Molecular basis of SERPINC1 mutations in Japanese patients with antithrombin deficiency.

BACKGROUND: Congenital antithrombin (AT) deficiency, which arises from various SERPINC1 defects, is an autosomal-dominant thrombophilic disorder associated with a high risk of recurrent venous thromboembolism. PATIENTS/METHODS: We investigated SERPINC1 defects in Japanese patients with congenital AT deficiency who developed venous thromboembolism or had a family history of deep vein thrombosis. We analyzed the full DNA sequences of SERPINC1 exons and exon-intron junctions by PCR-mediated direct sequencing. If no mutation was found, multiplex ligation-dependent probe amplification (MLPA) was conducted for the relative quantification of the copy number of all exons in SERPINC1. If splice-site mutations were detected, mRNA splicing abnormalities were further investigated using an in vitro cell-based exontrap assay. RESULTS: We identified 19 different SERPINC1 abnormalities, including 8 novel mutations, in 21 Japanese patients with AT deficiency. These abnormalities were distributed as follows: 9 missense mutations (42.9%), 3 nonsense mutations (14.3%), 1 splice-site mutation (4.8%), 2 small insertions (9.5%), 2 deletion mutations (9.5%) and 4 large deletions (19.0%). Cases with large deletions of SERPINC1 included Alu-mediated gene rearrangements and non-Alu-mediated complex gene rearrangements; the latter could conceivably be explained using the fork stalling and template switching (FoSTeS) model. CONCLUSIONS: We identified a variety of SERPINC1 defects in Japanese patients with AT deficiency. The SERPINC1 mutations detected in patients with type I AT deficiency included single nucleotide missense or nonsense mutations, small intragenic insertions or deletions, and large genomic structural deletions. Large deletions of SERPINC1 were caused by various recurrent or non-recurrent complex genomic rearrangement mutations.

Oxidative Stress Inhibits Healthy Adipose Expansion Through Suppression of SREBF1-Mediated Lipogenic Pathway.

Recent studies have emphasized the association of adipose oxidative stress (Fat reactive oxygen species [ROS]) with the pathogenesis of metabolic disorders in obesity. However, the causal roles of Fat ROS in metabolic disturbances in vivo remain unclear because no mouse model has been available in which oxidative stress is manipulated by targeting adipocytes. In this research, we generated two models of Fat ROS-manipulated mice and evaluated the metabolic features in diet-induced obesity. Fat ROS-eliminated mice, in which Cat and Sod1 were overexpressed in adipocytes, exhibited adipose expansion with decreased ectopic lipid accumulation and improved insulin sensitivity. Conversely, Fat ROS-augmented mice, in which glutathione was depleted specifically in adipocytes, exhibited restricted adipose expansion associated with increased ectopic lipid accumulation and deteriorated insulin sensitivity. In the white adipose tissues of these mice, macrophage polarization, tissue fibrosis, and de novo lipogenesis were significantly changed. In vitro approaches identified KDM1A-mediated attenuation of sterol-regulatory element-binding transcription factor 1 (SREBF1) transcriptional activities as the underlying mechanism for the suppression of de novo lipogenesis by oxidative stress. Thus, our study uncovered the novel roles of Fat ROS in healthy adipose expansion, ectopic lipid accumulation, and insulin resistance, providing the possibility for the adipocyte-targeting antioxidant therapy.

Effect of rebamipide on gastric bleeding and ulcerogenic responses induced by aspirin plus clopidogrel under stimulation of acid secretion in rats.

BACKGROUND AND AIM: We examined the prophylactic effect of rebamipide on gastric bleeding induced by the perfusion of aspirin (acetylsalicylic acid [ASA]) plus clopidogrel under the stimulation of acid secretion in rats. METHODS: Under urethane anesthesia, acid secretion was stimulated by the i.v. infusion of histamine (8 mg/kg/h), and the stomach was perfused with 25 mmol/L ASA at a rate of 0.4 mL/min. Gastric bleeding was evaluated as the concentration of hemoglobin in the perfusate. Clopidogrel (30 mg/kg) was given p.o. 24 h before the perfusion. Rebamipide (3-30 mg/kg) or other antiulcer drugs were given i.d. before the ASA perfusion. RESULTS: Slight gastric bleeding or damage was observed with the perfusion of ASA under the stimulation of acid secretion, whereas these responses were significantly increased in the presence of clopidogrel. Both omeprazole and famotidine inhibited acid secretion and prevented these responses to ASA plus clopidogrel. Rebamipide had no effect on acid secretion, but dose-dependently prevented gastric bleeding in response to ASA plus clopidogrel, with the degree of inhibition being almost equivalent to that of the antisecretory drugs, and the same effects were obtained with the gastroprotective drugs, irsogladine and teprenone. These agents also reduced the severity of gastric lesions, although the effects were less than those of the antisecretory drugs. CONCLUSIONS: These results suggest that the antiplatelet drug, clopidogrel, increases gastric bleeding induced by ASA under the stimulation of acid secretion, and the gastroprotective drug, rebamipide, is effective in preventing the gastric bleeding induced under such conditions, similar to antisecretory drugs.

Some characteristics of membrane Cd²+ transport in rat thymocytes: an analysis using Fluo-3.

Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd(2+) transport of rat thymocytes, the change in intracellular Cd(2+) concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd(2+) transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl(2). Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl(2), suggesting that the metabolic process can be involved in membrane Cd(2+) transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd(2+) transport into the cells. Bath application of CaCl(2) and ZnCl(2) equally decreased the augmentation, suggesting their competition with Cd(2+) at the membrane transport. The augmentation by CdCl(2) was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd(2+) transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd(2+) pathway, driven by electrochemical gradient of Cd(2+) and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd(2+) transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.

System identification of the mechanomyogram from single motor units during voluntary isometric contraction.

A mechanomyogram (MMG) from single motor units of the anconeus muscle in voluntary isometric contraction was recorded from seven subjects using a spike-triggered averaging technique. The MMG system, in which the input was an ideal impulse and the output was the MMG detected with an acceleration sensor, was identified as the fifth-order model by the subspace-based state-space model identification method. The transfer function of the MMG system was factorized to the second- and the first-order models. The second-order model was compared to the standard form of the second-order model, and its resonance frequency was calculated. The resonance frequencies of the second-order models were 166 ± 61 and 93 ± 27 Hz, which were within the range of the values estimated from mechanical impedance in the literature. The equivalent mechanical model of the MMG system of the single motor unit was proposed on the basis of the fifth-order model. The model might be useful to evaluate the visco-elastic properties of the anconeus muscle.

Cytometric analysis on cytotoxicity of curcumin on rat thymocytes: Proapoptotic and antiapoptotic actions of curcumin.

Curcumin exhibits various pharmacological actions including anti-inflammatory, anti-infectious, and anticancer actions. Furthermore, the supplements containing curcumin are supplied for persons consuming alcoholic beverage. A primary criterion for an ingredient ingested by general population is that it exerts no harmful effect. In this study, we examined the effect of curcumin on rat thymocytes to see if curcumin exerts cytotoxicity on normal cells. The incubation with 10 µM curcumin for 24h increased the population of dead cells while it was not the case for 5 µM or less. Curcumin at 5-10 µM increased the populations of shrunken cells and the cells positive to annexin V, phenomena for early stage of apoptosis. However, the incubation with 10 µM curcumin suppressed the increase in population of cells with hypodiploid DNA, a phenomenon for late stage of apoptosis. Thus, curcumin at 10 µM may show both proapoptotic and antiapoptotic actions. The simultaneous incubation with 5 µM, but not 3 µM, curcumin and 0.5% ethanol increased the population of shrunken cells. It is likely that curcumin at 5 µM or more exerts cytotoxic action on normal cells although many studies show some anticancer actions of curcumin at 10 µM or more on cancer cells.

Cytotoxic action of bisabololoxide A of German chamomile on human leukemia K562 cells in combination with 5-fluorouracil.

German chamomile (Matricaria recutita L.) is a popular ingredient in herbal teas. In previous study, micromolar bisabololoxide A, one of main constituents in German chamomile, exerted cytotoxic action on rat thymocyte, a normal non-proliferative cell. This result prompted us to study the effect of bisabololoxide A on proliferative cancer cells and to seek the possibility of its use with 5-fluorouracil, an anticancer agent. In this study, the effect of micromolar bisabololoxide A on human leukemia K562 cells was cytometrically examined. Although the incubation of K562 cells with 10 µM bisabololoxide A for 72h did not significantly increase the percentage populations of dead cells and shrunken cells, the inhibitory action on the growth was obviously observed. It was not the case for the concentrations of less than 5 µM. The threshold concentration of bisabololoxide A to exert the cytotoxic action on K562 cells was ascertained to be 5-10 µM. Bisabololoxide A at 5-10 µM did not exert cytotoxic action on normal non-proliferative cells (rat thymocytes) in our previous study. Since the antiproliferative action of micromolar bisabololoxide A on cancerous cells was expected to be beneficial to cancer treatment, the modification of antiproliferative action of 5-fluorouracil (3-30 µM) by bisabololoxide A was studied. The combination of 5-fluorouracil and bisabololoxide further inhibited the growth of K562 cells although the additive inhibition of growth by bisabololoxide A became smaller as the concentration of 5-fluorouracil increased. Therefore, it is suggested that the simultaneous application of German chamomile containing bisabololoxide A may reduce the dose of 5-fluorouracil.

Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: excessive increase in intracellular Zn2+ concentration.

The ability of zinc to retard oxidative processes has been recognized for many years. However, zinc is cytotoxic under certain oxidative stress. In this study, we investigated the effect of H2O2 on intracellular Zn2+ concentration of rat thymocytes and its relation to the cytotoxicity. Experiments were cytometrically performed by the use of fluorescent probes, propidium iodide, FluoZin-3-AM, and 5-chloromethylfluorescein diacetate. ZnCl2 potentiated cytotoxicity of H2O2 while TPEN, a chelator for intracellular Zn2+, attenuated it. Results suggested an involvement of intracellular Zn2+ in the cytotoxicity of H2O2. H2O2 at concentrations of 30microM or more (up to 1000microM) significantly increased intracellular Zn2+ concentration. There were two mechanisms. (1) H2O2 decreased cellular content of nonprotein thiols, possibly resulting in release of Zn2+ from thiols as cellular Zn2+ binding sites. (2) H2O2 increased membrane Zn2+ permeability because external ZnCl2 application further elevated intracellular Zn2+ concentration. Micromolar H2O2 may induce excessive elevation of intracellular Zn2+ concentration that is harmful to cellular functions. However, the incubation with micromolar ZnCl2 alone increased cellular content of nonprotein thiols, one of the factors protecting cells against oxidative stress. Though zinc is generally considered to be protective with its antioxidant property, this study reveals the toxic effect of zinc even in micromolar range under oxidative stress induced by H2O2.

Apoptosis-inducing action of two products from oxidation of sesamol, an antioxidative constituent of sesame oil: a possible cytotoxicity of oxidized antioxidant.

Many effects of sesamol, an antioxidative constituent of sesame oil, have been reported for human health benefits due to its antioxidative action. However, we recently isolated two cytotoxic products, trimer and tetramer of sesamol, from oxidation of sesamol by an assay-guided purification. In this study, we have revealed some cytotoxic characteristics of these products in rat thymocytes and human leukemia K562 cells. Incubation of cells with trimer or tetramer at 10-30 microM for 24h significantly increased cell lethality and population of rat thymocytes containing hypodiploid DNA, suggesting cell death with DNA fragmentation, while it was not the case for 30 microM sesamol. The cytotoxic action of tetramer was more potent than that of trimer in rat thymocytes when their concentrations were 10-30 microM. The incubation of cells with 10 microM tetramer for 24h increased the population of cells with exposed phosphatidylserine, the activity of caspases, and the nick of DNA. These results indicate tetramer-induced apoptosis. In K562 cells, the incubation with tetramer at 10 microM for 72 h significantly inhibited the growth without affecting the lethality. However, tetramer at 30 microM significantly increased cell lethality. It is likely that tetramer exerts more cytotoxic action on normal non-proliferative cells (rat thymocytes) rather than proliferative cancer cells (human leukemia K562 cells). It may be necessary to consider the condition for preservation of sesamol and the safety of products from in vivo oxidation of sesamol for human health.

Bisabololoxide A, one of the main constituents in German chamomile extract, induces apoptosis in rat thymocytes.

German chamomile (Matricaria recutita L.), one of the popular ingredients in herbal teas, has been traditionally used for medicinal purposes. Bisabololoxide A (BSBO) is one of the main constituents in this herb. BSBO is supposed to be principle in some bioactivities of German chamomile such as anti-inflammatory, gastrointestinal, and antipruritic actions. Although the use of German chamomile has spread, the information related to toxicity of BSBO is very limited. In present study, the cytotoxic effect of micromolar BSBO was cytometrically examined on rat thymocytes by using appropriate fluorescent dyes. When the cells were incubated with BSBO for 24 h, BSBO at concentrations of 30 microM or more significantly increased populations of dead cells, shrunken cells, and cells with phosphatidylserine exposed on membrane surface. Both cell shrinkage and externalization of membrane phosphatidylserine are general features in an early stage of apoptosis. In addition, BSBO significantly increased population of cells containing hypodiploid DNA, and the increase was completely attenuated by Z-VAD-FMK, a pan-inhibitor for caspases, indicating an involvement of caspase activation. Thus, it is likely that the type of cell death induced by BSBO is apoptosis. The significant changes in cellular parameters of rat thymocytes by BSBO were not observed when the concentration was 10 microM or less. Furthermore, the short incubation (3 h) of cells even with 30-100 microM BSBO did not significantly affect the cells. Therefore, it may be suggested that BSBO is practically safe when German chamomile is conventionally used.

Micromolar Zn(2+) potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: possible disturbance of intracellular Ca(2+) and Zn(2+) homeostasis.

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn(2+) at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn(2+) permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn(2+) at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn(2+), we have cytometrically examined the effects of econazole, ZnCl(2), and their combination on rat thymocytes. ZnCl(2) at concentrations ranging from 1 microM to 30 microM significantly increased the lethality induced by 10 microM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 microM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl(2), suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl(2). Econazole at 0.3 microM or more in the presence of ZnCl(2) increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca(2+). Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+), was also augmented by econazole at 0.1 microM or more in the presence of ZnCl(2). Results suggest that the combination of submicromolar econazole with micromolar ZnCl(2) may increase the intracellular concentration of Ca(2+) and Zn(2+), leading to disturbance of intracellular Ca(2+) and Zn(2+) homeostasis that triggers cytotoxic action.

Tri-n-butyltin increases intracellular Zn(2+) concentration by decreasing cellular thiol content in rat thymocytes.

Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn(2+) concentration was tested in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow cytometer and appropriate fluorescent probes. TBT at concentrations of 30 nM or more (up to 300 nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+) concentration, under external Ca(2+)- and Zn(2+)-free condition. Chelating intracellular Zn(2+) completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn(2+) from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indicator for cellular thiol content, were correlated with a coefficient of -0.962. Result suggests that the intracellular Zn(2+) release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn(2+) potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn(2+) concentration may be a type of stress responses to protect the cells.

Increase in intracellular Zn2+ concentration by thimerosal in rat thymocytes: intracellular Zn2+ release induced by oxidative stress.

Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn(2+) concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn(2+). To reveal the property of TMR-induced increase in intracellular Zn(2+) concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn(2+), of rat thymocytes was examined. TMR at concentrations ranging from 0.3 microM to 10 microM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca(2+)- and Zn(2+)-free condition. The threshold concentration was 0.3-1 microM. The increase in the intensity was significant when TMR concentration was 1 microM or more. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn(2+), completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H(2)O(2)) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was -0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H(2)O(2) and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn(2+) concentration via decreasing cellular thiol content.