Shinichi Miyairi

The Pseudomonas aeruginosa Autoinducer N-3-Oxododecanoyl Homoserine Lactone Accelerates Apoptosis in Macrophages and Neutrophils

ABSTRACT Quorum-sensing systems are critical regulators of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C12-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C12-HSL to induce apoptosis in certain types of cells. When bone marrow-derived macrophages were incubated with synthetic 3-oxo-C12-HSL, but when they were incubated not C4-HSL, significant loss of viability was observed in a concentration (12 to 50 μM)- and incubation time (1 to 24 h)-dependent manner. The cytotoxic activity of 3-oxo-C12-HSL was also observed in neutrophils and monocytic cell lines U-937 and P388D1 but not in epithelial cell lines CCL-185 and HEp-2. Cells treated with 3-oxo-C12-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C12-HSL-treated cells was confirmed by multiple criteria (caspases 3 and 8, histone-associated DNA fragments, phosphatidylserine expression). Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C12-HSL, the HSL backbone, and side chain length are required for maximal activity. These data suggest that Pseudomonas 3-oxo-C12-HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C12-HSL-induced cytotoxicity in macrophages and neutrophils. Our data suggest that the quorum-sensing molecule 3-oxo-C12-HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.

Metallothionein-3 is expressed in the brain and various peripheral organs of the rat

Metallothionein-3 (MT-3), also known as growth inhibitory factor (GIF), was originally identified in the brain. An essential step in elucidating the potential roles of MT-3 is to evaluate its expression levels in organs other than the brain. In this present study, we carried out RT-PCR, Western blot and immunohistochemical analyses to quantify MT-3 mRNA and its protein in the cerebrum, eye, heart, kidney, liver, prostate, testis, tongue, and muscle in male Wistar rats. MT-3 mRNA was detected in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue. Using a monoclonal anti-MT-3 antibody, we detected MT-3 in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue as a single band on an immunoblot. Immunohistochemical staining showed MT-3 in some astrocytes in the deep cortex, ependymal cells, and choroidal cells in the cerebrum. MT-3 was also detected in some cells of the glomerulus and the collective tubules in the kidney, some cells in the glandular epithelium of the dorsolateral lobe of the prostate, some Sertoli cells and Lydig cells in the testis, and taste bud cells in the tongue. Although MT-3 immunopositivity was obviously demonstrated in the kidney by the immnunohistochemical method, the expression of MT-3 was not fully detectable by RT-PCR and Western blot analyses. Interestingly, only a subset of cells showed positivity for MT-3, not all cells in all tissues. The localization of MT-3 in peripheral organs outside the brain suggests that MT-3 has roles in these tissues besides its role in growth inhibition of neurites.

GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae

Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L‐glutamine:D‐fructose‐6‐phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine‐6‐phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to />‐chloromer‐curibenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glu‐cosamine 6‐phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxic‐ity in eukaryotic cells.—Naganuma, A., Miura, N., Kaneko, S., Mishina, T., Hosoya, S., Miyairi, S., Furuchi, T., Kuge, S. GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae. FASEBJ. 14, 968–972 (2000)

RND type efflux pump system MexAB-OprM of pseudomonas aeruginosa selects bacterial languages, 3-oxo-acyl-homoserine lactones, for cell-to-cell communication

BackgroundBacteria release a wide variety of small molecules including cell- to- cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl- HSLs) as signal compounds for quorum sensing (QS) within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl- HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB- OprM of P. aeruginosa might function in the selection of acyl- HSLs, and we provide evidence to support this hypothesis.ResultsLoss of MexAB- OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR- 3- oxo- C12- HSL complex. Either complementation with a plasmid containing wild- type mexB or the addition of a LasR- specific inhibitor, patulin, repressed these high responses to 3- oxo- acyl- HSLs. Furthermore, it was shown that the acyl- HSLs- dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB- OprM deletion mutant showed a strong QS response to 3- oxo- C10- HSL produced by Vibrio anguillarum in a bacterial cross- talk experiment.ConclusionThis work demonstrated that MexAB- OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB- OprM not only influences multidrug resistance, but also selects acyl- HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB- OprM in P. aeruginosa.

Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species.

Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12)-HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C(12)-HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C(12)-HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C(12)-HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 microM 3-oxo-C(12)-HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C(12)-HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C(12)-HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone.

Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo

The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), β-naphthoflavone (β-NF), indirubin, indigo, 3,3′-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and β-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr−/−). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.

Partial purification of cytochrome P450 from rat brain and demonstration of estradiol hydroxylation

Cytochrome P450 was partially purified from brain microsomes of untreated rats. A difference spectrum of the dithionite-reduced CO-complex of the purified P450 showed essentially the hemeprotein absorbing exclusively at 449 nm. The purified brain P450 was able to catalyze estradiol (E2) hydroxylation leading to the formation of 6 alpha- and 6 beta-hydroxy(OH)E2, 4-OHE2, estrone, 6-oxoE2, 2-OHE2, 15 alpha-OHE2 and estriol. These results demonstrate that rat brain P450 is active in estradiol hydroxylation.

Anti-Clostridium difficile Potential of Tetramic Acid Derivatives from Pseudomonas aeruginosa Quorum-Sensing Autoinducers

ABSTRACT We have examined the potential bactericidal activities of several tetramic acids derived from Pseudomonas autoinducers against Clostridium difficile, a cause of antibiotic-associated pseudomembranous colitis. Clinical isolates of C. difficile (n = 4) were incubated in broth with a chemically synthesized Pseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and the mechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N-3-oxododecanoyl l-homoserine lactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 μM. The outer membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid, because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-staining characteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that the keto-enol structure and the length of the acyl side chain of tetramic acid may be essential for the antibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives to be novel agents with activity against C. difficile.

Structure of the adduct of 16α-hydroxyestrone with a primary amine : evidence for the Heyns rearrangement of steroidal D-ring α-hydroxyimines

Abstract 16α-Hydroxyestrone, a product of estrogen 16α-hydroxylotion in humans that is suspected to be implicated in cell transformation, has been found to form stable adducts with nuclear components. The stable covalent adduct formed from 16α-hydroxyestrone with 2-methoxyethylamine via the Heyns rearrangement of the α-hydroxyimine was identified as 3-hydroxy-17β-(2-methoxyethylamino)estra1, 3,5(10)-trien-16-one. Since the same product was obtained from 16β-hydroxyestrone with the amine, the α-hydroxyenamine is the most likely intermediate of the Heyns rearrangement. The adduct was fairly stable at 37 C in phosphate buffer (pH 7.4)/methanol (1:1 v/v), while the adduct formed from 16-oxoestradiol was disrupted reversely and completely within 6 hours. The evidence suggests that N-(3hydroxy-16-oxoestra-1,3,5 (10)-trien-17β-yI)amine is the partial structure of the stable adducts formed from D-ring α-ketol estrogens with proteins.

Radioimmunoassay of 16α-hydroxyestrone in human urine

Abstract A radioimmunoassay for the quantitation of the sum of free, glucuronidated and urine is described. The method is reliable and accurate. Using this method, urinary excretion of 16α-hydroxyestrone was determined in normal men, premenopausal women, and postmenopausal women. The values were compared to the urinary excretion of estrone and estradiol. In two women, the urinary excretion of the three estrogens was measured in daily samples throughout a normal menstrual cycle. We conclude that 16α-hydroxyestrone is a quantitatively important urinary estrogen. Inclusion of the measurement of 16α-hydroxyestrone should yield a more accurate assessment of estrogen metabolism.