Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population

Autoantibodies to myeloperoxidase (MPO) are a subset of anti-neutrophil cytoplasmic antibody (ANCA, MPO-ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO-ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO-ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO-ANCA in different vasculitic syndromes. We screened the sera of 148 MPO-ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I-CrGN), classic polyangiitis nodosa (cPAN), Churg-Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I-CrGN and MPA sera mainly reacted to the Ha epitope at the N-termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C-terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO-ANCA recognizing specific regions of the N-terminus of the MPO H-chain confer an increased risk of vasculitis RPGN, I-CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO-ANCA differentiates vasculitic from non-vasculitic syndromes associated with MPO-ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis.     

Synaptosomal-associated protein 25 gene expression is hormonally regulated during ovulation and is involved in cytokine/chemokine exocytosis from granulosa cells

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.     

Cloning and characterization of the gene encoding endo-beta-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-beta-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-beta-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-beta-1,3-glucanases, but GluA was partially similar to two fungal exo-beta-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two beta-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-beta-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

A preliminary genetic association study of GAD1 and GABAB receptor genes in patients with treatment-resistant schizophrenia

Molecular Biology Reports - GABAergic system dysfunction has been implicated in the etiology of schizophrenia and of cognitive impairments in particular. Patients with treatment-resistant...     

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.     

Lyconadins D and E, and complanadine E, new Lycopodium alkaloids from Lycopodium complanatum

Three new Lycopodium alkaloids, lyconadins D (1) and E (2), and complanadine E (3), were isolated from the club moss Lycopodium complanatum. Lyconadin D (1) was the first example of fastigiatine-type alkaloid isolated from Lycopodium complanatum. The structures and relative stereochemistry of 1-3 were elucidated on the basis of spectroscopic data. Complanadine E (3) enhanced mRNA expression for NGF.     

Control of cocklebur seed germination by nitrogenous compounds : Nitrite, nitrate, hydroxylamine, thiourea, azide and cyanide

Germination of non-dormant upper cocklebur (Xanthium pinsylvanicumWallr.) seeds was stimulated by not only CS(NH₂)₂ but also NH₂OH,KCN and NaN₃. This stimulation was not via the enhancement ofaerobic C₂H₄ production. NH₂OH, KCN and NaN₃ in certain concentrationspromoted the initial growth of axial and/or cotyledonary parts,but the degree of growth promotion by NH₂OH, NaN₃ and KCN wasslight compared with that by CS(NH₂)₂. As in the case of CS(NH₂)₂,however, the germinationstimulating effect of NH₂OH disappearedrapidly as the preceding imbibition period was prolonged. Incontrast, KCN and NaN₃ were still effective in stimulating thegermination of aged seeds maintained on a water substratum,as previously seen with anaerobiosis. Anaerobic induction wasenhanced not only by NaN₃ and KCN but also by NH₂OH, KNO₃, KNO₂CO(NH₂)₂ and CS(NH₂)₂ applied during the anaerobic treatment,but without causing an increase in anaerobic production of C₂H₄.Furthermore, KCN and NaN₃, given prior to the anaerobic treatmentacted additively with anaerobic induction. The germination-stimulatingactions of nitrogenous compounds are discussed in comparisonwith those of C₂H₄ and anaerobiosis. (Received May 6, 1978; )     

Preferential paternal origin of microdeletions caused by prezygotic chromosome or chromatid rearrangements in Sotos syndrome

Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.     

Effect of Exogenous Fatty Acids on the Cellular Fatty Acid Composition and Neomycin Formation in a Mutant Strain of Streptomyces fradiae

A mutant of Streptomyces fradiae which requires oleic acid for neomycin formation was isolated and the effects of exogenous fatty acids and other additives on the formation of neomycin were studied. Palmitic acid and high concentration of sodium ions could replace oleic acid in neomycin formation. The fatty acid spectrum of the mutant strain ST–5B was quite different from that of the parent strain 3123. The major fatty acid components of the mutant and the parent were anteiso 15:0 and iso 16: 0, respectively. However the fatty acid composition of the mutant was changed from the anteiso 15: 0-type to the parental iso 16: 0-type by the supplement of oleic acid or high concentration of sodium ions in the medium. In the case of palmitic acid, the major fatty acid component of the mutant cells was changed from anteriso 15: 0 to normal 16:0. The role of these additives in neomycin formation by the mutant is discussed.     

Protective efficacy of nonpathogenic nef-deleted SHIV vaccination combined with recombinant IFN-gamma administration against a pathogenic SHIV challenge in rhesus monkeys

We previously reported that a nef-deleted SHIV (SHIV-NI) is nonpathogenic and gave macaques protection from challenge infection with pathogenic SHIV-C2/1. To investigate whether IFN-gamma augments the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of recombinant human IFN-gamma (rIFN-gamma) in vaccinated and unvaccinated monkeys. Nine monkeys were vaccinated with nef-deleted nonpathogenic SHIV-NI. Four of them were administered with rIFN-gamma and the other five monkeys were administered with placebo. After the challenge with pathogenic SHIV-C2/1, CD4(+) T-cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were reduced to 100-fold in SHIV-NI vaccinated monkeys combined with rIFN-gamma compared with those in SHIV-NI vaccinated monkeys without rIFN-gamma. The peaks of plasma viral load were inversely correlated with the number of SIV Gag-specific IFN-gamma-producing cells. In SHIV-NI-vaccinated monkeys with rIFN-gamma, the number of SIV Gag-specific IFN-gamma-producing cells of PBMCs increased 2-fold compared with those in SHIV-NI-vaccinated monkeys without rIFN-gamma, and the NK activity and MIP-1alpha production of PBMCs were also enhanced. Thus, vaccination of SHIV-NI in combination with rIFN-gamma was more effective in modulating the antiviral immune system into a Th1 type response than SHIV-NI vaccination alone. These results suggest that IFN-gamma augmented the anti-viral effect by enhancing innate immunity and shifting the immune response to Th1.