Mannan-binding protein blocks the activation of metalloproteases meprin alpha and beta

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin alpha and beta (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.     

Effect of antithrombin III on glycoprotein Ib/IX/V activation in human platelets: Suppression of thromboxane A(2) generation

We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.     

Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

The GID1-mediated gibberellin perception mechanism is conserved in the Lycophyte Selaginella moellendorffii but not in the Bryophyte Physcomitrella patens

In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA(4), a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage.     

Linkage of Mercury, Cadmium, and Arsenate and Drug Resistance in Clinical Isolates of Pseudomonas aeruginosa

Of the 787 isolates, 99.8% were metal resistant, with most (99.5%) showing multiple resistance. Fifty-three percent of the isolates were both metal and drug resistant, whereas only 19% were metal resistant and drug sensitive.     

Redox-coupled conformational alternations in cytochrome c(3) from D. vulgaris Miyazaki F on the basis of its reduced solution structure

Heteronuclear NMR spectroscopy was performed to determine the solution structure of (15)N-labeled ferrocytochrome c(3) from Desulfovibrio vulgaris Miyazaki F (DvMF). Although the folding of the reduced cytochrome c(3) in solution was similar to that of the oxidized one in the crystal structure, the region involving hemes 1 and 2 was different. The redox-coupled conformational change is consistent with the reported solution structure of D. vulgaris Hildenborough ferrocytochrome c(3), but is different from those of other cytochromes c(3). The former is homologous with DvMF cytochrome c(3) in amino acid sequence. Small displacements of hemes 1 and 2 relative to hemes 3 and 4 were observed. This observation is consistent with the unusual behavior of the 2(1)CH(3) signal of heme 3 reported previously. As shown by the (15)N relaxation parameters of the backbone, a region between hemes 1 and 2 has more flexibility than the other regions. The results of this work strongly suggest that the cooperative reduction of hemes 1 and 2 is based on the conformational changes of the C-13 propionate of heme 1 and the aromatic ring of Tyr43, and the interaction between His34 and His 35 through covalent and coordination bonds.     

C-terminal cleavage of the LH1 <Emphasis Type="Italic">α</Emphasis>-polypeptide in the Sr<Superscript>2+</Superscript>-cultured <Emphasis Type="Italic">Thermochromatium tepidum</Emphasis>

The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q_y absorption at 915 nm and enhanced thermostability. Ca²⁺ can be biosynthetically replaced by Sr²⁺ in growing cultures of Tch. tepidum. However, the resulting Sr²⁺-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca²⁺-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca²⁺- and Sr²⁺-cultured cells. In this study, we examined the effects of the biosynthetic Sr²⁺-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr²⁺-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr²⁺-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr²⁺ substitutes for Ca²⁺ in the LH1 complex. Limited proteolysis of the native Ca²⁺-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr²⁺-substituted LH1-RC complexes in their Ca²⁺-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum.     

EGFR signaling pathway negatively regulates PSA expression and secretion via the PI3K-Akt pathway in LNCaP prostate cancer cells

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in hormone-refractory growth and poor prognosis of a subgroup of human prostate cancer. In this communication, we investigated the regulation of PSA by the EGFR signaling pathway using LNCaP C-81 prostate cancer cells. Administration of EGF stimulated the growth of LNCaP C-81 cells, however, PSA expression and secretion were suppressed. An EGFR inhibitor, AG1478, abrogated the PSA suppression effect by EGF, in concurrence with the suppression of tyro-phosphorylation levels of EGFR. Interestingly, the AR level was also decreased in EGF-treated LNCaP C-81 cells. Moreover, LY294002, but not PD98059, inhibited the PSA and AR suppression effect by EGF in concurrence with the suppression of phosphorylation levels of Akt. In conclusion, our results strongly suggest the existence of a novel androgen-independent PSA regulatory mechanism, i.e., the EGFR signaling pathway negatively regulates PSA expression which may be induced by the alteration of AR expression via the PI3K-Akt pathway in LNCaP C-81 cells.