Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification

The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.     

Xanthii Fructus Inhibits Inflammatory Responses in LPS-Stimulated Mouse Peritoneal Macrophages

Xanthii Fructus (XF) is an herb widely used in medicine for the treatment of a variety of inflammatory pathologies. In this study, using mouse peritoneal macrophages, we have examined whether XF affects nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-12p40 production induced by interferon (IFN)-γ and lipopolysaccharide (LPS). XF inhibits IFN-γ and LPS-induced NO production in a dose dependent manner. The decrease in NO synthesis was reflected as a decreased amount of inducible NO synthase protein. Furthermore, we also found that XF inhibits pro-inflammatory cytokine TNF-α production. However, treatment of XF in peritoneal macrophages had no effect on IL-12p40 production. These findings suggest that XF may be used in controlling macrophages-mediated inflammatory diseases.     

Clonorchiasis and cholangiocarcinoma: etiologic relationship and imaging diagnosis

Despite a gradual decrease in prevalence, clonorchiasis is still prevalent in East Asia. A large and compelling body of evidence links clonorchiasis and cholangiocarcinoma, although the mechanisms involved are not completely understood. Clonorchiasis induces biliary epithelial hyperplasia and metaplasia, and this could facilitate at least one stage of the carcinogenesis, which is promoting effect. In areas of endemic infection, more clonorchiasis cases are now diagnosed incidentally during radiological examinations such as cholangiography, ultrasonography, and computed tomography. Radiological findings are regarded as pathognomonic for clonorchiasis since they reflect the unique pathological changes of this disorder. These radiological examinations currently play important roles in the diagnosis, staging, and decision-making process involved in the treatment of cholangiocarcinoma. The morphological features and radiological findings of clonorchiasis-associated cholangiocarcinoma are essentially combinations of the findings for the two diseases. The morphological features of clonorchiasis- associated cholangiocarcinoma, observed in radiological examinations, do not differ from those of the usual cholangiocarcinoma. In patients diagnosed with or suspected to have clonorchiasis, radiological findings should be carefully scrutinized for occult cholangiocarcinoma.     

Metabolic changes after revascularization in a patient with innominate artery occlusion by localized in vivo proton magnetic resonance spectroscopy

Localized in vivo proton magnetic resonance spectroscopy ((1)H-MRS) has been used to measure the metabolic status of the human brain in a non-invasive manner; thus, it is often called "a non-invasive biochemical assay". MRS is more sensitive than magnetic resonance imaging (MRI) in detecting ischemic damage by measuring the metabolic changes that occur prior to the anatomic changes. We report a patient who presented with innominate artery occlusion and symptoms of posterior circulation insufficiency and showed favorable metabolic changes by (1)H-MRS after revascularization. He showed no visible lesion in brain MRI, but in (1)H-MRS, decreased N-acetylaspartate (NAA) signal was noted in a resting state.After revascularization, both symptomatic improvement and recovery of NAA signal were observed. (1)H-MRS may provide valuable clinical information in diagnosis and management of cerebral hypoperfusion at a much earlier stage prior to the anatomic changes.     

Conversion of leukotriene D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leucocytes

Leukotriene D4 (LTD4), the most active spasmogenic leukotriene constituent of the slow reacting substance of anaphylaxis was converted by suspended human polymorphonuclear leucocytes (PMNs) to a single, less polar metabolite which was not further catabolized. This product was identified as leukotriene E4 (LTE4) by its retention time during reverse phase-high performance liquid chromatography (RP-HPLC) and subsequent bioassay on the guinea-pig ileum. LTD4 with a retention time of 21 +/- 1.6 min (mean +/- SD) and a contractile activity of 5.0 +/- 0.4 u./pmol (mean +/- SD) was quantitatively converted extracellularly by PMNs to LTE4 with a retention time of 26 +/- 1.8 min and a contractile activity of 1.2 +/- 0.3 u./pmol. Subcellular fractionations of PMNs revealed the recovered LTD4-to-LTE4 converting activity, termed LTD4 dipeptidase, to be localized only in he granule fraction. There was a time- and calcium-dependent extracellular release of LTD4 dipeptidase in association with lysozyme (r = 0.97, n = 16, P less than 0.001), a constituent of both specific and azurophilic granules, in the absence of release of cytoplasmic lactate dehydrogenase (LDH) and of beta-glucuronidase from the azurophilic granule. Phorbol myristate acetate (PMA), which selectively induces secretion of specific granules, released lysozyme and the LTD4 dipeptidase in a constant dose-dependent manner from PMNs (r = 0.96, n = 8, P less than 0.001). Calcium ionophore A23187 at concentrations less than 10(-7) M stimulated the parallel secretion of LTD4 dipeptidase and lysozyme (r = 0.91, n = 9, P less than 0.005), dipeptidase and lysozyme (r = 0.91, n = 9, P less than 0.005), whereas higher concentrations resulted in secretion of beta-glucuronidase and additional lysozyme without further release of dipeptidase. Thus, human PMNs can convert LTD4 to LTE4, a less vasoactive and spasmogenic leukotriene, via the secretion of a dipeptidase associated with the specific granules.     

Ultrastructural studies of the hepatocytes after chronic exposure to low levels of halothane.

Adult rats were exposed to 10 ppm or 500 ppm halothane 8 hr/day and 5 days/wk for 8 wk or 4 wk, respectively. In the liver from animals which were exposed to 10 ppm of halothane, the rough endoplasmic reticulum in some hepatocytes accumulated a floccular, electron-dense material which gave the hepatocytes a dense and dark appearance. Increase in the matrical density and C-shaped transformation were observed in the mitochondria of some hepatocytes. In addition to these findings, areas of focal cytoplasmic degradation, dilatation of the bile canaliculi, peribiliary accumulation of lysosomes, and extensive dilatation of the smooth endoplasmic reticulum to form large cytoplasmic vacuoles were also observed in the hepatocytes of animals which had been exposed to 500 ppm halothane. Toxic potential of halothane upon chronic exposure is suggested.     

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.     

A polymorphic major histocompatibility complex class II‐like locus maps outside of both the chicken B‐system and Rfp‐Y‐system

Chickens have two major regions encoding major histocompatibility complex (MHC) class Iα genes and MHC class IIß genes, the serological and functional B‐system and the Rfp‐Y‐system. Recently, they have been shown to assort in a genetically independent way although still located on the same microchromosome. Moreover, the monomorphic MHC class IIα gene maps at a third locus located 5 c m from the nearest class IIß genes, located in the B‐system ( Kaufman et al., 1995 ). A pedigree family was studied in three generations in order to assign MHC class IIß restriction fragments observed in Southern blot analyses to either the B‐system, the Rfp‐Y‐system or the B‐Lα locus. In this study, we demonstrate by classical genetic testing of chickens within this fully pedigreed family the existence of an MHC class II‐like polymorphic restriction fragment that segregates independently of the B‐system, the Rfp‐Y‐system and of the B‐Lα locus.     

Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.