Analysis of tissue cadmium distribution in chronic cadmium-exposed mice using in-air micro-PIXE

This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE) and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with 3 mg/kg body weight of CdCl₂ thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity. The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced toxicity.     

Transcriptional Induction of Two Genes for CCaPs, Novel Cytosolic Proteins, in Arabidopsis thaliana in the Dark

Ca2+-signaling in downstream effectors is supported by many kinds of Ca2+-binding proteins, which function as a signal mediator and a Ca2+-buffering protein. We found in Arabidopsis thaliana a new type of Ca2+-binding protein, CCaP1, which consists of 152 amino acid residues, and binds (45)Ca2+ even in the presence of a high concentration of Mg2+. We found two other proteins with similar motifs, CCaP2 and CCaP3. These three proteins had no organelle localization signal and their green fluorescent protein (GFP) fusions were detected in the cytosol. Real-time PCR and histochemical analysis of promoter-beta-glucuronidase fusions revealed that CCaP1 was predominantly expressed in petioles while CCaP2 was expressed in roots. CCaP3 was hardly expressed. Expression of CCaP1 and CCaP2 was enhanced in darkness and became maximal after 24 h. Immunoblotting revealed petiole-specific accumulation of CCaP1. Expression of CCaP1 and CCaP2 was suppressed by a high concentration of Ca2+ and other metal ions. Deletion of sucrose from the medium markedly increased the mRNA levels of CCaP1 and CCaP2 within 2 h. Gibberellic acid enhanced the expression of CCaP1 and CCaP2 by 5- and 2.5-fold, respectively, after 6 h. CCaP1 and CCaP2 were suppressed in the petiole and the root, respectively, by light and the product of photosynthesis (sucrose) or both. These results suggest that CCaP1 functions as a mediator in response to continuous dark or gibberellic acid.     

Regulation of leukocyte degranulation by cGMP-dependent protein kinase and phosphoinositide 3-kinase: potential roles in phosphorylation of target membrane SNARE complex proteins in rat mast cells

We examined the roles of cGMP-dependent protein kinase (PKG) and PI3K in degranulation induced by fMLF and by FcepsilonRI cross-linking. In rat basophilic leukemia-2H3 cells expressing formyl peptide receptor, the PKG inhibitors KT5823 and Rp-8-Br-PET-cGMP, as well as the PI3K inhibitor LY294002, reduced agonist-stimulated beta-hexosaminidase release in a dose-dependent manner. These inhibitors also abolished vesicular fusion with the plasma membrane, as evidenced by diminished annexin V staining. Agonist-induced degranulation was completely blocked when LY294002 was applied together with one of the PKG inhibitors, suggesting an additive and possibly synergistic effect. In contrast, the PKG inhibitors did not affect fMLF-induced intracellular calcium mobilization and Akt phosphorylation. Likewise, LY294002 did not alter fMLF-induced elevation of intracellular cGMP concentration, and the inhibitory effect of LY294002 was not reversed by a cell-permeable analog of cGMP. Treatment with fMLF induced phosphorylation of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP)-23, syntaxins 2, 4, and 6, and Monc18-3. The induced phosphorylation of SNAP-23 and syntaxins 2 and 4 was blocked by Rp-8-Br-PET-cGMP and LY294002. However, LY294002 was less effective in inhibiting Munc18-3 phosphorylation. The induced phosphorylation of syntaxin 6 was not effectively blocked by either Rp-8-Br-PET-cGMP or LY294002. Treatment of human neutrophils with the PKG inhibitors and LY294002 reduced enzyme release from primary, secondary, and tertiary granules. These results suggest that PKG and PI3K are involved in degranulation, possibly through phosphorylation of target membrane SNAP receptor proteins and their binding proteins.     

Schnurri-2 controls memory Th1 and Th2 cell numbers in vivo

Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kappaB was up-regulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kappaB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent Ab responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.     

Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning

We have previously reported that the prolonged transient acidosis during early reperfusion mediates the cardioprotective effects in canine hearts. Recently, postconditioning has been shown to be one of the novel strategies to mediate cardioprotection. We tested the contribution of the prolonged transient acidosis to the cardioprotection of postconditioning. Open-chest anesthetized dogs subjected to 90-min occlusion of the left anterior descending coronary artery and 6-h reperfusion were divided into four groups: 1) control group; no intervention after reperfusion (n = 6); 2) postconditioning (Postcon) group; four cycles of 1-min reperfusion and 1-min reocclusion (n = 7); 3) Postcon + sodium bicarbonate (NaHCO(3)) group; four cycles of 1-min reperfusion and 1-min reocclusion with the administration of NaHCO(3) (n = 8); and 4) NaHCO(3) group; administration of NaHCO(3) without postconditioning (n = 6). Infarct size, the area at risk (AAR), collateral blood flow during ischemia, and pH in coronary venous blood were measured. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK) in ischemic myocardium was assessed by Western blot analysis. Systemic hemodynamic parameters, AAR, and collateral blood flow were not different among the four groups. Postconditioning induced prolonged transient acidosis during the early reperfusion phase. Administration of NaHCO(3) completely abolished the infarct size-limiting effects of postconditioning. Furthermore, the phosphorylation of Akt and ERK in ischemic myocardium induced by postconditioning was also blunted by the cotreatment of NaHCO(3). In conclusion, postconditioning mediates its cardioprotective effects possibly via prolonged transient acidosis during the early reperfusion phase with the activation of Akt and ERK.     

Muscarinic potassium channels augment dynamic and static heart rate responses to vagal stimulation

Vagal control of heart rate (HR) is mediated by direct and indirect actions of ACh. Direct action of ACh activates the muscarinic K(+) (K(ACh)) channels, whereas indirect action inhibits adenylyl cyclase. The role of the K(ACh) channels in the overall picture of vagal HR control remains to be elucidated. We examined the role of the K(ACh) channels in the transfer characteristics of the HR response to vagal stimulation. In nine anesthetized sinoaortic-denerved and vagotomized rabbits, the vagal nerve was stimulated with a binary white-noise signal (0-10 Hz) for examination of the dynamic characteristic and in a step-wise manner (5, 10, 15, and 20 Hz/min) for examination of the static characteristic. The dynamic transfer function from vagal stimulation to HR approximated a first-order, low-pass filter with a lag time. Tertiapin, a selective K(ACh) channel blocker (30 nmol/kg iv), significantly decreased the dynamic gain from 5.0 +/- 1.2 to 2.0 +/- 0.6 (mean +/- SD) beats.min(-1).Hz(-1) (P < 0.01) and the corner frequency from 0.25 +/- 0.03 to 0.06 +/- 0.01 Hz (P < 0.01) without changing the lag time (0.37 +/- 0.04 vs. 0.39 +/- 0.05 s). Moreover, tertiapin significantly attenuated the vagal stimulation-induced HR decrease by 46 +/- 21, 58 +/- 18, 65 +/- 15, and 68 +/- 11% at stimulus frequencies of 5, 10, 15, and 20 Hz, respectively. We conclude that K(ACh) channels contribute to a rapid HR change and to a larger decrease in the steady-state HR in response to more potent tonic vagal stimulation.     

Simulation study on the disordered state of an Alzheimer's beta amyloid peptide Abeta(12 36) in water consisting of random-structural, beta-structural, and helical clusters

The monomeric Alzheimer's beta amyloid peptide, Abeta, is known to adopt a disordered state in water at room temperature, and a circular dichroism (CD) spectroscopy experiment has provided the secondary-structure contents for the disordered state: 70% random, 25% beta-structural, and 5% helical. We performed an enhanced conformational sampling (multicanonical molecular dynamics simulation) of a 25-residue segment (residues 12-36) of Abeta in explicit water and obtained the conformational ensemble over a wide temperature range. The secondary-structure contents calculated from the conformational ensemble at 300 degrees K reproduced the experimental secondary-structure contents. The constructed free-energy landscape at 300 degrees K was not plain but rugged with five clearly distinguishable clusters, and each cluster had its own characteristic tertiary structure: a helix-structural cluster, two beta-structural clusters, and two random-structural clusters. This indicates that the contribution from the five individual clusters determines the secondary-structure contents experimentally measured. The helical cluster had a similarity with a stable helical structure for monomeric Abeta in 2,2,2-trifluoroethanol (TFE)/water determined by an NMR experiment: The positions of helices in the helical cluster were the same as those in the NMR structure, and the residue-residue contact patterns were also similar with those of the NMR structure. The cluster-cluster separation in the conformational space indicates that free-energy barriers separate the clusters at 300 degrees K. The two beta-structural clusters were characterized by different strand-strand hydrogen-bond (H-bond) patterns, suggesting that the free-energy barrier between the two clusters is due to the H-bond rearrangements.     

Possible immune functions of congerin, a mucosal galectin, in the intestinal lumen of Japanese conger eel

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.     

Purification, cloning and characterization of egg lectins from the teleost Tribolodon brandti

Three L-rhamnose-binding egg lectins, TBL1, TBL2 and TBL3, were isolated from the eggs of the Far East dace Tribolodon brandti by a combination of affinity chromatography on L-rhamnose-Sepharose 6B gel and reversed-phase HPLC. L-rhamnose is a common inhibitor of the purified lectins and strongly inhibited the hemagglutinating activity of TBL2 and TBL3, but less weakly that of TBL1. L-arabinose, which has the same hydroxyl group orientation at C2 and C4 as L-rhamnose, and D-galactose showed no inhibitory activity against TBL1 but showed weak inhibitory activity against TBL2 and TBL3. The open reading frames of the cDNAs of TBL1, TBL2 and TBL3 encoded for mature proteins of 207, 189, and 293 amino acid residues, respectively. A BLAST homology search showed that the TBLs have about 40% homology to the carbohydrate recognition domains of rhamnose-binding lectins in salmonid eggs. The tandem repeated domains present in TBL1, TBL2 and TBL3 were two, two and three, respectively. TBL2 was exclusively expressed in ovary, while TBL1 and TBL3 were expressed mainly in ovary and weakly in various tissues including gill, heart, kidney, liver, spleen and testis.