Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.     

Role of mitochondrial phosphate carrier in metabolism-secretion coupling in rat insulinoma cell line INS-1

In pancreatic β-cells, glucose-induced mitochondrial ATP production plays an important role in insulin secretion. The mitochondrial phosphate carrier PiC is a member of the SLC25 (solute carrier family 25) family and transports Pi from the cytosol into the mitochondrial matrix. Since intramitochondrial Pi is an essential substrate for mitochondrial ATP production by complex V (ATP synthase) and affects the activity of the respiratory chain, Pi transport via PiC may be a rate-limiting step for ATP production. We evaluated the role of PiC in metabolism-secretion coupling in pancreatic β-cells using INS-1 cells manipulated to reduce PiC expression by siRNA (small interfering RNA). Consequent reduction of the PiC protein level decreased glucose (10 mM)-stimulated insulin secretion, the ATP:ADP ratio in the presence of 10 mM glucose and elevation of intracellular calcium concentration in response to 10 mM glucose without affecting the mitochondrial membrane potential (Δψm) in INS-1 cells. In experiments using the mitochondrial fraction of INS-1 cells in the presence of 1 mM succinate, PiC down-regulation decreased ATP production at various Pi concentrations ranging from 0.001 to 10 mM, but did not affect Δψm at 3 mM Pi. In conclusion, the Pi supply to mitochondria via PiC plays a critical role in ATP production and metabolism-secretion coupling in INS-1 cells.     

Requirement of a relatively high threshold level of Mg(2+) for cell growth of a rhizoplane bacterium, Sphingomonas yanoikuyae EC-S001

Mg(2+) is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg(2+)) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg(2+) levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg(2+) or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg(2+) requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg(2+)-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg(2+) for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg(2+) to Mg(2+)-free HSG medium. Our studies concluded that Mg(2+) is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg(2+) or another specific essential element for their growth.     

Functional and Structural Characterization of Neutralizing Epitopes of Measles Virus Hemagglutinin Protein

Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.     

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

Background

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.

Methods

The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1⁺NIL were also compared to identify pathogen-responsive proteins.

Results

Eight proteins were either induced or upregulated in inoculated Fhb1⁺NIL when compared with mock-inoculated Fhb1⁺NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1⁺NIL when compared with Fusarium-inoculated Fhb1⁻NIL. Proteins that were differentially expressed in the Fhb1⁺NIL, not in the Fhb1⁻NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.

Conclusions

Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1⁺NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.     

Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.     

Brünnich's guillemots (Uria lomvia) maintain high temperature in the body core during dives

A major challenge for diving birds, reptiles, and mammals is regulating body temperature while conserving oxygen through a reduction in metabolic processes. To gain insight into how these needs are met, we measured dive depth and body temperatures at the core or periphery between the skin and abdominal muscles simultaneously in freely diving Brünnich's guillemots (Uria lomvia), an arctic seabird, using an implantable data logger (16-mm diameter, 50-mm length, 14-g mass, Little Leonardo Ltd., Tokyo). Guillemots exhibited increased body core temperatures, but decreased peripheral temperatures, during diving. Heat conservation within the body core appeared to result from the combined effect of peripheral vasoconstriction and a high wing beat frequency that generates heat. Conversely, the observed tissue hypothermia in the periphery should reduce metabolic processes as well as heat loss to the water. These physiological effects are likely one of the key physiological adaptations that makes guillemots to perform as an efficient predator in arctic waters.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Relationships between Micro-Vascular and Iodine-Staining Patterns in the Vicinity of the Tumor Front of Superficial Esophageal Squamous Carcinoma

Objective

The aim of the present study was to clarify differences between micro-vascular and iodine-staining patterns in the vicinity of the tumor fronts of superficial esophageal squamous cell carcinomas (ESCCs).

Methods

Ten consecutive patients with ESCCs who were treated by endoscopic submucosal dissection (ESD) were enrolled. At the edge of the iodine-unstained area, we observed 183 sites in total using image-enhanced magnifying endoscopy. We classified the micro-vascular and iodine-staining patterns into three types: Type A, in which the line of vascular change matched the border of the iodine-unstained area; Type B, in which the border of the iodine-unstained area extended beyond the line of vascular change; Type C, in which the line of vascular change extended beyond the border of the iodine-unstained area. Then, by examining histopathological sections, we compared the diameter of intra-papillary capillary loops (IPCLs) in cancerous areas and normal squamous epithelium.

Results

We investigated 160 sites that the adequate quality of pictures were obtained. There was no case in which the line of vascular change completely matched the whole circumference of the border of an iodine-unstained area. Among the 160 sites, type A was recognized at 76 sites (47.5%), type B at 79 sites (49.4%), and type C at 5 sites (3.1%). Histological examination showed that the mean diameter of the IPCLs in normal squamous epithelium was 16.2±3.7μm, whereas that of IPCLs in cancerous lesions was 21.0±4.4μm.

Conclusions

The development of iodine-unstained areas tends to precede any changes in the vascularity of the esophageal surface epithelium.     

Significant antitumor effect of a synthetic lipid A analogue,DT-5461, on murine syngeneic tumor models

Summary The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.