrations higher than 6.25 microg/ml when the cell numbers, cellular MTT reduction, and cell proliferation by 3H-thymidine incorporation decreased in a dose-dependent manner (IC50=15 microg/ml=48 microM). Morphological studies of cells by fluorescence and electron microscopy did not show features for apoptosis but only large vacuoles, swollen mitochondria and dense cytoskeletal filaments bunching in the cytoplasm. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of topoisomerase II expression at 25 microg/ml tangutorine, thereby impeding cell progression from S to G2/M phase. Cells accumulated at G1 phase of the cell cycle at concentrations > or =50 microg/ml tangutorine. Interestingly, some cells escaped from prolonged growth arrest without cell division and resulted in binucleated and polyploid G1 cells. Taken all results together, tangutorine induced a p21 suppression of all cyclins and their associated kinases, such as the topoisomerase II, and thus inhibited normal DNA replication and mitosis.
Four isomalabaricane triterpenes were isolated from marine sponge Geodia japonica [W.H. Zhang, C.T. Che, Isomalabaricane-type nortriterpenoids and other constituents of the marine sponge Geodia japonica, J. Nat. Prod. 64 (2001) 1489-1492. ] and their cytotoxicity was evaluated using a human promyelocytic leukemia HL60 cell line. Of the four triterpenes tested, geoditin A was the most cytotoxic to HL60 cells [IC50=3 microg/ml (<6.6 microM)], followed by stellettins A and B, whereas geoditin B exhibited relatively weak cytotoxicity. The treated cells manifested nuclear changes characteristic for apoptosis, and associated with dissipation of mitochondrial membrane potential, activation of caspase 3, and decrease of cytoplasmic proliferating cell nuclear antigen (PCNA), as demonstrated by fluorescence and immunofluorescence microscopy. When the HL60 cells were exposed to geoditin A ranging from 1.25 to 25 microg/ml, a dose-dependent increase of reactive oxygen species, a progressive dissipation of mitochondrial membrane potential, and an increase in annexin V-FITC binding were measured by flow cytometry. Taken together our results suggest that geoditin A markedly induced reactive oxygen species, decreased mitochondrial membrane potential and mediated a caspases 3 apoptosis pathway.
Using immunohistochemistry, Fas and caspase 8 positive sites were localized in the brains of Alzheimer patients and those with multiple infarcts as well as age-matched normal. The frontal, occipital and hippocampal regions were dissected out from these patients and normal individuals shortly after death. Positive Fas neurons were found in all these regions of normal individuals and Alzheimer patients, but not in the brains of patients with multiple infarct. Activated caspase 8 protein was found only in the brains of Alzheimer patients and not in the other groups. Plaques in the Alzheimer patients were positive for Fas and activated caspase 8. The former was localized in the membrane and the inside of the plaque whereas activated caspase 8 was only present in the interior of the plaque, as revealed by confocal microscopy. Our study shows that caspase 8 was activated in the Alzheimer brain and the presence of Fas and activated caspase 8 points to a possibility that at least some plaques were of intracellular origin as Fas was a cell membrane receptor and caspase 8 was a intracellular enzyme downstream to Fas. Furthermore, we had confirmed that in most neurons Fas and caspase 8 did not exist concomitantly and that the glial cells (astrocytes) did not express caspase 8 in Alzheimer patients.
The immunomodulatory lectin, Volvariella volvacea lectin (VVL), isolated from the edible mushroom, Volvariella volvacea, has been shown to stimulate the expression of Th1 cytokines and the proliferative activity of mouse splenocytes (She et al. [1998]: Biochem Biophys Res Comm 247:106-111). In order to elucidate the mechanisms underlying these activities, we conducted a kinetic analysis of the early and late activation markers in mouse T lymphocytes: (1) flow cytometric analysis of calcium influx, (2) induction of activation molecules (CD25 and CD69), (3) expression and DNA-binding activity of the nuclear factor of activated T cells (NFAT), NFkappaB, and activation protein-1 (AP-1), (4) translational production of cytokines (interleukin-2 (IL-2) and interferon-gamma (IFNgamma)), and (5) cell proliferation by expression of proliferating cell nuclear antigen (PCNA) and tritiated thymidine incorporation. All results showed that VVL induced a rapid expression of CD69, CD25, NFAT, IL-2, and PCNA in a dose- and time-dependent manner, leading to lymphocyte proliferation. These effects brought about by VVL were more potent than those stimulated by equimolar concentrations of mitogenic lectin, concanavalin A (Con A). Cell activation and proliferation were mediated through a calcium-dependent pathway as demonstrated by a VVL-induced increase of intracellular calcium influx, and a proliferation inhibition by the Ca-dependent phosphatase calcineurin blocker-cyclosporin A (CsA). Taken all data together, VVL is a lectin which activates lymphocyte through successive calcium influx, nuclear localization of NFAT transcription factor, induction of activation markers, CD25 and CD69, intracellular cytokine production, and cell proliferation.
Angiogenesis is crucial to tumor growth and metastasis, and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. The present study has made use of the S180 tumor-bearing mouse model to investigate the polysaccharopeptide, PSP, isolated from the edible mushroom Coriolus versicolor, a herbal medicine known for its anti-angiogenesis properties. Quantitative analysis of microcorrosion casting of the tumor tissue showed more angiogenic features such as dense sinusoids and hot spots, in control (untreated) than in PSP-treated animals. Immunostaining of tumor tissues with antibody against the endothelial cell marker (Factor VIII) demonstrated a positive correlation in that both the vascular density and tumor weight were lower in mice treated with PSP. Morphometric analysis of corrosion casts revealed that, even though the total amount of new vessel production was reduced, the basic tumor type-specific vascular architecture was retained. However, the expression of vascular endothelial cell growth factor (VEGF) in these tumors was suppressed. In conclusion, anti-angiogenesis should be one of the pathways through which PSP mediated its anti-tumor activity.
Wheat germ lectin (WGA) is a cytotoxic lectin for many cell lines [Wang et al., 2000], but its underlying mechanism is not clear. In this report, we found that incubation of synchronized mouse L929 fibroblasts with WGA resulted in a dose-dependent reduction of intracellular incorporation of 3H-thymidine and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)-conversion activity (IC50 congruent with 0.4 microM). Fluorescein-conjugated WGA was demonstrated to transport from the cell surface into the paranuclear region of cultured L929 cells within 30 min, and subsequently evoked lipid peroxidation of plasma membrane and vacuolation in the cytoplasm of these cells. Studies with tritiated thymidine incorporation, immunofluorescence microscopy, immunoblotting analysis and flow cytometry revealed that WGA inhibited cell cycle progression after one replication, resulting in G2/M arrest and alteration of cell cycle regulatory proteins, particularly activation of p21Cip1/WAF1 and suppression of cyclin B and cdc 2. Although there was an increase of cytosolic caspase 3 and bax protein expression, no apoptotic bodies were observed by both fluorescence and transmission electron microscopy. These results suggest that WGA arrested L929 proliferation after one cell cycle in the G2/M phase through activation of the p21Cip1/WAF1 and suppression of Cyclin B-Cdc2.
A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway.
Euphorbia fischeriana is a Chinese herbal medicine which has been reported to possess chemotherapeutic effects, yet the underlying mechanism is unclear. In order to understand its possible anti-tumor property, we have isolated a number of chemical compounds from the roots of this plant [Phytochemistry 52 (1999) 117] and studied their in vitro effects by using human prostate LNCaP cancer cell line. Among the six compounds tested, jolkinolide B exhibited the most potent anti-proliferative activity (IC(50)=12.5 microg/mL=40 microM) and it inhibited DNA synthesis by down-regulating bromodeoxyuridine (BrdU) incorporation in LNCaP cells in a dose-dependent manner. Jolkinolide B, at concentrations up to 25 microg/mL, induced G1 arrest and neuroendocrine differentiation of LNCaP cells. Immunoblotting analysis confirmed the increased expression of neuroendocrine markers, keratin 8/18 (K8/18) and neuron specific enolase (NSE), in these cells. Apoptotic bodies and DNA fragmentation were observed by fluorescence microscopy and flow cytometry when the cells were exposed to a concentration higher than 25 microg/mL jolkinolide B. Taken all data together, jolkinolide B seems to play a role in the regulation of proliferation, differentiation, and apoptosis of LNCaP cells.
Induction of apoptosis and androgen ablation are two major approaches for treating human prostate carcinoma. In a study of the bioactive components of the soft coral Nephthea chabroli, we found that lemnabourside is a 5alpha-reductase inhibitor, as shown by its ability to inhibit the conversion of testosterone into the more potent dihydrotestosterone in rat prostate homogenate. The compound also inhibited the incorporation of tritiated thymidine into human prostate androgen-dependent carcinoma LNCaP cells, and thus blocking the cell proliferation (IC50 = 37.5 microM). The expression of prostate marker genes, including 5alpha-reductase, prostate-specific antigen, prostatic acid phosphatase and androgen receptor, and the anti-apoptotic bcl-2 gene were markedly reduced, but the transcription of apoptosis-related caspase 3 gene showed a dose-dependent increase in lemnabourside-treated LNCaP cells. Immunofluorescent microscopy and flow cytometric analysis further demonstrated apoptotic changes in these cells. Taken all results together, a relatively weak 5alpha-reductase inhibitory activity on LNCaP cells (EC50 > 250 microM), and a similar growth inhibitory activity on both androgen dependent- and independent-prostate cells (IC50 approximately 37.5 microM) indicated that caspase-3 apoptosis pathway is one of the possible antiproliferative activities mediated by lemnabourside.
Two extended haplotypes of the tau gene (H1 and H2) have been described. The frequency of H1 haplotype is increased in progressive supranuclear palsy (PSP). PSP is associated with filamentous tau lesions in neurons and glia, which are reportedly composed exclusively of tau isoforms with four repeats in the microtubule-binding domain (4R tau). To determine the influence of the tau haplotype on tau isoform composition and neuropathology, we studied 25 PSP cases and 6 Alzheimer's disease patients matched for age, sex, and postmortem delay. In the basal ganglia, tau and amyloid burdens were determined to see if there was an effect of concurrent Alzheimer-type pathology, and the ratio of 4R to 3R tau was measured in detergent-insoluble tau fractions. Insoluble tau from PSP was not composed exclusively of 4R tau. All brains had a mixture of 4R and 3R tau, but the ratio was different in Alzheimer's disease and PSP. In Alzheimer's disease there was less 4R than 3R tau, whereas the ratio was reversed in PSP. In PSP cases with concurrent Alzheimer-type pathology, the ratio of 4R to 3R was intermediate between Alzheimer's disease and PSP. The H1 haplotype had no effect on the 4R to 3R ratio or on tau and amyloid burdens. In summary, the H1 haplotype does not have a major influence on the pathological or biochemical phenotype of PSP.
Ginseng is a medicinal herb widely used in Asian countries, and many of its pharmacological actions are attributed to the ginsenosides. In a study of the anti-proliferative activity of ginsenosides using human prostate carcinoma LNCaP cell line, ginsenoside Rg3 displayed growth inhibitory activity. The cells lost its adherent property after incubation in the presence of 250 microM of ginsenoside for 48h. The expression of biomarker genes, including prostate specific antigen (PSA), androgen receptor (AR) and 5alpha-reductase (5alphaR), and that of the proliferating cell nuclear antigen (PCNA), were suppressed. Ginsenoside Rg3 induced classic apoptotic morphology and interfered with the expression of apoptosis-related genes, bcl-2 and caspase-3, in LNCaP cells, as demonstrated by fluorescence microscopy, flow cytometry and reverse transcriptase-polymerase chain reaction. Taken our results together, we suggested that ginsenoside Rg3 activated the expression of cyclin-kinase inhibitors, p21 and p27, arrested LNCaP cells at G1 phase, and subsequently inhibited cell growth through a caspase3-mediated apoptosis mechanism.
The effects of lectins with different carbohydrate-binding specificities on human hepatoma (H3B), human choriocarcinoma (JAr), mouse melanoma (B16) and rat osteosarcoma (ROS) cell lines were investigated. Cell viability was estimated by uptake of crystal violet. Wheat germ lectin was the lectin with the most deleterious effect on the viability of H3B, JAr and ROS cell lines. The cytotoxicity of lectins with similar sugar-binding specificity to wheat germ lectin, including Maackia amurensis lectin and Solanum tuberosum lectin, was weaker than that of wheat germ lectin. N-acetylgalactosamine-and galactose-binding Tricholoma mongolicum lectin ranked third, after wheat germ lectin and Maackia amurensis lectin, with regard to its effect on H3B, and ranked, together with Maackia amurensis lectin, as the lectins with the second most pronounced effects on ROS. However, the cytotoxic effects of Tricholoma mongolicum lectin on JAr were much weaker than those of Maackia amurensis lectin, Solanum tuberosum lectin and Anguilla anguilla lectin. Artocarpus integrifolia lectin, Lens culinaris lectin and Anguilla anguilla lectin possessed milder cytotoxicity than the remaining lectins. which were approximately equipotent. The mannose-binding Narcissus pseudonarcissus and Lens culinaris lectins were only weakly cytotoxic, the exception being a stronger effect on H3B. The N-acetylgalactosamine-binding Glycine max lectin and methylgalactose-binding Artocarpus integrifolia lectin similarly exhibited low cytotoxicity. It can thus be concluded that in general the ranking was wheat germ lectin > Maackia amurensis lectin approximately Trichloma mongolicum lectins > other aforementioned lectins in cytotoxicity. A particular lectin may manifest more conspicuous toxicity on certain cell lines and less on others.
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Recently, mutations within the tau gene have been associated with some familial forms of frontotemporal dementia. To investigate whether tau gene mutations are also associated with Pick's disease, we analyzed the tau gene in 30 cases of pathologically confirmed Pick's disease. Two coding mutations were identified in separate cases of Pick's disease. A glycine-to-arginine mutation at codon 389 was detected in 1 case and a lysine-to-threonine mutation at codon 257 was identified in another. Analysis of dephosphorylated tau from the brain of the patient with the codon 389 mutation revealed a prominent band representing tau, with four microtubule-binding domains and no amino terminal inserts. This is in contrast to Pick's disease without any tau gene mutations, which consist of tau with mainly three microtubule-binding domains and only a trace of tau, with four microtubule-binding domains. Functional analysis of tau with these two mutations demonstrated a reduced ability of tau to promote microtubule assembly. Surprisingly, these mutations increased tau's susceptibility to calpain I digestion, suggesting that this feature may be related to the formation of a Pick type of histology. Moreover, these data suggest that Pick's disease is not a separate entity but part of the frontotemporal dementia disease spectrum.
Glycation is a non-enzymatic posttranslational modification that involves a covalent linkage between a sugar and an amino group of protein molecule forming ketoamine. Subsequent oxidation, fragmentation and/or crosslinking of ketoamine leads to the production of advanced glycation endproducts (AGEs). Formation of AGEs causes detrimental effects on the structure and function of affected proteins. Accumulation of AGEs has been implicated in normal aging and in the pathogenesis of diabetes-associated complications and Alzheimer's disease (AD). Of all AGEs, Nepsilon-(carboxymethyl)lysine (CML) is a major glycoxidation product known to be stable and accumulate progressively in vivo. In order to determine if tau is glycated in AD, we raised a rabbit antibody to CML that demonstrated its usefulness in detecting glycation of different proteins in vitro, including BSA, ribonuclease, lysozyme and recombinant tau. Immunochemical analyses indicated that ribose and glucose-6-phosphate are more effective than glucose in generating CML formation in these proteins. We used this antibody to probe for glycation in the following human tau preparations: tau of normal brains and preparations of soluble PHF-tau as well as insoluble PHF from AD brains. All three principal tau components resolved from PHF-tau on Western blots showed CML immunoreactivity indicating that tau is glycated in PHF-tau; and insoluble PHF exhibited prominent CML immunoreactivity on top of the stacking gel. Moreover, immunoelectron microscopic analyses indicate that the anti-CML antibody labels predominantly PHF in aggregates. Taken together, these results suggest that tau becomes glycated in PHF-tau and glycation may play a role in stabilizing PHF aggregation leading to tangle formation in AD.