Molecular cloning and characterization of novel splicing variants of human decay-accelerating factor

Decay-accelerating factor (DAF) is one of the complement regulatory proteins. Two isoforms of DAF have been identified in humans. In this study, we isolated novel cDNAs encoding five isoforms of DAF from the human lung, which were generated by insertion of new exonic sequences. RT-PCR revealed that all isoforms were expressed in almost all tissues tested, although the expression patterns and levels differed among the tissues. Transfection of isoform vDAF1, 2, and 3 cDNAs into CHO cells showed that these molecules are soluble forms secreted after glycosylation. Isoform vDAF4 and vDAF5 cDNAs included a part of and the entire intron 7 sequence, respectively, and the transfection of vDAF4 cDNA produced a large, glycosylated, membrane-bound form. These results suggest that more than seven isoforms of human DAF are involved in the regulation of complement activation under physiological conditions through their specific structures and localization.     

Genetic dissection of vasculitis, myeloperoxidase-specific antineutrophil cytoplasmic autoantibody production, and related traits in spontaneous crescentic glomerulonephritis-forming/Kinjoh mice

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a model of human crescentic glomerulonephritis and vasculitis associated with the production of the myeloperoxidase (MPO)-specific antineutrophil cytoplasmic autoantibody (MPO-ANCA). Although the disease is mediated initially by mutation of the Fas gene (lpr), SCG/Kj mice also have non-Fas predisposing genetic factors. To define these factors, genome-wide quantitative trait locus (QTL) mapping was performed on female (B(6)x SCG/Kj) F(2) intercross mice. Fourteen non-Fas QTLs were identified. QTLs of glomerulonephritis were located on chromosomes 1, 10, 13, 16, and 17, vasculitis on chromosomes 1 and 17, splenomegaly on chromosome 1, hypergammaglobulinemia on chromosomes 1, 2, 4, 6, 7, 11, 13, and 17, antinuclear Ab on chromosomes 1, 8, 10, and 12, and MPO-ANCA production on chromosomes 1 and 10. Significant QTLs derived from SCG/Kj on chromosomes 1, 2, 7, and 13 were designated Scg-1 to Scg-5, respectively, and those derived from B(6) on chromosomes 4, 6, 17, and 10 were designated Sxb-1 to Sxb-4, respectively. Two loci linked to MPO-ANCA production on chromosomes 1 and 10 were designated Man-1 and Man-2 (for MPO-ANCA), respectively. Although both Scg-1 and Scg-2 were on chromosome 1 and shared several functions, it was of interest that aberrant MPO-ANCA production was exclusively controlled by Man-1, the centromeric half region of the Scg-2 chromosomal segment. We also examined the epistatic effects between the lpr mutation and non-Fas susceptibility genes. QTLs are discussed in relation to previously described loci, with emphasis on their candidate genes.     

Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio)

The lectin pathway of complement is considered to be the most ancient complement pathway as inferred from identification of ancient homologs of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) in some invertebrates. MBL homologs with galactose selectivity and an MASP3-like sequence also occur in bony fish, linking the evolution of the lectin complement pathway from invertebrates to higher vertebrates. However, these cannot be considered authentic complement components until confirmatory functional evidence is obtained. Here, we report the isolation and characterization of two MBL homologs from a cyprinid teleost, the common carp, Cyprinus carpio. One, designated GalBL, corresponds to the MBL-like molecule with the galactose specificity. The other is an authentic MBL with mannose specificity. Both were found to associate with a serine protease that cleaves native human C4 into C4b but not C4i with a hydrolyzed thioester. Molecular cloning and phylogenetic analysis revealed this C4-activating protease to be carp MASP2, indicating that MASP2 arose before the emergence of bony fish. Database mining of MBL-like genes reveals that MBL and GalBL genes are arranged in tandem in the zebrafish genome and that both lectins are conserved in the distantly related puffer fish. These results imply that bony fish have developed a diverged set of MBL homologs that function in the lectin complement pathway.     

Spectroscopic and substrate binding properties of heme-containing aldoxime dehydratases, OxdB and OxdRE

Aldoxime dehydratase (Oxd) is a novel hemeprotein that catalyzes the dehydration reaction of aldoxime to produce nitrile. In this study, we studied the spectroscopic and substrate binding properties of two Oxds, OxdB from Bacillus sp. strain OxB-1 and OxdRE from Rhodococcus sp. N-771, that show different quaternary structures and relatively low amino acid sequence identity. Electronic absorption and resonance Raman spectroscopy revealed that ferric OxdRE contained a six-coordinate low-spin heme, while ferric OxdB contained a six-coordinate high-spin heme. Both ferrous OxdRE and OxdB included a five-coordinate high-spin heme to which the substrate was bound via its nitrogen atom for the reaction to occur. Although the ferric Oxds were inactive for catalysis, the substrate was bound to the ferric heme via its oxygen atom in both OxdB and OxdRE. Electronic paramagnetic resonance (EPR) and rapid scanning spectroscopy revealed that the flexibility of the heme pocket was different between OxdB and OxdRE, which might affect their substrate specificity.     

The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.     

Discovery of 4,5-diphenyl-1,2,4-triazole derivatives as a novel class of selective antagonists for the human V(1A) receptor

In the search for a novel class of selective antagonists for the human V(1A) receptor, high-throughput screening (HTS) of the Yamanouchi chemical library using CHO cells expressing the cloned human V(1A) (hV(1A)) receptor led to the discovery of 5-(4-biphenyl)-4-(2-methoxyphenyl)-3-methyl-1,2,4-triazole (3) which possessed the novel 4,5-diphenyl-1,2,4-triazole structure. Subsequent structure-activity relationships studies on a series of the 4,5-diphenyl-1,2,4-triazole derivatives related to 3 revealed that the 4,5-diphenyl-1,2,4-triazole structure played an essential role in exerting high affinity for the hV(1A) receptor and that introduction of a basic amine moiety to the methoxy part of the 4-phenyl ring was effective in the improvement of both affinity for the hV(1A) receptor and selectivity versus the hV(2) receptor. Compound 3 and the 2-(morphorino)ethoxy derivative (11b) were shown to be antagonists for the hV(1A) receptor, from their effects on AVP-induced [Ca(2+)](i) response in CHO cells expressing the hV(1A) receptor.     

Caveosomal Oxidative Stress Causes Src-p21ras Activation and Lysine 63 TRAF6 Protein Polyubiquitination in Iron-induced M1 Hepatic Macrophage Activation

Proinflammatory M1 activation of hepatic macrophages (HM) is critical in pathogenesis of hepatitis, but its mechanisms are still elusive. Our earlier work demonstrates the role of ferrous iron (Fe(2+)) as a pathogen-associated molecular pattern-independent agonist for activation of IκB kinase (IKK) and NF-κB in HM via activation and interaction of p21(ras), transforming growth factor β-activated kinase-1 (TAK1), and phosphatidylinositol 3-kinase (PI3K) in caveosomes. However, iron-induced signaling upstream of these kinases is not known. Here we show that Fe(2+) induces generation of superoxide anion (O(2)()) in endosomes, reduces protein-tyrosine phosphatase (PTP) activity, and activates Src at 2～10 min of Fe(2+) addition to rat primary HM culture. Superoxide dismutase (SOD) blocks O(2)() generation, PTP inhibition, and Src activation. Fe(2+)-induced p21(ras) activity is abrogated with the Src inhibitor PP2 and SOD. Fe(2+) stimulates Lys(63)-linked polyubiquitination (polyUb) of TRAF6 in caveosomes, and a dominant negative K63R mutant of ubiquitin or SOD prevents iron-induced TRAF6 polyUb and TAK1 activation. These results demonstrate that Fe(2+)-generated O(2)() mediates p21(ras) and TAK1 activation via PTP inhibition and Lys(63)-polyUb of TRAF6 in caveosomes for proinflammatory M1 activation in HM.     

Photochemical nature of parietopsin

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV-visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an "evolutionary intermediate" between invertebrate and vertebrate visual opsins.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.