Tacrolimus-Induced Apoptosis is Mediated by Endoplasmic Reticulum-derived Calcium-dependent Caspases-3,-12 in Jurkat Cells.

Apoptotic signal pathways are delivered to caspase-3, caspase-9, or both in different cells via the death receptor pathway, mitochondrial pathway, or by the endoplasmic reticulum (ER) pathway through initiators of caspase-3, -8, -9, or -12. Tacrolimus (Tac)-induced apoptosis was characterized by nuclear fragmentation and caspase-3 activation. We examined the effect of tacrolimus on ER-derived calcium and caspase-3,-12-mediated apoptosis on Jurkat human T lymphocyte. Tac decreased the viability of Jurkat cells in a dose-dependent manner. Tac also increased continuously intracellular concentration of calcium from 24 hours to 72 hours. We did not find intracellular calcium changes on the treatment of calcium ionorpore (A23187) regardless of 1 nmol/L Tac concentration level. However, calcium adenosine triphosphatase inhibitor (thapsigargin) increased intracellular calcium accumulation and co-treating 1 nmol/L Tac further induced intracellular calcium accumulation. Interestingly, we found that 1 nmol/L Tac treatment induced activation of caspase-12 protease as well as the catalytic activity of caspase-3 but not catalytic activation of caspase-6, -8, and -9 proteases in Jurkat cells. These data advance our understanding of Tac-induced apoptosis is ER-derived calcium and caspases-3,-12- mediated apoptosis in human Jurkat cell line.

Expression of Endoplasmic Reticulum-Mediated Stress Proteins in FK506-Treated T-Lymphocytes.

BACKGROUND: FK506-induced apoptotic endoplasmic reticulum (ER)-mediated stress protein expression was investigated in Jurkat human T-lymphocytes. METHODS: The effect of FK506 on apoptosis and cell viability were examined. FK506-induced apoptosis was confirmed by nuclear fragmentation after DAPI staining. Expression of apoptotic ER-mediated stress proteins was examined by means of Western blotting of Grp78/BiP, Grp94, double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK), phosphor-PERK, CHOP/GADD153, and Bak. A flow cytometry analysis was performed after DAF-DA or DCF-DA staining. FK506-induced apoptosis was dose-dependent (10 nmol/L) and time-dependent (72 hours). RESULTS: Grp78/BiP and Grp94 expressions were increased 36 hours after FK506 treatment. Increased phospho-PERK expression was observed 6 hours after FK506 treatment and peak activation of phospho-PERK was observed at 36 hours. CHOP/GADD153 expression was increased 48 hours after FK506 treatment. Expression of iNOS after FK506 treatment began to increase at 12 hours, peaked at 24 hours, and decreased after 36 hours. CONCLUSIONS: From these results, we confirmed that FK506 induces apoptosis and acts dose- and time-dependently to decrease the viability of Jurkat cells through activation of apoptosis signaling and expression of apoptotic ER-mediated stress proteins.

Transcriptional activation of nuclear-related factor 2 by FK506 in Jurkat T cells.

BACKGROUND: We investigated the effect of FK506 in transcriptional activation of nuclear factor (erythroid-derived 2)-like2 (Nrf2) in human Jurkat T cells. METHODS: FK506 treatment increased the generation of reactive oxygen species and reactive nitrogen species in Jurkat cells in a dose-dependent manner. Generation of nitric oxide was also increased after treatment with FK506 in Jurkat cells. Peak levels of endothelial nitricoxide synthase expression occurred at 24 hours and then decreased after 48 hours. RESULTS: We found that a marked dissociation of Nrf 2 from Kelch-like ECH-associated protein-1 and subsequently Nrf 2 nuclear translocation occurred in Jurkat cells treated with FK506 during 48 hours. Immunohistochemistry and Western blot analysis data revealed that the FK506 treatment increased expression of heme oxygenase-1 (HO-1) in Jurkat cells in a dose-dependent manner. HO-1 expression was induced after 6 hours of treatment of FK506 to Jurkat cells, peaked at 24 hours, and then decreased after 48 hours. CONCLUSIONS: These results suggest that FK506 induces Nrf 2-driven transcriptional activation of the antioxidant response element by activating HO-1 and free radicals such as reactive oxygen species and nitric oxide.

Lack of association between ACE and bradykinin B2 receptor gene polymorphisms and ACE inhibitor-induced coughing in hypertensive Koreans.

BACKGROUND & OBJECTIVE: Angiotensin converting enzyme (ACE) inhibitors are used widely in therapy for hypertension, congestive heart failure and myocardial infarction. However, coughing, one of their major adverse effects limits their use. It is documented that Asians are more liable to coughing than Europeans. The aim of this study was to investigate genetic polymorphism involved in ACE inhibitor-induced coughing. METHODS: We monitored hypertensive subjects (n = 110) treated with ACE inhibitors, and tested for any associations between ACE inhibitor-induced coughing and polymorphisms in the genes for ACE and the bradykinin B2 receptor, which are suspected to be related to coughing. RESULTS & DISCUSSION: We found no significant differences between the groups with coughing and without coughing in the frequency of ACE I/D (Insertion/Deletion) polymorphisms. One single nucleotide polymorphism was discovered in the promoter (-58T/C) and, one in intron-exon junction upsteam of exon 2 (-59C/A), of the bradykinin B2 receptor gene. However, no significant correlation was found between those genotypes or allele distributions and ACE inhibitor-induced coughing. CONCLUSION: We found no significant links between polymorphisms of the ACE gene or bradykinin B2 receptor gene with ACE inhibitor-induced coughing in hypertensive Koreans. But, the topic remains controversial and requires more study.

Influence of CYP2D6*10 on the pharmacokinetics of metoprolol in healthy Korean volunteers.

BACKGROUND AND OBJECTIVE: Genetic polymorphism of CYP2D6 leads to differences in pharmacokinetics of CYP2D6 substrates. The CYP2D6*10 allele is clinically important in Koreans because of its high frequency in Asians. We investigated whether the pharmacokinetics of metoprolol was altered by the presence of the CYP2D6*10 allele in Korean subjects. METHODS: One hundred and seven volunteers were recruited and grouped as CYP2D6*1/*1, CYP2D6*1/*10 and CYP2D6*10/*10 according to their genotypes. Metoprolol tartrate 100 mg (Betaloc) was administered orally once to each subject in these three groups (n = 6, 7 and 5, respectively). The pharmacokinetic parameters of metoprolol and its metabolite, alpha-hydroxymetoprolol, and the metabolic ratio for the three groups were estimated and compared. RESULTS AND DISCUSSION: The area under the plasma concentration-time curve (AUC(0-->infinity)), the maximum plasma concentration (C(max)) and the elimination half-life (T(1/2)) of metoprolol and alpha-hydroxymetoprolol for the CYP2D6*10/*10 group were all significantly different from those of the CYP2D6*1/*1 group (P < 0.05). The AUC(0-->infinity)s of metoprolol were 443.7 +/- 168.1, 995.6 +/- 321.4 and 2545.3 +/- 632.0 ng.h/mL, and the AUC(0-->infinity)s of alpha-hydroxymetoprolol were 1232.0 +/- 311.2, 1344.0 +/- 288.1 and 877.4 +/- 103.4 ng.h/mL for groups CYP2D6*1/*1, *1/*10 and *10/*10, respectively. The corresponding T(1/2) values of metoprolol were 2.7 +/- 0.5, 3.2 +/- 1.3 and 5.0 +/- 1.1 h, while those of alpha-hydroxymetoprolol were 5.4+/-1.5, 6.0 +/- 1.4 and 10.5 +/- 4.2 h, respectively. The metabolic ratios of the three groups were significantly different (P < 0.05). CONCLUSION: The CYP2D6*10 allele altered the pharmacokinetics of metoprolol in Korean subjects and is likely to affect other drugs metabolized by the CYP2D6 enzyme, similarly.

Emergent patterns of feedback strategies in performing a closed motor skill.

This sturdy identified self-chosen patterns of feedback usage and how they influenced accuracy and consistency in performing a closed motor skill. Learners completed 100 practice trials with 25 opportunities to receive knowledge of results (KR), the timing of which was self-chosen, followed by 25 no KR retention trials. The study identified two patterns of self-chosen KR, namely, a faded frequency pattern and a reversed faded frequency pattern. Analyses of variance indicated a significant trial block effect for accuracy in acquisition and no significant main effects or interactions associated with self-chosen patterns of KR. Quantification of regularity of KR patterns yielded correlations indicating that less regularity was associated with greater accuracy in performing the task. These results were congruent with previous research on self chosen feedback.

Effect of low back posture on the morphology of the spinal canal.

OBJECTIVE: To define the possible mechanism of posture-dependent symptoms of spinal stenosis by measuring the effect of low back posture on morphologic changes of the intervertebral discs and spinal canal in healthy young people. DESIGN: Twenty healthy young volunteers underwent magnetic resonance imaging while supine with their spine in neutral, flexed, extended, and right and left rotational positions. The axial MR images at the middle of the intervertebral discs of L3-4 and L4-5 were analyzed to measure the difference in the size and shape of the intervertebral discs and spinal canal in each posture. RESULTS: Extension or rotation decreased the sagittal diameters and cross-sectional areas of the dural sac and spinal canal and increased the thickness of the ligamentum flavum, whereas flexion had the opposite effects. The gap between the convex posterior disc margin and the anterior margin of the facet joint on each side, represented as the subarticular sagittal diameter, increased with flexion and decreased with extension or rotation. The direction of rotation did not result in asymmetry of the subarticular sagittal diameter, but right rotation caused thickening of the right ligamentum flavum, and vice versa. The shape and dimensions of the disc did not change significantly according to the positions of the low back. CONCLUSIONS: With extension or rotation, the thickness of the ligamentum flavum increased and the posterior margin of the intervertebral disc was approximated to the facet joint without any change in shape and size of the disc. These phenomena result in a decrease in the size of the spinal canal and dural sac in extension or rotation postures in young healthy people without disc degeneration, and may explain the posture-dependent symptom of spinal stenosis.

Protective effects of bergenin, the major constituent of Mallotus japonicus, on D-galactosamine-intoxicated rat hepatocytes.

This study was designed to investigate the effects of bergenin against D-galactosamine-induced injury in primary cultured rat hepatocytes. Bergenin (100 microM) decreased the release of glutamic pyruvic transaminase and sorbitol dehydrogenase by 62 and 50%, respectively, into hepatocyte medium incubated for 14 h with 1.5 mM galactosamine. Decreased RNA synthesis by 1.5 mM galactosamine was recovered 2.5 times compared with that of control hepatocytes at 100 microM bergenin. Therefore, the present results suggest that bergenin show hepatoprotective effects against galactosamine-intoxicated rat hepatocytes by inhibiting the release of glutamic pyruvic transaminase and sorbitol dehydrogenase as well as by increasing RNA synthesis.

Anti-inflammatory effects of fangchinoline and tetrandrine.

Fangchinoline and tetrandrine are the major alkaloids from Stephania tetrandrae S. Moore which has been used traditionally for the treatment of inflammatory diseases in oriental countries including Korea. Both fangchinoline and tetrandrine showed anti-inflammatory effects on mouse ear edema induced by croton oil. In addition, the effects of fangchinoline and tetrandrine on cyclooxygenase, murine interleukin-5 (mIL-5) and human interleukin-6 (hIL-6) were examined in vitro to investigate the anti-inflammatory action mechanisms. One hundred micromolar of fangchinoline showed 35% of inhibition on cyclooxygenase, but the same concentration of tetrandrine did not show any inhibition. On the other hand, 12.5 microM of tetrandrine exhibited 95% of inhibition on mIL-5 activity, while fangchinoline did not show any effects. However, 4 microM of fangchinoline and 6 microM of tetrandrine showed 63 and 86% of inhibitions on hIL-6 activity, respectively. These results suggest that biochemical mechanisms of fangchinoline and tetrandrine on anti-inflammation are significantly different even though they are similar in chemical structure.

Antihepatotoxic activity of bergenin, the major constituent of Mallotus japonicus, on carbon tetrachloride-intoxicated hepatocytes.

To determine the antihepatotoxic activity of bergenin from Mallotus japonicus, carbon tetrachloride (CCl4)-induced cytotoxicity in primary cultured rat hepatocytes has been adopted as an assay system. Bergenin significantly reduced the activities of glutamic pyruvic transaminase and sorbitol dehydrogenase released from the CCl4-intoxicated hepatocytes. The antihepatotoxicity of bergenin was also evidenced by elevating the activities of glutathione S-transferase and glutathione reductase, and content of glutathione in the CCl4-intoxicated hepatocytes. From these results, it is assumed that bergenin exerted antihepatotoxicity against CCl4-induced cytotoxicity through glutathione-mediated detoxification as well as free radical suppressing activity.

Modulation of NMDA receptor subunit mRNA in butorphanol-tolerant and -withdrawing rats.

The NMDA receptor has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of NMDA receptor subunit NR1, NR2A, NR2B, and NR2C gene expression were investigated by using in situ hybridization technique. Continuous intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/microl/h) resulted in significant modulations in the NRI, NR2A, and NR2B mRNA levels. The level of NR1 mRNA was significantly decreased in the cerebral cortex, thalamus, and CA1 area of hippocampus in butorphanol tolerant and withdrawal (7 h after stopping the infusion) rats. The NR2A mRNA was significantly decreased in the CA1 and CA3 of hippocampus in tolerant rats and increased in the cerebral cortex and dentate gyrus in butorphanol withdrawal rats. NR2B subunit mRNA was decreased in the cerebral cortex, caudate putamen, thalamus, CA3 of hippocampus in butorphanol withdrawal rats. No changes of NR1, NR2A, NR2C subunit mRNA in the cerebellar granule cell layer were observed in either butorphanol tolerant or withdrawal rats. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [3H]MK-801 was increased significantly in all brain regions except in the thalamus and hippocampus, at the 7 hr after stopping the butorphanol infusion. These results suggest that region-specific changes of NMDA receptor subunit mRNA (NR1 and NR2) as well as NMDA receptor binding ([3H]MK-801) are involved in the development of tolerance to and withdrawal from butorphanol.

Suloctidil-induced hepatotoxicity.

Suloctidil is a new drug that is currently being evaluated in many clinical trials for use in dementia and thrombotic disorders. Hepatotoxicity has to date been reported exclusively in the European literature, and the few available histologic descriptions have been reported in the French language. We report a case of suloctidil-induced hepatotoxicity documented by serum liver biochemical tests and liver biopsy. Histologic features included focal necrosis of hepatocytes, mild hyperplasia of Kupffer cells, and other features suggestive of mild acute hepatitis.

Substance data bases and right to know laws: application to occupational epidemiology.

The Illinois Right to Know (RTK) law included requirements for substance lists to be submitted by companies to the Illinois Department of Labor (IDOL). This provided an opportunity to test the feasibility of identifying workplaces utilizing common chemicals for future epidemiologic investigations. A sample of IDOL files (n = 115) was obtained, and relevant data elements were coded. A second sample of substance names within these files (n = 1,015) was selected, and searches in three standard references were conducted to identify chemical descriptions. Equal proportions of employers and manufacturer/suppliers were in general compliance with RTK law reporting requirements (58%). Forty-five percent of substances sampled from employers (chemical users) and 71% of substances sampled from manufacturer/suppliers (chemical producers and distributors) could be identified. The ability to identify substances reported using chemical names was approximately equal across companies (90%), while the ability to identify substances with nonchemical names was greater in manufacturer (59%) than in employer (32%) files. This study suggests that the ability to identify potential occupationally exposed groups using this resource may be greater among manufacturers than among employers. Recognition of substances used in the workplace could be improved if companies were required to report chemical names.