Inhibitor of IκB kinase activity,BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells

Introduction

Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.

Methods

We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.

Results

Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.

Conclusions

These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.     

The roles of Klenow processing and flap processing activities of DNA polymerase I in chromosome instability in Escherichia coli K12 strains.

The sequences of spontaneous mutations occurring in the endogenous tonB gene of Escherichia coli in the DeltapolA and polA107 mutant strains were compared. Five categories of mutations were found: (1) deletions, (2) minus frameshifts, (3) plus frameshifts, (4) duplications, and (5) other mutations. The DeltapolA strain, which is deficient in both Klenow domain and 5' --> 3' exonuclease domain of DNA polymerase I, shows a marked increase in categories 1-4. The polA107 strain, which is deficient in the 5' --> 3' exonuclease domain but proficient in the Klenow domain, shows marked increases in categories 3 and 4 but not in 1 or 2. Previously, we reported that the polA1 strain, which is known to be deficient in the Klenow domain but proficient in the 5' --> 3' exonuclease domain, shows increases in categories 1 and 2 but not in 3 or 4. The 5' --> 3' exonuclease domain of DNA polymerase I is a homolog of the mammalian FEN1 and the yeast RAD27 flap nucleases. We therefore proposed the model that the Klenow domain can process deletion and minus frameshift mismatch in the nascent DNA and that flap nuclease can process plus frameshift and duplication mismatch in the nascent DNA.     

Isolation and Partial Properties of a Porin-Like Protein from Vibrio parahaemolyticus Cell Envelope

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 m NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.     

Electron microscopic demonstration and isolation of ribosomes in mesosomes from Staphylococcus aureus

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple-layered membrane and contained ribosome-like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X-100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mM Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mM. These findings indicate that the mesosomal tubule is packed with ribosomes.     

A simplified and efficient system for separating proteins by preparative polyacrylamide electrophoresis. With notes on horse heart myoglobin

A simplified but effective method for preparative polyacrylamide gel electrophoresis is presented, suitable for single runs of ∼15 mg of protein, which allows: (1) a gel casting shaped and cooled so that proteins do not assume curvature as they migrate down the column; (2) direct contact of the bottom of the gel with buffer; (3) use of reduced electrical field since anode and cathode are spaced only 9.5 cm apart; (4) increased stability of the anodal face of the gel, by employing a dilute running buffer and consequently a still lower current (7 to 8 mA at 150 V); (5) obtaining successive effluents in separate Visking sacs, at appropriate time intervals, and removal of proteins in absence of an electrical field; (6) rinsing of the column between collections; (7) advance planning of the actual times of collection, calculated from the rates of migration of proteins of interest relative to the time of passage of (yellow) “myoglobin standard” determined in analytical columns run under the same conditions.     

Autolytic Enzyme System of Clostridium botulinum

The mode of action of the autolytic enzymes of Clostridium botulinum type A strain 190L was investigated using a partially purified autolysin. The autolysin completely solubilized SDS-treated cell walls of the organism, liberating 1.2 moles of NH₂-terminal-L-alanine and 0.6 moles of reducing groups per mole of glutamic acid. Neither the NH₂-termini of other amino acids nor COOH-termini of any amino acids were released. These results show that the autolysin contains an N-acetylmuramyl-L-alanine amidase and a hexosaminidase. A disaccharide and peptides were isolated from the wall lysate in a chromatographically homogeneous state. The reducing end of the disaccharide was elucidated to be N-acetylglucosamine by borohydride reduction. This fact indicates that the hexosaminidase is likely to be an endo-β-N-acetylglucosaminidase. A possible structure of the cell wall peptidoglycan is proposed.     

A preliminary report on the treatment of ovulation failure in cows with gonadotropin-releasing hormone analog or human chorionic gonadotropin combined with insemination

Seventy-two cows which did not ovulate within 24–36 h after insemination were reinseminated and treated intramuscularly at the same time with either 50, 100 or 200 μg of gonadotropin-releasing hormone (GnRH) analog (49 cows) or 2000 MU of human chorionic gonadotropin (hCG) (23 cows). One hundred and seventy-seven other cows, which were not considered to ovulate within 24–36 h unless treated with hormones having LH activity because of abnormalities in Graafian follicles detected by rectal palpation at insemination or because of repeat breeding, were also treated with either GnRH (144 cows) or hCG (33 cows) at the same time of initial service. The ovulation rate within 24–36 h after treatment and the conception rate were 79.6% (39 cows of 49) and 46.9% (23 cows of 49) in cows treated with GnRH and 87.0% (20 cows of 23) and 26.1% (6 cows of 23) in cows treated with hCG, respectively. Prophylactic application of GnRH for ovulation failure-predicted cases resulted in ovulation and conception rates of 86.8% (125 cows of 144) and 51.4% (74 cows of 144), and the results of hCG treatment were 78.8% (26 cows of 33) and 45.5% (15 cows of 33). Ovulation rates and conception rates did not differ following treatment with GnRH or hCG, but the lack of untreated controls leaves some uncertainty as to the effectiveness of either of these prophylactic treatments.     

How genes causing unfit hybrids evolve within populations: a review of models of postzygotic isolation

 The main subject for models of postzygotic isolation has been how reproductive isolation genes (RI genes) which cause hybrid inviability or sterility spread within populations despite their deleterious effects. The models are divided into three categories according to the within-population effect of RI genes in their fixation process. (1) The beneficial effect model, where RI genes are assumed to spread within populations by a positive selective force via natural or sexual selection. (2) The neutral effect model, where RI genes are assumed not to affect the fitness of individuals in their fixation process and to be spread by genetic drift. (3) The deleterious effect model, where RI genes are assumed to exhibit some (slightly) deleterious effects in their fixation process and to be spread by genetic drift. Factors that affect the applicability of these models are discussed. If a selective force such as sexual conflict or natural selection facilitates the evolution of RI genes, the beneficial effect model should be applied. Many empirical studies have suggested that positive selection plays an important role in the evolution of hybrid male sterility. If the mutation rates of RI genes are low, and the specificity of epistatic interaction causing hybrid inviability or sterility is high, the neutral effect model should be applied. However, if the opposite condition applies, the deleterious effect model should be applied. Received: February 7, 2002 / Accepted: October 17, 2002 Acknowledgments We are grateful to two anonymous reviewers and the editor for helpful comments and suggestions. Correspondence to:T.I. Hayashi     

Social organization of the voleClethrionomys rufocanus and its demographic and genetic consequences: A review

Recent findings on the relationship between social interaction and demographic process in the gray-sided voleClethrionomys rufocanus are reviewed with reference to the findings in other microtine rodents. Social behavior was particularly focused on spacing and dispersal, and their effects on population dynamics are discussed. Female territoriality can limit a population abundance as a density-dependent factor, although its regulatory effect is controversial. Female philopatry and male-biased dispersal should bring about the clumped distribution of female relatives and genetically random distribution of males during the breeding season. The sexual difference in dispersal patterns can contribute to the mating behavior of the vole; promiscuous mating and low frequency of incestuous mating. However, effects of social structure, including kinship, on reproduction and survival of individuals still remains to be clarified. Molecular markers may help to solve these issues and provide new field of population ecology in microtine rodents.