Insight into innate immune response in “Yusho”: The impact of natural killer cell and regulatory T cell on inflammatory prone diathesis of Yusho patients

Background: In 1968 in western Japan, polychlorinated biphenyl-contaminated “Kanemi rice oil” was used in cooking, causing food poisoning in many people. More than 50 years have passed since the Yusho incident, and although inflammatory disorders such as suppuration have been observed in Yusho patients, the etiology of this inflammation susceptibility remains obscure. Objectives: To investigate the mechanisms of susceptibility to inflammation in Yusho patients, peripheral immune cell fractions and concentrations of inflammatory cytokines were evaluated in blood samples collected from both Yusho patients and age-matched healthy subjects undergoing medical examination in Nagasaki. Methods: To exclude diagnostic uncertainty, serum levels of polychlorinated biphenyl (PCB), polychlorinated quarterphenyl (PCQ), and polychlorinated dibenzofuran (PCDF) were measured. Immune cell (e.g. natural killer and regulatory T cell) populations were analyzed by flow cytometry. Serum cytokines involved in immune cell activation were measured by ELISA. Results: The relative proportion of natural killer cells was higher in Yusho patients than in healthy subjects, while the proportion of regulatory T cells did not differ between groups. Serum concentrations of IL-36 and IFN-γ were significantly lower in Yusho patients than in healthy subjects. Conversely, serum cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which is a cytokine related to activated NK cells, was higher in Yusho patients than in healthy subjects and was positively correlated with PCDF blood levels. Conclusion: Increased numbers of NK cells in Yusho patients suggests that the innate immune response has been activated in Yusho patients. The seemingly paradoxical results for CTLA-4 and IFN-γ may reflect counterbalancing mechanisms preventing excessive NK cell activation. This dysregulation of innate immunity might contribute to the inflammation observed in Yusho patients

Purification, characterization and amplification of a 1.8 kbp fragment of xylanase 5 from <i>Aeromonas caviae </i>W-61

179-184Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1,2,3,4, and 5. In this study, we purified and characterized the xylanase 5 of A. caviae W-61, and amplified a part of xylanase 5 gene (xyn5). The purified xylanase 5 was found to be a single polypeptide with molecular mass of 140 kDa. It was an endo-β-1 ,4-xylanase showing optimum temperature 40oC and optimum pH 6.0. Xylobiose, xylotriose, xylotetrose, xylopentose, xylohexose and a small amount of xylose were detected as the hydrolysis products. The N-terminal amino acid sequence and several internal amino acid sequences of xylanases 5 were determined. From the sequence, a 1.8 kbp fragment was amplified by PCR using forward and reverse primers. DNA sequencing confirmed the presence of nucleotide sequences corresponding to the N-terminal amino acid sequence and the internal amino acid sequences of xylanase 5

Single-molecule imaging of cooperative assembly of γ-hemolysin on erythrocyte membranes

Single-molecule fluorescence imaging was used to investigate assembly of Staphylococcus aureus LukF and HS monomers into pore-forming oligomers (γ-hemolysin) on erythrocyte membranes. We distinguished the hetero-oligomers from the monomers, as indicated by fluorescence resonance energy transfer between different dyes attached to monomeric subunits. The stoichiometry of LukF (donor) and HS (acceptor) subunits in oligomers was deduced from the acceptor emission intensities during energy transfer and by direct acceptor excitation, respectively. Based on populations of monomeric and oligomeric intermediates, we estimated 11 sequential equilibrium constants for the assembly pathway, beginning with membrane binding of monomers, proceeding through single pore oligomerization, and culminating in the formation of clusters of pores. Several stages are highly cooperative, critically enhancing the efficiency of assembly

Two Segments in Bacterial Antizyme P22 Are Essential for Binding and Enhance Degradation of Lysine/Ornithine Decarboxylase in Selenomonas ruminantium▿

In Selenomonas ruminantium, a strictly anaerobic and gram-negative bacterium, the degradation of lysine/ornithine decarboxylase (LDC/ODC) by ATP-requiring protease(s) is accelerated by the binding of P22, which is a ribosomal protein of this strain. Amino acid sequence alignment of S. ruminantium P22 with the L10 ribosomal proteins of gram-positive and -negative bacteria showed that P22 has a 5-residue K101NKLD105 segment and an 11-residue G160VIRNAVYVLD170 segment, both of which are lacking in L10 in any other gram-positive and gram-negative bacteria reported. To elucidate whether the two segments are involved in P22 function, a series of mutant genes of P22 were constructed and expressed in Escherichia coli. The proteins were isolated and assayed for their function with respect to S. ruminantium LDC/ODC and mouse ODC. The results indicated that the two segments of P22 are crucial for P22 binding to both enzymes and also accelerated degradation of both decarboxylases

Stochastic Assembly of Two-Component Staphylococcal γ-Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3

Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively

Preliminary X-ray crystallographic study of staphylococcal alpha-haemolysin monomer

Staphylococcal alpha-haemolysin is a beta-barrel pore-forming toxin expressed by Staphylococcus aureus. alpha-Haemolysin is secreted as a water-soluble monomeric protein which binds to target membranes and forms membrane-inserted heptameric pores. Although the crystal structures of the heptameric pore and monomer bound to an antibody have been determined, that of monomeric alpha-haemolysin without binder has yet to be elucidated. Previous mutation studies showed that mutants of His35 retain the monomeric structure but are unable to assemble into heptamers. Here, alpha-haemolysin H35A mutants were expressed, purified and crystallized. Diffraction data were collected to 2.90 angstrom resolution. The crystals belonged to space group P6(1), with unit-cell parameters a = b = 151.3, c = 145.0 angstrom. Molecular replacement found four molecules in an asymmetric unit. The relative orientation among molecules was distinct from that of the pore, indicating that the crystal contained monomeric alpha-haemolysin