Yoshihisa Iwamoto

Sparfloxacin phototoxicity: Potential photoaugmentation by ultraviolet A and B sources

Sparfloxacin, a quinolone antibacterial agent, frequently elicits photosensitive skin reactions. Our clinical studies of patients treated with sparfloxacin have demonstrated that this photosensitivity is primarily phototoxic and that a marked erythematous response is induced by sequential irradiation with ultraviolet A (UVA) and B (UVB) but not UVA or UVB alone, suggesting potential synergism between UVA and UVB. We evaluated the phototoxicity of this agent using in vitro DNA breaking activity and in vivo murine cutaneous response. Sparfloxacin induced DNA strand breaks in vitro and converted the supercoiled closed circular form of plasmid DNA to the open circular form by its photodynamic action. In mice, the topical application of sparfloxacin and subsequent irradiation with UVB, but not UVA, induced ear swelling responses. However, the UVB-induced ear swelling response was augmented by irradiation with UVA before or after UVB exposure. Such interaction between UVA and UVB in the production of ear swelling was further confirmed by systemic administration of sparfloxacin. Our study suggests that sparfloxacin is a unique phototoxic agent in that photosensitivity dermatitis is evoked by photoaugmentation between UVA and UVB.

Vibrio trachuri sp. nov., a new species isolated from diseased Japanese horse mackerel.

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4‐diamino‐6,7‐diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.

Biological activities and antitumor effects of synthetic lipid a analog linked n-acylated serine

The mitogenicity, lethal toxicity, production of nitric oxide (NO), induction of tumor necrosis factor (TNF) and antitumor activity against Meth A fibrosarcoma by chemically synthesized N-acylated serine-linked non-phosphorylated (A-606 and A607) and phosphorylated (A-608) acylglucosamine-derived lipid A analog were determined. Compounds A-606, A-608 and A-103 [with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3 positions] induced significant incorporation of [3H]thymidine into splenocytes of C3H/He mice at concentrations ranging from 3.13 to 50 microM. However, A-607 [with (R)-3-tetradecanoyloxytetradecanoyl and with tetradecanoyl at the C-2 and C-3 positions] showed most significant incorporation of [3H]thymidine. The compounds A-606, A-608 and A-103 did not exhibit the lethality at doses of 30 and 300 nmol/kg in C57BL/6 mice loaded with D-galactosamine, whereas A-607 caused the death of two out of six mice at a dose of 300 nmol/kg. These compounds, except A-607, exhibited little NO production by macrophages, but did cause NO production in the presence of interferon-gamma (IFN-gamma). Peritoneal macrophages, stimulated with A-606-A-608, caused production of TNF which induce L929 cell lysis in vitro, and A-608 showed high production of TNF. NO-inducible activity and induction of TNF by compound A-103 seemed to be lower than that of serine-linked derivatives. A-607, A-608 and A-103 showed antitumor activity against Meth A fibrosarcoma in BALB/c mice, and furthermore, the enhancement of antitumor activity by a combination of A-608 with muramyl dipeptide (MDP) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and Sensitive PCR Detection of Vibrio trachuri Pathogenic to Japanese Horse Mackerel (Trachurus japonicus)

A polymerase chain reaction (PCR) method was performed for rapid and sensitive detection of pathogenic Vibrio trachuri isolated from cultured Japanese horse mackerel. A set of primers was selected from the base sequence of the Pst I fragment of T9210 chromosomal DNA and used for PCR detection of T9210. This PCR specifically amplified the DNAs from V. trachuri T9210, T9213, and T9216 but not of those other bacterial strains. PCR using a Pst I‐1 primer set made it possible to detect 100 fg of T9210 DNA. The PCR method reported here may be useful for detection and identification of V. trachuri pathogenic to Japanese horse mackerel.

Epitope analysis of Aztreonam by antiaztreonam monoclonal antibodies and possible consequences in beta-lactams hypersensitivity

Three cell lines producing monoclonal antibodies, Az-1 (IgG1), Az-2 (IgG1) and Az-3 (IgM) against aztreonam were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams, which were conjugated with human serum albumin (HSA), were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. In ELISA, Az-1 and Az-2 reacted only with aztreonam and ceftazidime, which have the same acyl side chain. Furthermore, Az-2 showed a strong cross-reaction with carumonam. In the ELISA inhibition test, Az-1 and Az-2 were inhibited from binding to aztreonam-HSA by aztreonam, ceftazidime, aztreonam hydrolysate, aztreonam-epsilon-amino-n-caproic acid (EACA) and ceftazidime-EACA. Az-2 was also inhibited with carumonam. From the above results, it seems that Az-1 can recognize only the degraded structure of monobactam nucleus, and Az-2 can recognize the degraded nucleus moiety and the acyl side chain. On the other hand, Az-3 displayed broad cross-reaction to various beta-lactams in ELISA. Furthermore, the MAb showed no inhibitory reaction with various beta-lactams except aztreonam- and ceftazidime-EACA conjugates in the ELISA inhibition test, suggesting that Az-3 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. The above results indicate that antibodies can recognize at least three epitopes of the degraded product(s) of aztreonam nucleus, acyl side chain and NAD in aztreonam-protein conjugate.

Photodynamic DNA-breaking activity of serum from patients with various photosensitivity dermatoses

Various drugs and chemicals break the DNA strand under ultraviolet irradiation. This study aimed to clarify the DNA-breaking activity (DBA) of serum from 39 patients with various photosensitivity disorders and that from eight normal subjects. A mixture of serum and circular plasmid DNA was exposed to longwave ultraviolet radiation, and the photoinduced cleavage of plasmid DNA was examined by electrophoretic analysis. DBA was found in serum from patients with erythropoietic protoporphyria (2 of 2), drug-induced photosensitivity (3 of 5), chronic actinic dermatitis (1 of 12) and hydroa vacciniforme (1 of 1). DBA was not found in serum from patients with porphyria cutanea tarda, collagen diseases with photosensitivity, papulovesicular light eruption or pellagra. The inhibition profile of DBA by active oxygen scavengers was different between afloqualone- and tetracycline-induced photosensitivity and chronic actinic dermatitis. The present method was useful for the detection of serum phototoxicity and the investigation of the pathomechanisms of photosensitivity.

Biological activities of chemically synthesized N-acylated serine-linked lipid A analog in mice

The mitogenicity, lethal toxicity and antitumor activity against Meth A fibrosarcoma and the induction of tumor necrosis factor (TNF) of chemically synthesized N-acylated serine-linked nonphosphorylated acylglucosamine-derived lipid A analog (A-601, A-602 and A-603) were determined. Compounds A-603 (with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 position) and A-103 (2,3-acyloxyacylglucosamine-4-phosphate) induced significant incorporations of [3H]thymidine into splenocytes of C3H/He mice at concentrations ranging from 6.25 to 100 microM. The mitogenicity of A-601 and A-602 (with tetradecanoyl at the C-2 position) exhibited a lower activity than of A-603. Compounds A-601 and A-603 showed almost the same lethality at doses from 1 to 50 nmol/mouse in C57BL/6 mice loaded with D-galactosamine, whereas A-103 caused the death of two out of six mice at a dose of 25 nmol/mouse. A-601 and A-603 showed weak antitumor activity against Meth A fibrosarcoma in BALB/c mice, but there was no enhancement of antitumor activity by a combination of A-603 with muramyl dipeptide. Peritoneal macrophages, stimulated with A-601, A-602 or A-603, caused production of TNF which induces L929 cell lysis in vitro. But the activity of A-603 among the compounds on TNF-production was the highest. These findings indicate that the linkage of nonphosphorylated acylglucosamine and N-acylated serine affects the expression of the biological activity.

COMPARISON OF THE BIOLOGICAL ACTIVITY OF SYNTHETIC N-ACYLATED ASPARAGINE OR SERINE LINKED MONOSACCHARIDE LIPID A ANALOGS

The mitogenicity, lethal toxicity, induction of tumor necrosis factor (TNF), production of nitric oxide (NO) and antitumor activity against Meth A fibrosarcoma by chemically synthesized N-acylated asparagine-linked (A-701, A-702 and A-703) or N-acylated serine-linked (A-607) nonphosphorylated acylglucosamine and 4-0-phosphorylated acylglucosamine (A-103) derived lipid A analogs were determined. compound A-607 (with tetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3 positions) induced a significant incorporation of 3H-thymidine into splenocytes of C3H/He mice at concentrations ranging from 3.13 to 50 microM, but the mitogenic activity of A-701 (2-N-acetylglucosamine), A-702 (tetradecanoyl at the C-2), and A-703 (with (R)-tetradecanoyloxytetradecanoyl and tetradecanoyl at the C-2 and C-3) was very weak. The lethality of A-703 and A-103 (with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3) was weaker than that of A-607 at doses of 300 and 750 nmol/kg in C57BL/6 mice loaded with D-galactosamine. Peritoneal macrophages, stimulated with A-701-A-703, caused production of TNF which induce L929 cell lysis in vitro, and A-703 showed a high production of TNF. The compounds, except for A-607, exhibited little NO production by macrophages, but did induce the NO production in the presence of interferon gamma. Induction of TNF and NO inducible activity by A-703 was lower than that of A-607. A-703, A-607 and A-103 showed antitumor activity against Meth A fibrosarcoma in BALB/c mice. When A-703 or A-103 with muramyl dipeptide was administered, A-703 failed to show combined effects, but A-103 did. We concluded from these findings that the biological potency of asparagine compounds appears to be placed between serine- and amino-free compounds.

Phototoxicity of Quinolones: Measurement by UV-A Inhibition of Concanavalin A-Stimulated Lymphocyte Mitogenic Response

Phototoxicity of ten quinolones was measured by inhibition of concanavalin A-stimulated mitogenesis (measured by [3H]-thymidine uptake) of guinea pig splenocytes after UV-A irradiation in the presence of drug. At a drug concentration of 2.5μg/ml and a radiation dose of 3.9J/cm2, inhibition rates (expressed as percentage of uptake, without irradiation) were: lomefloxacin (LFLX), 51%; fleroxacin (FLRX), 34%; ciprofloxacin (CPFX) and enoxacin (ENX), 27%; norfloxacin (NFLX), 25%; ofloxacin (OFLX), 19%; piromidic acid (PA), 13% tosufloxacin (TFLX), 12%; pipemidic acid (PPA), 11%; and levofloxacin (LVFX, an optical isomer of OFLX), 8%. At a concentration, of 25 μg/ml, on the other hand the phototoxic activity of quinolones decreased in the order LFLX, CPFX, ENX, FLRX>NFLX>LVFX, PA, OFLX>PPA. These results suggest that LFLX, with a fluorine attached to the 8 position, is strongly photoxic, although it shows low cytotoxicity (measured by inhibition of incorporation in the dark), against splenocytes. UV-A irradiation of solutions of LFLX and FLRX blue-shifted the absorption maximum from 280 nm to 270 nm and decreased the optical density between 325 and 340 nm. Although CPFX, ENX, NFLX, OFLX, LVFX and PA also showed optical density decreases at 260–290 nm and 325–350 nm, the maximal absorption did not shift. There were no changes in the absorption spectra of PPA and TFLX even after UV-A irradiation at a dose of 7.8J/cm2. Thus, quinolones phototoxicity and the spectral changes produced by UV-A irradiation appear to be related.

INHIBITORY EFFECTS OF ETHANOL ON THE PHOTODYNAMIC CELL INACTIVATION AND PETITE INDUCTION BY EUFLAVINE IN YEAST, Saccharomyces cerevisiae

Abstract— Effects of active oxygen scavengers on cell inactivation and petite induction of yeast by the photodynamic action of euflavine were examined. Histidine, sodium azide, 1,3‐diphenyl‐isobenzofuran and p‐carotene, which are singlet oxygen scavengers, inhibited photodynamic cell killing. Histidine and sodium azide inhibited petite induction, too. These results suggested that photobiological effects of euflavine are induced via singlet oxygen‐mediated Type II reaction process. In this work, however, we found that ethanol, which is reported to be a hydroxyl radical scavenger, notably inhibited photodynamic cell inactivation and petite induction by euflavine. Inhibition of petite induction was increased with increasing concentration of ethanol. Decrease of absorbance of euflavine by irradiation was also inhibited by the addition of ethanol.