Irreversible hyperoxidation of peroxiredoxin 2 is caused by tert-butyl hydroperoxide in human red blood cells.

Peroxiredoxin 2 (Prx2) is the third most abundant protein in red blood cells (RBCs). In this study, we have succeeded in implementing the rapid and simultaneous detection of the hyperoxidized (Prx2-SO2/3) and reduced (Prx2-SH) forms of Prx2 in human RBCs using reverse phase high-performance liquid chromatography. The detection of a peak corresponding to Prx2-SO2/3 was clearly observed following treatment of tert-butyl hydroperoxide (t-BHP), but not H2O2, and was found to be dose-dependent. The identity of the peak was confirmed as Prx2 by immunoblotting and mass spectrometry analysis. Our results suggest that t-BHP hyperoxidizes cysteine residues in Prx2 more readily than H2O2, and that accumulation of hyperoxidized Prx2 might reflect disruption of redox homeostasis in RBCs.

Effect of portal haemodynamics on liver graft and intestinal mucosa after small-for-size liver transplantation in swine.

BACKGROUND: After small-for-size graft (SFSG) transplantation, elevated portal venous pressure (PVP) may lead to postoperative liver damage. Herein we evaluated the impact of portocaval shunt (PCS) to control PVP on liver grafts and intestine following SFSG transplantation. METHODS: Nineteen SFSG transplantations were performed with 30% of native liver in swine. Swine were divided into 3 groups: a high-flow shunt group (HS: n = 7), in which portal venous flow (PVF) was reduced with a 10-mm diameter PCS; a low-flow shunt group (LS: n = 6), in which PVF was reduced with a 5-mm diameter PCS, and a no-shunt group (NS: n = 6), in which no PCS was placed. RESULTS: Seven-day survivals were 83.3% in NS, 100% in LS and 0% in HS (p = 0.0088). PVP was significantly higher in the NS group (p = 0.0001; mean ± SEM NS/LS/HS: 20.5 ± 0.7/14.0 ± 1.2/11.6 ± 0.5 mm Hg). The LS group exhibited the highest compliance (PVF/PVP; NS/LS/HS 42.7 ± 10.9/44.6 ± 4.9/37.7 ± 8.3 ml/min/mm Hg; p = 0.009), the lowest aspartate aminotransferase (NS/LS/HS 562 ± 18/370 ± 55/720 ± 130 IU/l; p = 0.0493), and suppressed deleterious alternations of the hepatic parenchyma and intestinal mucosa. CONCLUSIONS: Portal hypertension after SFSG transplantation impaired liver and intestinal mucosa; however, inadequate portal flow impaired not only the liver, but also survival.

Involvement of tumour necrosis factor-alpha in Clostridium perfringens beta-toxin-induced plasma extravasation in mice.

BACKGROUND AND PURPOSE: Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK(1) receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation. EXPERIMENTAL APPROACH: Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA. KEY RESULTS: Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-alpha and interleukin (IL)-1beta levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-alpha and IL-1beta. The plasma extravasation and the release of TNF-alpha induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-alpha. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-alpha, but not anti-IL-1beta. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody. CONCLUSIONS AND IMPLICATIONS: These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-alpha via the mechanism involving tachykinin NK(1) receptors, but not via TLR4.

The application of an institutional clinical data warehouse to the assessment of adverse drug reactions (ADRs). Evaluation of aminoglycoside and cephalosporin associated nephrotoxicity.

OBJECTIVES: To apply an institutional clinical data warehouse (CDW) to the assessment of adverse drug reactions (ADRs) and demonstrate its utility through a specific example. METHODS: We modeled the process for assessing ADRs through retrospective cohort design by using CDW at the Osaka University Hospital as follows: 1) We defined a drug X, an adverse drug reaction (ADR) Y, and a laboratory measurement Z to assess Y during a given study period; 2) we excluded those whose Z value exceeded the defined criteria or were not available at the inception of the cohort; 3) we divided the patients into two groups based on exposure or non-exposure to X; 4) we matched the patient characteristics between the two groups through stratification and randomization; and 5) we compared the frequency of patients who presented Y during the study period between the two groups. Aminoglycoside and Cephalosporin associated nephrotoxicity in pediatric inpatients was used as an example to demonstrate the usefulness of this approach. RESULTS: Our evaluation indicates that there is an increased risk of nephrotoxicity for pediatric inpatients who were prescribed cephalosporin either alone or in combination with aminoglycoside; further, aminoglycoside tends to increase the cephalosporin-associated nephrotoxicity. CONCLUSIONS: Our findings are consistent with those drawn from other studies, indicating that the method of applying an institutional CDW is useful for assessing ADRs.

Down-regulation of an inhibitor of cell growth, transmembrane protein 34 (TMEM34), in anaplastic thyroid cancer.

PURPOSE: Ansaplastic thyroid cancer (ATC) is one of the most lethal malignancies, but the carcinogenic mechanism of ATC has not been clarified. Recently, we performed a cDNA microarray analysis and identified transmembrane protein 34 (TMEM34) that down-regulated in anaplastic thyroid cancer cell lines (ACL)s as compared to normal thyroid tissues. METHODS: To investigate the role of TMEM34 in ATC carcinogenesis, we examined expression levels of TMEM34 in ACLs as well as differentiated thyroid cancers (DTC)s and normal human tissues. To explore the effect of TMEM34 in ATC development, cell-growth assays with KTA2 cells were performed. RESULTS: Expression of TMEM34 was down-regulated in all 11 ACLs, as compared to either normal thyroid tissues or cell lines derived from papillary or follicular thyroid cancers. TMEM34 was expressed ubiquitously in normal human tissues tested. Transfection of TMEM34 into KTA2 cells led to inhibition of cell growth. CONCLUSIONS: Our findings suggest that TMEM34 might be a tumor suppressor gene, associated with the development of ATC from DTC.

Chemical evolution of RNA under hydrothermal conditions and the role of thermal copolymers of amino acids for the prebiotic degradation and formation of RNA.

The roles of thermal copolymers of amino acids (TCAA) were studied for the prebiotic degradation of RNA. A weak catalytic ability of TCAA consisted of Glu, L-Ala, L-Val, L-Glu, L-Asp, and optionally L-His was detected for the cleavage of the ribose phosphodiester bond of a tetranucleotide (5'-dCrCdGdG) in aqueous solution at 80 degees C. The rate constants of the disappearance of 5'-dCrCdGdG were determined in aqueous solutions using different pH buffer and TCAA. The degradation rates were enhanced 1.3-3.0 times in the presence of TCAA at pH 7.5 and 8.0 at 80 degrees C, while the hydrolysis of oligoguanylate (oligo(G)) was accelerated about 1.6 times at pH 8.0. A weak inhibitory activity for the cleavage of oligo(G) was detected in the presence of 0.055 M TCAA-Std. On the other hand, our recent study on the influences of TCAA for the template-directed reaction of oligo(G) on a polycytidylic acid template showed that TCAA has an acceleration activity for the degradation of the activated nucleotide monomer and an acceleration activity for the formation of G5' ppG capped oligo(G). This series of studies suggest that efficient and selective catalytic or inhibitory activities for either the degradation or formation of RNA under hydrothermal conditions could have hardly emerged from the simple thermal condensation products of amino acids. A scenario is going to be deduced on the chemical evolution of enzymatic activities and RNA molecules concerning hydrothermal earth conditions.

Comprehensive gene expression profiling of anaplastic thyroid cancers with cDNA microarray of 25 344 genes.

Little is known about the genetic mechanisms of anaplastic thyroid cancer (ATC). This is the most virulent of all human malignancies, and it is believed to result from transformation of differentiated thyroid cancers. To identify a set of genes involved in the development of ATC, we investigated expression profiles of 11 cell lines derived from ATC using a cDNA microarray representing 25 344 genes. Semi-quantitative RT-PCR experiments carried out for some genes that had shown altered expression on the microarray verified frequent over-expression of destrin, HSPA8, stathmin, LDH-A, ATP5A1, PSMB6, B23, HDP-1 and LDH-B, and frequent under-expression of thyroglobulin, PBP and c-FES/FPS genes among the cell lines and also among ten primary ATCs. In addition to mRNA expression studies, up-regulation of GDI2, destrin and stathmin were confirmed with immunohistochemical analysis. The extensive list of genes identified provides valuable information towards understanding the development of ATC, and provides a source of possible biomarkers for diagnosis and/or molecular targets for the development of novel drugs to treat ATC.

Male siblings with dyserythropoiesis, microcephaly and intrauterine growth retardation.

Male siblings with intrauterine growth retardation, hydrops, mild liver dysfunction, chronic diarrhoea, failure to thrive and microcephaly are reported. In both patients, the intrauterine growth retardation was detected in the second trimester of pregnancy. Relatively severe early onset neonatal jaundice, microcytosis, anisocytosis and abnormal iron metabolism were also seen. Bone marrow examination in the second sibling showed marked ringed sideroblasts and multilobulated erythroblasts in late developmental stages. The brain was very small with enlarged cerebrospinal fluid space, a reduced number of gyri and a thin cortex. The clinical and laboratory findings in these patients appear to be unique.

The first direct oxidative conversion of a selenol to a stable selenenic acid: experimental demonstration of three processes included in the catalytic cycle of glutathione peroxidase.

[structure: see text]. A stable selenenic acid was synthesized by direct oxidation of a selenol bearing a novel bowl-type substituent with H2O2, and its structure was established by X-ray crystallographic analysis. Selenenyl sulfides obtained by the reaction of the selenenic acid with 1,4-dithiols were reduced to the corresponding selenol by treatment with a tertiary amine, thus achieving the experimental demonstration of three processes included in the catalytic cycle of glutathione peroxidase.

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum.

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.

Distribution of peripheral nerve terminals in the small and large intestine of congenital aganglionosis rats (Hirschsprung's disease rats).

The congenital aganglionosis rat is considered to be an animal model of Hirschsprung's disease. The mutants had a long constricted segment (from distal ileum to rectum) below the dilated distal ileum. In the dilated region, synaptophysin-immunoreactivity (IR) was almost preserved in all layers of the intestinal wall. In the constricted distal ileum and oral proximal colon, synaptophysin-IR was scarce in all layers, including the circular and longitudinal muscle layers. In the anal proximal and distal colon, synaptophysin-IR was almost scarce in the circular muscle layer (CML). An ultrastructural study confirmed that almost no terminals were found in the CML of any regions of constricted intestine. Therefore, the CML in any region of a constricted segment, is presumed to be poor innervation. However, a few synaptophysin-IR were found in the longitudinal muscle layer (LML) of an anal part of a constricted segment. An ultrastructural study also confirmed that some terminals were observed in the LML of this segment. The present study suggested that denervated CML is related to the production of constricted segment, irrespective of the presence or absence of terminals in the LML.

Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.

Improved hepatic microcirculation by human soluble urinary thrombomodulin in the xeno-perfused porcine liver.

PURPOSE: Both the protein C/thrombomodulin system and the heparin/anti-thrombin III system are major physiological anticoagulant systems, which may also play a major role in preserving the hepatic microcirculation in xenogeneic liver transplantation. To compensate for the functional incompatibilities of the porcine thrombomodulin (TM)-cofactor activity beyond species for human thrombin, soluble human TM protein was tested in xenogeneic perfusion of the porcine liver. MATERIALS AND METHODS: The livers were harvested from adult female pigs and perfused through the portal vein (PV) and hepatic artery (HA) for 2 hr, with fresh human blood in group 1 (n=5), fresh porcine blood (10 units/ml) in group 2 (n=5), and fresh human blood with TM (50,000 units/1.5 l) in group 3 (n=5). The tissue PO2 level, tissue blood flow, PV and HA pressures were all continuously monitored. Circulating perfusate and liver tissue samples were periodically obtained for blood chemistry and histologic analyses. RESULTS: The activated protein C (aPC) level was significantly elevated in the TM-treated group 3 (47.5%+/-3.5% at preperfusion and 51%+/-2.8% after 120 min of perfusion) in comparison to group 1 (32.3%+/-7.2% and 35.3+/-12.0%). The hepatocyte enzyme release of aspartate aminotransferase (AST) was suppressed significantly more in group 3 (238.2+/-107 IU/l), than in group 1 (672.3+/-160 IU/l) at 2 hr after reperfusion. In group 3, the tissue PO2 levels and tissue blood flow also remained significantly higher throughout the perfusion. The platelet counts in the perfusate remained significantly higher in group 3 (37.1% to 74.3% of the preperfusion level) than in group 1 (4.4% to 14.7%), after 0 to 80 min of perfusion. According to the histologic findings, the degree of interlobular hemorrhaging and congestion decreased remarkably more in group 3 than in group 1. CONCLUSION: These findings thus indicated that soluble thrombomodulin protein extracted from human urine remarkably improved hepatic microcirculation in the xenoperfused porcine liver. The thrombomodulin/protein C system might, thus, play an important role in restoring the physiological anticoagulant system in the xenoperfused porcine liver.

Effect of bezafibrate on soluble adhesion molecules and platelet activation markers in patients with connective tissue diseases and secondary hyperlipidemia.

We evaluated the plasma concentrations of soluble adhesion molecules, platelet activation markers, and platelet-derived microparticles (PMPs) in patients with connective tissue diseases who had secondary hyperlipidemia caused by long-term steroid administration (n = 22) before and after treatment with bezafibrate. There were differences in levels of platelet activation markers both before and after treatment (platelet CD62p: 15.11+/-2.03 vs 10.38+/-8.53%, P < 0.05; platelet CD63: 12.12+/-9.17 vs 9.90+/-7.20%, P < 0.05). There were also differences in the levels of PMPs and soluble adhesion molecules both before and after treatment (PMP: 514+/-273 vs 401+/-201 /10(4) platelet. P < 0.05; soluble vascular cell adhesion molecule-1: 724+/-191 vs 666+/-157 ng/mL, P < 0.01). After 6 months of treatment, serum lipid concentrations were reduced by 9% for total cholesterol (TC) and 32% for triglyceride (TG). The level of PMPs, activated platelets, and soluble adhesion molecules were all significantly decreased after treatment with bezafibrate. These findings suggest that bezafibrate may be useful for inhibiting both PMP-dependent and -independent vascular damage in patients with connective tissue diseases complaining of secondary hyperlipidemia.

Platelet activation markers and soluble adhesion molecules in patients with systemic lupus erythematosus.

We assessed the role of platelet activation markers (PMPs, Annexin V and CD62P on activated platelets), cytokines (IL-1 beta, IL-4, IL-6, IFN- gamma, GM-CSF, and TNF alpha ), and soluble factors (sIL-2R, TM, sHLA-1, beta(2) -m, sVCAM-1, sPECAM-1, sP-selectin and sE-selectin) in vascular damage related to SLE. There were differences in the levels of PMPs and platelet activation markers between the SLE patients and controls (PMPs: 493+/-82 vs. 328+/-36, p<0.05; plt-CD62P; 8.5%+/-1.2 % vs. 4.6%+/-0.7 %, p<0.05; plt-Annexin V: 11.3%+/-2.1 % vs. 4.9%+/-0.6 %, p<0.01). There were no differences in the levels of IFN- gamma between the groups. However, the levels of IL-1 beta, IL-4, IL-6, GM-CSF, TNF alpha, and soluble factors were higher in the SLE patients than in the controls. The levels of IL-4, IL-6, beta2 -m, sIL-2R, sVCAM-1, sP-selectin, and sE-selectin in SLE patients with elevated sTM levels were higher than those in the SLE patients without elevated sTM levels. On the other hand, elevations of sIL-2R, sVCAM-1, and sP-selectin were not found in patients with Behçet disease or rheumatoid arthritis. The levels of platelet CD62P, platelet annexin V, and PMP were significantly elevated in high-sTM patients. These findings suggest the possibility that activated platelets and cytokines participate in the pathogenesis of SLE in patients with elevated sTM levels.

[Systemic inflammatory response syndrome triggered by necrotizing cholecystitis after treatment of underlying low titer cold agglutinin disease].

In April 1996, a 77-year-old man initially presented with fever, rash and polyarthralgia, and was diagnosed as having low titer cold agglutinin disease with acute hemolytic anemia. The patient's condition and laboratory findings improved after administration of corticosteroid (prednisolone 60 mg). In June 1996, however, he developed acute cholecystitis and died due to sepsis, disseminated intravascular coagulation and multiple organ failure. During the course, the levels of inflammatory cytokines such as TNF-alpha and IL-6 were correlated with the pathology, and the disease was diagnosed as systemic inflammatory response syndrome (SIRS). Autopsy revealed necrotizing cholecystitis, erythrophagocytosis in the liver, and cytomegalovirus infection in the lung and gall bladder. This was considered to be a rare case of low titer cold agglutinin disease complicated by SIRS.

Determination of functional regions of p125, a novel mammalian Sec23p-interacting protein.

The Sec23p-Sec24p complex is a component of coat protein II-coated vesicles involved in protein export from the endoplasmic reticulum. We previously identified a novel Sec23p-interacting protein, p125, which consists of 1000 amino acids and comprises a proline-rich region and a phospholipase A(1) homology region. p125, when ectopically expressed in cultured cells, localizes to endoplasmic reticulum-Golgi intermediate regions. In the present study we showed that expressed p125 principally colocalizes with p115 and GM130, both of which are involved in vesicle tethering to Golgi membranes. Next, we determined the functional regions of p125 by expressing a p125 series with deletions. The results showed that the proline-rich region (residues 135-259) is responsible for the binding to Sec23p. For the correct localization of p125, a region (residues 135-1000) comprising both the proline-rich and phospholipase A(1) homology regions was required.