Crystal structure of rat heme oxygenase-1 in complex with heme bound to azide. Implication for regiospecific hydroxylation of heme at the alpha-meso carbon

Heme oxygenase (HO) catalyzes physiological heme degradation consisting of three sequential oxidation steps that use dioxygen molecules and reducing equivalents. We determined the crystal structure of rat HO-1 in complex with heme and azide (HO-heme-N(3)(-)) at 1.9-A resolution. The azide, whose terminal nitrogen atom is coordinated to the ferric heme iron, is situated nearly parallel to the heme plane, and its other end is directed toward the alpha-meso position of the heme. Based on resonance Raman spectroscopic analysis of HO-heme bound to dioxygen, this parallel coordination mode suggests that the azide is an analog of dioxygen. The azide is surrounded by residues of the distal F-helix with only the direction to the alpha-meso carbon being open. This indicates that regiospecific oxygenation of the heme is primarily caused by the steric constraint between the dioxygen bound to heme and the F-helix. The azide interacts with Asp-140, Arg-136, and Thr-135 through a hydrogen bond network involving five water molecules on the distal side of the heme. This network, also present in HO-heme, may function in dioxygen activation in the first hydroxylation step. From the orientation of azide in HO-heme-N(3)(-), the dioxygen or hydroperoxide bound to HO-heme, the active oxygen species of the first reaction, is inferred to have a similar orientation suitable for a direct attack on the alpha-meso carbon.     

Endosomal/lysosomal targeting of a single helper T-cell epitope of an intracellular bacterium by DNA immunisation induces a specific T-cell subset and partial protective immunity in vivo

We evaluated here the effect of the intracellular targeting of a helper T-cell (Th) epitope, literiolysin O 215-226 derived from Listeria monocytogenes, on induction of a specific Th by gene gun immunisation. Immunisation of C3H/He mice with pE215LAMP plasmid encoding the Th epitope fused with the endosomal/lysosomal targeting signal of lysosome-associated membrane protein (LAMP)-1 gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pE215LAMP-immunised mice showed moderate, but significant protective immunity against listerial challenge. These results suggest that the intracellular targeting of a Th epitope to endosomal/lysosomal compartments by DNA immunisation is useful for eliciting a specific Th subset in vivo.     

Highly potent inhibitors of TNF-alpha production. Part II: metabolic stabilization of a newly found chemical lead and conformational analysis of an active diastereoisomer

Design and synthesis of metabolically stabilized inhibitors of TNF-alpha production, which could be new drug candidates, are reported. Conformational analysis of an active diastereoisomer was performed based on biological evaluations of the conformationally fixed indane derivatives 17 and 18. Structure-activity relationships (SARs) based on biological evaluations of the optically active derivatives are also discussed. Full details including chemistry are reported.     

Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads

A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.     

Math3 and NeuroD regulate amacrine cell fate specification in the retina

The basic helix-loop-helix genes Math3 and NeuroD are expressed by differentiating amacrine cells, retinal interneurons. Previous studies have demonstrated that a normal number of amacrine cells is generated in mice lacking either Math3 or NEUROD: We have found that, in Math3-NeuroD double-mutant retina, amacrine cells are completely missing, while ganglion and Müller glial cells are increased in number. In the double-mutant retina, the cells that would normally differentiate into amacrine cells did not die but adopted the ganglion and glial cell fates. Misexpression studies using the developing retinal explant cultures showed that, although Math3 and NeuroD alone only promoted rod genesis, they significantly increased the population of amacrine cells when the homeobox gene Pax6 or Six3 was co-expressed. These results indicate that Math3 and NeuroD are essential, but not sufficient, for amacrine cell genesis, and that co-expression of the basic helix-loop-helix and homeobox genes is required for specification of the correct neuronal subtype.     

RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene,conferring resistance to cucumber mosaic virus requires salicylic acid,ethylene and a novel signal transduction mechanism

The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently.     

Highly potent inhibitors of TNF-alpha production. Part 1: Discovery of chemical leads

The discovery of 2-acylamino-2-phenylethyl disodium phosphates and as structurally novel inhibitors of TNF-alpha production is reported. Structure-activity relationships (SARs) are also discussed.     

Relationship between gastric ulcer and Helicobacter pylori VacA detected in gastric juice using bead-ELISA method

Background. VacA is an important pathogenetic factor produced by Helicobacter pylori. VacA has often been detected in supernatants of liquid cultures or lysates of whole bacterial cells. However, no studies have ever tried to assay VacA produced in the human stomach. We applied a very sensitive and simple method, bead‐ELISA, to detect VacA in gastric juice. Materials and Methods. Forty‐eight H. pylori‐positive patients (16 nonulcer dyspepsia, 16 gastric ulcer, and 16 duodenal ulcer) and four H. pylori‐negative nonulcer dyspepsia patients had endoscopy performed and gastric juice were aspirated. Polystyrene beads coated with the antibody to VacA, were used in this bead‐ELISA method. The nucleotide sequences of vacA in the signal and middle regions were investigated. Results. Of the 48 samples that were positive for H. pylori, 21 [43.8%] were found to be VacA positive in gastric juice. The average and maximum concentrations of detected VacA in gastric juice were 143.2 ± 216.5 and 840 pg/ml, respectively. The average density of VacA from gastric ulcer patients (227.5 ± 276.7 pg/ml) was higher than that found in nonulcer dyspepsia (51.8 ± 39.8 pg/ml) and duodenal ulcer (49.2 ± 21.5 pg/ml) patients. There was no relationship between VacA in gastric juice and vacA genotype. Conclusions. VacA in gastric juice could be directly detected by bead‐ELISA. In this study, the diversity of disease outcome was associated with not the quality but the quantity of VacA. Therefore, not only the quality but also the quantity of VacA is important etiological factors in the pathogenesis of mucosal damage.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

Spatial raft coalescence represents an initial step in Fc gamma R signaling

Characterization of lipid rafts as separated membrane microdomains consist of heterogeneous proteins suggesting that lateral assembly of rafts after Ag receptor cross-linking represents the earliest signal generating process. In line with the concept, cross-linked Ag receptors have been shown to associate with detergent-insoluble raft fraction without the aid of Src family kinases. However, it has not been established whether spatial raft coalescence could also precede Src family kinase activation. In this study, we showed that spatial raft coalescence after low-affinity FcgammaR cross-linking in RAW264.7 macrophages is independent of Src family kinase activity. The lateral raft assembly was found to be ascribed to the action of ligand-binding subunits, rather than to immunoreceptor tyrosine-based activation motif-bearing signal subunits, because monomeric murine FcgammaRIIb expressed in rat basophilic leukemia cells successfully induced spatial raft reorganization after cross-linking. We also showed that extracellular and transmembrane region of FcgammaRIIb is sufficient for raft stabilization. Moreover, this receptor fragment triggers rapid calcium mobilization and linker for activation of T cells phosphorylation, in a manner sensitive to Src family kinase inhibition and to cholesterol depletion. Presence of immunoreceptor tyrosine-based inhibitory motif and addition of immunoreceptor tyrosine-based activation motif to the receptor fragment abolished and enhanced the responses, respectively, but did not affect raft stabilization. These findings support the concept that ligand-binding subunit is responsible for raft coalescence, and that this event triggers initial biochemical signaling.