Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (^?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against ^?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H²O²), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a ^?OH-trapping agent after UVB exposure (305?nm) to H²O² for 1?min in the presence of rebamipide. The signal intensity of ^?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10¹⁰, 8.16?×?10⁹ and 1.65?×?10¹⁰?M^-1?s^-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the ^?OH generated by UVB/H²O². Specific formation of this product explained the molecular characteristics of rebamipide as a potential ^?OH scavenger.     

Longer uncommon polyamines have a stronger defense gene-induction activity and a higher suppressing activity of Cucumber mosaic virus multiplication compared to that of spermine in Arabidopsis thaliana

Key message

Our work suggests that long chain polyamines and their derivatives are potential chemicals to control viral pathogens for crop production.

Abstract

Previously we showed that two tetraamines, spermine (Spm) and thermospermine (T-Spm), induce the expression of a subset of defense-related genes and repress proliferation of Cucumber mosaic virus (CMV) in Arabidopsis. Here we tested whether the longer uncommon polyamines (LUPAs) such as caldopentamine, caldohexamine, homocaldopentamine and homocaldohexamine have such the activity. LUPAs had higher gene induction activity than Spm and T-Spm. Interestingly the genes induced by LUPAs could be classified into two groups: the one group was most responsive to caldohexamine while the other one was most responsive to homocaldopentamine. In both the cases, the inducing activity was dose-dependent. LUPAs caused local cell death and repressed CMV multiplication more efficiently as compared to Spm. LUPAs inhibited the viral multiplication of not only avirulent CMV but also of virulent CMV in a dose-dependent manner. Furthermore, LUPAs can activate the systemic acquired resistance against CMV more efficiently as compared to Spm. When Arabidopsis leaves were incubated with LUPAs, the putative polyamine oxidase (PAO)-mediated catabolites were detected even though the conversion rate was very low. In addition, we found that LUPAs induced the expression of three NADPH oxidase genes (rbohC, rbohE and rbohH) among ten isoforms. Taken together, we propose that LUPAs activate two alternative reactive oxygen species evoked pathways, a PAO-mediated one and an NADPH-oxidase-mediated one, which lead to induce defense-related genes and restrict CMV multiplication.     

Association between alkaline phosphatase and hypertension in a rural Japanese population: The Nagasaki Islands study

Background

Although serum alkaline phosphatase (ALP) levels have been associated with hypertension, and ALP is known as an enzyme affected by alcohol consumption, no study has been published on the associations between ALP and the risk of hypertension in relation to drinking status.

Methods

We conducted a cross-sectional study of 2,681 participants (837 men and 1,846 women) aged 30 to 89 years undergoing a general health check-up to investigate the associations between ALP and hypertension in relation to drinking status.

Results

Of the 2,681 participants, 1,549 (514 men and 1,035 women) were diagnosed with hypertension. A sex difference was observed for the relationship between ALP and hypertension. While no significant association was observed for men, the association was significantly positive for women. The multivariable adjusted odds ratio and 95% coincidence interval (CI) of hypertension per increment of 1-log ALP were 0.95 (95% CI: 0.56 to 1.59) for men and 1.57 (95% CI: 1.07 to 2.33) for women. When this analysis was restricted to nondrinkers, a significantly elevated risk of hypertension was observed for men and remained significant for women; that is, 3.32 (95% CI: 1.38 to 8.02) for men and 1.68 (95% CI: 1.11 to 2.55) for women.

Conclusion

ALP is associated with hypertension for both male and female nondrinkers, but not for drinkers. For analyses of associations between ALP and blood pressure, alcohol consumption should thus be considered a potential confounder.     

Chain elongation of pectic β-(1→4)-galactan by a partially purified galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) hypocotyls

Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified a β-(1→4)-galactosyltransferase (GalT) involved in the synthesis of the β-(1→4)-galactan side chains of pectin. The enzyme protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized β-(1→4)-galactoheptaose (Gal₇-AB), leading to the formation of Gal_8–11-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for 2-aminopyridine-derivatized β-(1→4)-galactooligomer acceptors (Konishi et al. in Planta 218:833–842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains to about Gal₃₅-AB, thus almost reaching the length (43–47 Gal units) of native β-(1→4)-galactan side chains found in pectic polysaccharides from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301–1313, 2002). Enzyme activity increased with increasing chain length of β-(1→4)-galactooligomers and reached maximal activity at heptaose, whereas galactooligomers higher than heptaose showed lower acceptor efficiency. Sugars described in this paper belong to the d-series unless otherwise noted.     

Increased myeloid cell responses to M-CSF and RANKL cause bone loss and inflammation in SH3BP2 "cherubism" mice

While studies of the adaptor SH3BP2 have implicated a role in receptor-mediated signaling in mast cells and lymphocytes, they have failed to identify its function or explain why SH3BP2 missense mutations cause bone loss and inflammation in patients with cherubism. We demonstrate that Sh3bp2 "cherubism" mice exhibit trabecular bone loss, TNF-alpha-dependent systemic inflammation, and cortical bone erosion. The mutant phenotype is lymphocyte independent and can be transferred to mice carrying wild-type Sh3bp2 alleles through mutant fetal liver cells. Mutant myeloid cells show increased responses to M-CSF and RANKL stimulation, and, through mechanisms of increased ERK 1/2 and SYK phosphorylation/activation, they form macrophages that express high levels of TNF-alpha and osteoclasts that are unusually large. M-CSF and RANKL stimulation of myeloid cells that overexpress wild-type SH3BP2 results in similar large osteoclasts. This indicates that the mutant phenotype reflects gain of SH3BP2 function and suggests that SH3BP2 is a critical regulator of myeloid cell responses to M-CSF and RANKL stimulation.     

Oryza sativa polyamine oxidase 1 back-converts tetraamines, spermine and thermospermine, to spermidine

Key message

Oryza sativa polyamine oxidase 1 back-converts spermine (or thermospermine) to spermidine. Considering the previous work, major path of polyamine catabolism in rice plant is suggestive to be back-conversion but not terminal catabolism.

Abstract

Rice (Oryza sativa) contains seven genes encoding polyamine oxidases (PAOs), termed OsPAO1 to OsPAO7, based on their chromosomal number and gene ID number. We previously showed that three of these members, OsPAO3, OsPAO4 and OsPAO5, are abundantly expressed, that their products localize to peroxisomes and that they catalyze the polyamine back-conversion reaction. Here, we have focused on OsPAO1. The OsPAO1 gene product shares a high level of identity with those of Arabidopsis PAO5 and Brassica juncea PAO. Expression of OsPAO1 appears to be quite low under physiological conditions, but is markedly induced in rice roots by spermine (Spm) or T-Spm treatment. Consistent with the above finding, the recombinant OsPAO1 prefers T-Spm as a substrate at pH 6.0 and Spm at pH 8.5 and, in both cases, back-converts these tetraamines to spermidine, but not to putrescine. OsPAO1 localizes to the cytoplasm of onion epidermal cells. Differing in subcellular localization, four out of seven rice PAOs, OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze back-conversion reactions of PAs. Based on the results, we discuss the catabolic path(s) of PAs in rice plant.     

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C₁₈ reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 °C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.     

Acquired resistance to the rice blast in transgenic rice accumulating the antimicrobial peptide thanatin

Thanatin is an antimicrobial peptide with a strong and wide-ranging antimicrobial spectrum, including certain species of fungi and Gram-negative and Gram-positive bacteria. To evaluate the application of thanatin to the generation of disease-resistant plants, we introduced a synthetic thanatin gene into rice. Several transformants that expressed the introduced gene showed significant level of antimicrobial activity. The substances showing antimicrobial activity were partially purified from these transformants and their properties were determined. The molecule with characteristics similar to those of native thanatin on the elution pattern in HPLC analysis had an identical molecular mass to that of native molecule. It should also be noted that the transformant acquired a sufficient level of resistance to the rice blast fungus, Magnaporthe oryzae, presumably due to the repressive activity of thanatin to its initial stage of infection. This result demonstrates that thanatin has antifungal activity for M. oryzae and that the introduction of the thanatin gene into rice is effective in generating a plant resistant to rice blast disease.