Dietary flavonoid apigenin is a potential inducer of intracellular oxidative stress: the role in the interruptive apoptotic signal

Apigenin is a representative dietary flavone (2-phenyl-4H-1-benzopyran-4-one) inhibiting cancer cell growth both in cell culture systems and in vivo. The prooxidant potential of apigenin was confirmed by the observations using flowcytometric and immunoblotting techniques that the intracellular accumulations of reactive oxygen species (ROS) and protein carbonyls were detected in the cells treated with apigenin in a dose-dependent manner. Conversely, chrysin (5,7-dihydroxyflavone) did not show any prooxidant effect. A structure-activity relationship data thus indicated that a 4'-monohydroxyl group, which can be oxidized to semiquinone radical but not up to quinone-like metabolite, is essential for prooxidant effect. When HL-60 cells were treated with not only a heme synthesis inhibitor succinyl acetone (SA) but also myeloperoxidase (MPO) inhibitors, the ROS level enhanced by apigenin was significantly reduced. The gathered data suggested that peroxidase-catalyzed production of apigenin B-ring phenoxyl radicals might be responsible for the prooxidant effect. This is supported by the observation that MPO is able to catalyze production of apigenin phenoxyl radicals, detected by an electron spin resonance-spin trapping technique. We also reveal that both SA and alpha-tocopherol enhance cellular susceptibility to apoptosis-inducing stimuli by apigenin. In conclusion, the prooxidant effect of apigenin is likely to oxidize a variety of thiols through the formation of phenoxyl radicals and thus seems to play a significant role in the abortive apoptotic pathway switching to necrotic cell death.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme.

4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent.Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.     

Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of<Emphasis Type="Italic"> Vallisneria gigantea</Emphasis>

In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts.Preliminary results of part of the local irradiation study have already been reported in abstract form [N. Sakurai et al. (2002) J Photosci 9:326–328].     

Anti-allodynic effects of intrathecally and intracerebroventricularly administered 26RFa, an intrinsic agonist for GRP103, in the rat partial sciatic nerve ligation model

26RFa and QRFP are endogenous ligands of GPR103. 26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain. In the present study, the effects of intrathecal and intracerebroventricular applications of 26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal and intracerebroventricular injection of 26RFa attenuated the level of mechanical allodynia. 26RFa has been reported to activate not only GPR103 but also neuropeptide FF2 receptor and the effect of intrathecally and intracerebroventricularly administered 26RFa was not antagonized by BIBP3226, an antagonist of neuropeptide FF receptor. Immunohistochemical examination revealed that QRFP-like immunoreactivity (QRFP-LI) was expressed mainly in the small to medium sized neurons in the L5 dorsal root ganglion (DRG) and that partial sciatic nerve injury increased the percentage of QRFP-LI positive neurons. 7 days after the nerve injury, QRFP-LI positive neurons in the L5 DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5 DRG ipsilateral to the sham operation. These data suggest that (1) exogenously applied 26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model, (2) the mechanism 26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103, and (3) partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.     

Intensity of larval diapause in the bamboo borer,Omphisa fuscidentalis

Larvae of the bamboo borer, Omphisa fuscidentalis, enter larval diapause in September and pupate in the following June (Singtripop et al., 1999). We examined the changes in the responses of larvae to exogenous 20-hydroxyecdysone (20E) in order to estimate the progress of diapause development. In this respect, we adopted two terms, responsiveness and sensitivity of larvae to 20E. Responsiveness was estimated by the percentage of larvae that pupated, and sensitivity was evaluated by the duration from the day of 20E injection to pupation. The responsiveness of larvae declined gradually from September to November when larvae were least responsive to 20E, and then increased markedly from January to February. This indicates that the intensity of diapause increases from September to November and terminated gradually thereafter. Thus the sequence of events as the larval responses to 20E is characterized by a V-shaped curve. Sensitivity of larvae to 20E was at the same level from September to December, and increased remarkably from December to January. The abrupt increase in the sensitivity occurred one month earlier than the bottom of the V-shaped curve of larval responsiveness, suggesting that the increases in the responsiveness and sensitivity in the latter half of diapause may be brought about by respective mechanisms.     

Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).     

Structural factors contributing to the Abl/Lyn dual inhibitory activity of 3-substituted benzamide derivatives

To investigate why 3-substituted benzamide derivatives show dual inhibition of Abl and Lyn protein tyrosine kinases, we determined their inhibitory activities against Abl and Lyn, carried out molecular modeling, and conducted a structure-activity relationship study with the aid of a newly determined X-ray structure of the Abl/Lyn dual inhibitor INNO-406 (formerly known as NS-187) bound to human Abl. We found that this series of compounds interacted with both kinases in very similar ways, so that they can inhibit both kinases effectively.     

First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan

Schizophyllan is a natural beta-(1-->3)-D-glucan that exists as a triple helix in H(2)O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homo-polynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H(2)O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H(2)O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.