Biomedical subjects
Publications and source records attributed to R Yu.
We report a delayed "choice" quantum eraser experiment of the type proposed by Scully and Druhl (where the "choice" is made randomly by a photon at a beam splitter). The experimental results demonstrate the possibility of delayed determination of particlelike or wavelike behavior via quantum entanglement. The which-path or both-path information of a quantum can be marked or erased by its entangled twin even after the registration of the quantum.
Green tea polyphenols (GTP) have been demonstrated to suppress tumorigenesis in several chemical-induced animal carcinogenesis models, and predicted as promising chemopreventive agents in human. Recent studies of GTP extracts showed the involvement of mitogen-activated protein kinases (MAPKs) in the regulation of Phase II enzymes gene expression and induction of apoptosis. In the current work we compared the biological actions of five green tea catechins: (1) induction of ARE reporter gene, (2) activation of MAP kinases, (3) cytotoxicity in human hepatoma HepG2-C8 cells, and (4) caspase activation in human cervical squamous carcinoma HeLa cells. For the induction of phase II gene assay, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) potently induced antioxidant response element (ARE)-mediated luciferase activity, with induction observed at 25 microM with EGCG. The induction of ARE reporter gene appears to be structurally related to the 3-gallate group. Comparing the activation of MAPK by the five polyphenols, only EGCG showed potent activation of all three MAPKs (ERK, JNK and p38) in a dose- and time-dependent manner, whereas EGC activated ERK and p38. In the concentration range of 25 microM to 1 mM, EGCG and ECG strongly suppressed HepG2-ARE-C8 cell-growth. To elucidate the mechanisms of green tea polyphenol-induced apoptosis, we measured the activation of an important cell death protein, caspase-3 induced by EGCG, and found that caspase-3 was activated in a dose- and time-dependent manner. Interestingly, the activation of caspase-3 was a relatively late event (peaked at 16 h), whereas activation of MAPKs was much earlier (peaked at 2 h). It is possible, that at low concentrations of EGCG, activation of MAPK leads to ARE-mediated gene expression including phase II detoxifying enzymes. Whereas at higher concentrations of EGCG, sustained activation of MAPKs such as JNK leads to apoptosis. These mechanisms are currently under investigation in our laboratory. As the most abundant catechin in GTP extract, we found that EGCG potently induced ARE-mediated gene expression, activated MAP kinase pathway, stimulated caspase-3 activity, and induced apoptosis. These mechanisms together with others, may contribute to the overall chemopreventive function of EGCG itself as well as the GTP
Many natural products elicit diverse pharmacological effects. Using two classes of potential chemopreventive compounds, the phenolic compounds and the isothiocyanates, we review the potential utility of two signaling events, the mitogen-activated protein kinases (MAPKs) and the ICE/Ced-3 proteases (caspases) stimulated by these agents in mammalian cell lines. Studies with phenolic antioxidants (BHA, tBHQ), and natural products (flavonoids; EGCG, ECG, and isothiocyanates; PEITC, sulforaphane), provided important insights into the signaling pathways induced by these compounds. At low concentrations, these chemicals may activate the MAPK (ERK2, JNK1, p38) leading to gene expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detoxifying enzymes; GST, QR) resulting in survival and protective mechanisms (homeostasis response). Increasing the concentrations of these compounds will additionally activate the caspase pathway, leading to apoptosis (potential cytotoxicity). Further increment to suprapharmacological concentrations will lead to nonspecific necrotic cell death. The wider and narrow concentration ranges between the activation of MAPK/gene induction and caspases/cell death exhibited by phenolic compounds and isothiocyanates, respectively, in mammalian cells, may reflect their respective therapeutic windows in vivo. Consequently, the studies of signaling pathways elicited by natural products will advance our understanding of their efficacy and safety, of which many may become important therapeutic drugs of the future.
The eicosapentaenoic acid (EPA) synthesis gene cluster from an EPA-producing bacterium, Shewanella sp. SCRC-2738, was cloned into a broad-host range vector, pJRD215, and then introduced into a marine cyanobacterium, Synechococcus sp. NKBG15041c, by conjugation. The transconjugant cyanobacteria produced 3.7 +/- 0.2% (2.24 +/- 0.13 mg/L) EPA (n-3) and 2.5 +/- 0.2% (1.49 +/- 0.06 mg/L) eicosatetraenoic acid (n-3) of the total fatty acids when the cells were cultured at 23 degrees C at a light intensity of 1,000-1,500 Lux. The EPA and eico-satetraenoic acid contents of the cells were increased to 4.6 +/- 0.6% (3.86 +/- 1.11 mg/L) and 4.7 +/- 0.3% (3.86 +/- 0.82 mg/L), and 7.5 +/- 0.3% (1.76 +/- 0.10 mg/L) and 5.1 +/- 0.2% (1.19 +/- 0.06 mg/L) when they were cultured at low temperature (18 degrees C) and at lower light intensity (40 Lux), respectively.
We present a global optimization strategy that incorporates predicted restraints in both a local optimization context and as directives for global optimization approaches, to predict protein tertiary structure for alpha-helical proteins. Specifically, neural networks are used to predict the secondary structure of a protein, restraints are defined as manifestations of the network with a predicted secondary structure and the secondary structure is formed using local minimizations on a protein energy surface, in the presence of the restraints. Those residues predicted to be coil, by the network, define a conformational sub-space that is subject to optimization using a global approach known as stochastic perturbation that has been found to be effective for Lennard-Jones clusters and homo-polypeptides. Our energy surface is an all-atom 'gas phase' molecular mechanics force field, that is combined with a new solvation energy function that penalizes hydrophobic group exposure. This energy function gives the crystal structure of four different alpha-helical proteins as the lowest energy structure relative to other conformations, with correct secondary structure but incorrect tertiary structure. We demonstrate this global optimization strategy by determining the tertiary structure of the A-chain of the alpha-helical protein, uteroglobin and of a four-helix bundle, DNA binding protein.
Aberrant apoptosis-mediated cell death is believed to result in a number of different human diseases. For example, excessive apoptosis in the liver can result in fulminant and autoimmune forms of hepatitis. We have explored the possibility that inhibition of Fas expression in mice would reduce the severity of fulminant hepatitis. To do this, we have developed a chemically modified 2'-O-(2-methoxy)ethyl antisense oligonucleotide (ISIS 22023) inhibitor of mouse Fas expression. In tissue culture, this oligonucleotide induced a reduction in Fas mRNA expression that was both concentration- and sequence-specific. In Balb/c mice, dosing with ISIS 22023 reduced Fas mRNA and protein expressions in liver by 90%. The ID50 for this response was 8-10 mg kg-1 daily dosing, and the reduction was highly dependent on oligonucleotide sequence, oligonucleotide concentration in liver, and treatment time. Pretreatment with ISIS 22023 completely protected mice from fulminant hepatitis induced by agonistic Fas antibody, by a mechanism entirely consistent with an oligonucleotide antisense mechanism of action. In addition, oligonucleotide-mediated suppression of Fas expression reduced the severity of acetaminophen-mediated fulminant hepatitis, but was without effect on concanavalin A-mediated hepatitis. Our results demonstrate that 2'-O-(2-methoxy)ethyl containing antisense oligonucleotides targeting Fas can exert in vivo pharmacological activity in liver, and suggest that oligonucleotide inhibitors of Fas may be useful in the treatment of human liver disease.
Preliminary evaluation of recently developed large-area flat panel detectors (FPDs) indicates that FPDs have some potential advantages: compactness, absence of geometric distortion and veiling glare with the benefits of high resolution, high detective quantum efficiency (DQE), high frame rate and high dynamic range, small image lag (< 1%), and excellent linearity (approximately 1%). The advantages of the new FPD make it a promising candidate for cone-beam volume computed tomography (CT) angiography (CBVCTA) imaging. The purpose of this study is to characterize a prototype FPD-based imaging system for CBVCTA applications. A prototype FPD-based CBVCTA imaging system has been designed and constructed around a modified GE 8800 CT scanner. This system is evaluated for a CBVCTA imaging task in the head and neck using four phantoms and a frozen rat. The system is first characterized in terms of linearity and dynamic range of the detector. Then, the optimal selection of kVps for CBVCTA is determined and the effect of image lag and scatter on the image quality of the CBVCTA system is evaluated. Next, low-contrast resolution and high-contrast spatial resolution are measured. Finally, the example reconstruction images of a frozen rat are presented. The results indicate that the FPD-based CBVCT can achieve 2.75-lp/mm spatial resolution at 0% modulation transfer function (MTF) and provide more than enough low-contrast resolution for intravenous CBVCTA imaging in the head and neck with clinically acceptable entrance exposure level. The results also suggest that to use an FPD for large cone-angle applications, such as body angiography, further investigations are required.
Butylated hydroxyanisole (BHA), a commonly used food preservative, is reported to have anticarcinogenic properties in some animal models. However, the use of BHA as a chemopreventive agent against cancer in human has been challenged by the observation that BHA may exert toxic effect in some tissues of animals. Therefore, it is of great significance to understand the mechanism of BHA-induced toxicity. Here, we report that BHA induces apoptosis in freshly isolated rat hepatocytes. Treatment of hepatocytes with BHA also induced loss of mitochondrial transmembrane potential (Deltapsi(m)), cytochrome c, and activation of caspase-3, -8, and -9 but not caspase-1. Pretreatment with cyclosporin A, an agent that stabilizes mitochondrial permeability transition pore, inhibited BHA-induced loss of Deltapsi(m), cytochrome c release, caspase activation, and apoptosis. Interestingly, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone failed to prevent these mitochondrial events, although it blocked caspase activation and apoptosis. Furthermore, BHA-induced apoptosis appeared to be independent of formation of reactive intermediates, as evidenced by the lack of effects of antioxidants N-acetyl-L-cysteine and ascorbic acid. Indeed, direct incubation of BHA with isolated mitochondria triggered cytochrome c release. Thus, these results indicate that the cytotoxicity of BHA is due to the induction of apoptosis that is mediated by the direct release of cytochrome c and the subsequent activation of caspases.
The pituitary transforming gene, PTTG, is abundantly expressed in endocrine neoplasms. PTTG has recently been recognized as a mammalian securin based on its biochemical homology to Pds1p. PTTG expression and intracellular localization were therefore studied during the cell cycle in human placental JEG-3 cells. PTTG mRNA and protein expressions were low at the G1/S border, gradually increased during S phase, and peaked at G2/M, but PTTG levels were attenuated as cells entered G1. In interphase cells, wild-type PTTG, an epitope-tagged PTTG, and a PTTG-EGFP conjugate all localized to both the nucleus and cytoplasm, but in mitotic cells, PTTG was not observed in the chromosome region. PTTG-EGFP colocalized with mitotic spindles in early mitosis and was degraded in anaphase. Intracellular fates of PTTG-EGFP and a conjugate of EGFP and a mutant inactivated PTTG devoid of an SH3-binding domain were observed by real-time visualization of the EGFP conjugates in live cells. The same cells were continuously observed as they progressed from G1/S border to S, G2/M, and G1. Most cells (67%) expressing PTTG-EGFP died by apoptosis, and few cells (4%) expressing PTTG-EGFP divided, whereas those expressing mutant PTTG-EGFP divided. PTTG-EGFP, as well as the mutant PTTG-EGFP, disappeared after cells divided. The results show that PTTG expression and localization are cell cycle-dependent and demonstrate that PTTG regulates endocrine tumor cell division and survival.
OBJECTIVE: To study the relationship between the lipoprotein lipase(LPL) gene and type 2 diabetes mellitus (DM) and its lipid metabolism. METHODS: PCR-RFLP method was used to determine the DNA polymorphism of 6th intron at LPL gene in 127 DM patients and 125 controls. RESULTS: The allele frequencies in case and control groups were 69.3%, 63.6% for "P+" and 30.7%, 36.4% for "P-", respectively; the frequencies of genotypes were 48.0%, 44.8% for P+/P+, 42.5%, 37. 6% for P+/P- and 9.45%, 17.6% for P-/P-, respectively. There was no significant difference in frequencies of genotypes or alleles between the two groups. The LPL Pvu II polymorphism in Chinese population was similar to that in Japanese population, and was different from that in European and American population. CONCLUSION: The LPL-Pvu II-RFLP in Chinese population is not significantly associated with type 2 diabetes mellitus. The significant difference of Pvu II-RFLP among various races was observed.
OBJECTIVE: To evaluate the value of X-ray imaging for diagnosing abdominal tuberculous lymphadenopathy. METHODS: 27 cases of tuberculous lymphadenopathy proved by clinical or pathohistological evidence were collected, all of which were examined with radiological imaging methods. RESULTS: The typical CT findings enlarged lymph nodes with peripheral or multilocular enhancement. Enlarged lymph nodes with multiple flecked or massive calcifications were important for diagnosis. Ultrasound examination showed enlarged lymph nodes with uniform echo (less than 2 cm) and with heterogeneous echo (larger than 2 cm). The aim of the barium meal examination of the gastrointestinal tract was to find tuberculous lymphadenopathy encroaching on intestine or merged with intestinal tuberculosis. Abdominal X-ray plain film was helpful to find the calcified tuberculous lymphadenopathy and intestinal obstruction. CONCLUSIONS: Various imaging methods have shown their diagnostic value, but CT was the most important tool for definite diagnosis of tuberculous lymphadenopathy. Ultrasound was more useful for follow-up cases.
OBJECTIVE: To study the method of quantitative evaluation of dyspnea and to investigate the effects of M-receptor antagonist inhalation on dyspnea in patients with chronic obstructive pulmonary disease (COPD) during exercise. METHODS: Cycle exercise test was used in 27 patients with COPD and the effects of ipratropium bromide, a M-receptor antagonist, by inhalation on dyspnea and exercise capacity in the patients were observed. RESULTS: (1) Dyspnea index as Borg scale was closely correlated with oxygen uptake (V(O2)) during exercise in all the patients. Three variables for quantitative evaluation of dyspnea could be derived from the linear regression line. They were Borg scale slope (BSS), threshold load of dyspnea (TLD), and breakpoint load of dyspnea(BLD). (2) BSS was negatively correlated with forced expiratory volume in one second to forced vital capacity ratio (r = -0.519, P < 0.01) and diffusing capacity of the lungs for carbon monoxide (r = -0.497, P < 0.01) but positively correlated with residual volume/total lung capacity (r = 0.396, P < 0.05). (3) Inhalation of ipratropium bromide could significantly improve BSS [from (12.09 +/- 3.41) BS x L(-1) x min(-1) to (8.57 +/- 2.79) BS x L(-1) x min(-1), P < 0.01] and BLD [from (0.77 +/- 0.19) L/min to (0.81 +/- 0.14) L/min, P < 0.05] during exercise. CONCLUSIONS: (1) BSS, TLD and BLD could be used as reliable and convenient variables in quantitative evaluation of dyspnea in patients with COPD. BSS and BLD could to some extent reflect the severity of the disease. (2) M-receptor antagonists could alleviate dyspnea and increase exercise capacity in patients with COPD.
Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers.
N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a potent inducer of cellular stress leading to chromosomal aberrations, point mutations, and cell death. To study the effect of capsaicin on c-jun expression when given with MNNG to rats, Fisher-344 rats that had been administered MNNG were treated with capsaicin in their diet and organs were removed for measuring c-jun transcripts. We show that pre- or post-treatment of capsaicin relative to MNNG administration up- or down-regulates (depending on the organ) c-jun expression in a consistent pattern in most organs. In fact, we found in this study that capsaicin inhibits c-jun induction, stimulated by MNNG, in the spleen, heart, stomach and lung. Since MNNG, a methylating agent, is a powerful carcinogen that is very effective in the induction of c-jun mRNA, the results suggest that capsaicin uptake in the diet could play a role in inhibition of tumorigenesis induced by MNNG.
The thyrotropin-releasing hormone (TRH) receptor was expressed in embryonic fibroblasts from mice lacking the alpha subunits of Gq and G11 (Fq/11 cells) to determine whether G protein coupling is necessary for agonist-dependent receptor internalization. Neither TRH nor agonists acting on endogenous receptors increased intracellular calcium unless the cells were co-transfected with the alpha subunit of Gq. In contrast, temperature-dependent internalization of [3H]MeTRH in Fq/11 cells was the same whether Gqalpha was expressed or not. A rhodamine-labeled TRH analog and fluorescein-labeled transferrin co-localized in endocytic vesicles in Fq/11 cells, indicating that endocytosis took place via the normal clathrin pathway. Cotransfection with beta-arrestin or V53D beta-arrestin increased TRH-dependent receptor sequestration. Fq/11 cells were co-transfected with the TRH receptor and a green fluorescent protein (GFP)-beta-arrestin conjugate. GFP-beta-arrestin was uniformly distributed in the cytoplasm of untreated cells and quickly translocated to the periphery of the cells when TRH was added. A truncated TRH receptor that lacks potential phosphorylation sites in the cytoplasmic carboxyl terminus signaled but did not internalize or cause membrane localization of GFP-beta-arrestin. These results prove that calcium signaling by the TRH receptor requires coupling to a G protein in the Gq family, but TRH-dependent binding of beta-arrestin and sequestration do not.
Serum antioxidative vitamin levels and lipid peroxidation in gastric cancer patients were compared with values for age-matched healthy subjects. Blood samples were collected from the stomach of cancer patients scheduled for surgical removal of their tumor. Serum ascorbic acid, alpha-tocopherol, beta-carotene, and retinol in serum were determined by high-performance liquid chromatography, and malondialdehyde levels were analyzed spectrophotometrically. General health characteristics and taste preference for spicy and/or salty food were assessed by a self-administered questionnaire. Significant decreases in serum ascorbic acid and beta carotene were observed in stomach cancer patient as compared to the control group. The levels of ascorbic acid in patients with gastric carcinoma were less than one-fifth of the control. Beta carotene and alpha-tocopherol levels in the serum of the cancer patients were significantly decreased compared to the control group, but there were no differences in retinol between the groups. Serum malondialdehyde levels were significantly higher in the cancer patients than in controls. The levels of ascorbic acid and alpha-tocopherol tend to be lower in the patient with a preference for spicy and salty food than in others without the preference. Our results demonstrate that a correlation existed between the levels of serum ascorbic acid and beta carotene, alpha-tocopherol and lipid peroxidation in gastric carcinoma.
Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and beta-lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNKI or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicin-induced JNKI activity, but had no effect on JNKI activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a specific intracellular Ca2+ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no effect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The differential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of antimicrotubule drugs.
The purpose of this study was to examine the effects of feeding docosahexaenoic acid (DHA) as triacylglycerol on the fatty acid composition, eicosanoid production, and select activities of human peripheral blood mononuclear cells (PBMNC). A 120-d study with 11 healthy men was conducted at the Metabolic Research Unit of Western Human Nutrition Reach Center. Four subjects (control group) were fed the stabilization diet throughout the study; the remaining seven subjects were fed the basal diet for the first 30 d, followed by 6 g DHA/d for the next 90 d. DHA replaced an equivalent amount of linoleic acid; the two diets were comparable in their total fat and all other nutrients. Both diets were supplemented with 20 mg D alpha-tocopherol acetate per day. PBMNC fatty acid composition and eicosanoid production were examined on day 30 and 113; immune cell functions were tested on day 22, 30, 78, 85, 106, and 113. DHA feeding increased its concentration from 2.3 to 7.4 wt% in the PBMNC total lipids, and decreased arachidonic acid concentration from 19.8 to 10.7 wt%. It also lowered prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production, in response to lipopolysaccharide, by 60-75%. Natural killer cell activity and in vitro secretion of interleukin-1beta and tumor necrosis factor alpha were significantly reduced by DHA feeding. These parameters remained unchanged in the subjects fed the control diet. B-cell functions as reported here and T-cell functions that we reported previously were not altered by DHA feeding. Our results show that inhibitory effects of DHA on immune cell functions varied with the cell type, and that the inhibitory effects are not mediated through increased production of PGE2 and LTB4.