EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M₄PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H₂O₂, which is the recombination product of ·OH, and OCl^- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Molecular Epidemiology of Rabies Viruses Circulating in Two Rabies Endemic Provinces of Laos, 2011–2012: Regional Diversity in Southeast Asia

BackgroundAlthough rabies is endemic in Laos, genetic characterization of the viruses in this country is limited. There are growing concerns that development in the region may have increased transport of dog through Laos for regional dog meat consumption, and that this may cause spillover of the viruses from dogs brought here from other countries. This study was therefore undertaken to evaluate the current rabies situation and the genetic characteristics of rabies viruses currently circulating in Laos.MethodsWe determined the rate of rabies-positive samples by analyzing data from animal samples submitted to the Lao Ministry of Agriculture and Forestry’s National Animal Health Centre rabies laboratory from 2004 through 2011. Twenty-three rabies-positive samples were used for viral genetic characterization. Full genome sequencing was performed on two rabies viruses.ResultsRabies-positive samples increased substantially from 40.5% in 2004 to 60.2% in 2009 and continued at this level during the study period. More than 99% of the samples were from dogs, followed by cats and monkeys. Phylogenetic analyses showed that three rabies virus lineages belonging to the Southeast Asian cluster are currently circulating in Laos; these are closely related to viruses from Thailand, Cambodia and Vietnam. Lineages of the circulating Laos rabies viruses diverged from common ancestors as recently as 44.2 years and as much as 55.3 years ago, indicating periodic virus invasions.ConclusionThere is an increasing trend of rabies in Laotian animals. Similar to other rabies-endemic countries, dogs are the main viral reservoir. Three viral lineages closely related to viruses from neighboring countries are currently circulating in Laos. Data provide evidence of periodic historic exchanges of the viruses with neighboring countries, but no recent invasion.     

IL-38: A new factor in rheumatoid arthritis

The newly characterized cytokine IL-38 (IL-1F10) belongs to the IL-1 family of cytokines. Previous work has demonstrated that IL-38 inhibited Candida albicans-induced IL-17 production from peripheral blood mononuclear cells. However, it is still unclear whether IL-38 is an inflammatory or an anti-inflammatory cytokine. We generated anti-human IL-38 monoclonal antibodies in order to perform immunohistochemical staining and an enzyme-linked immunosorbent assay. While human recombinant IL-38 protein was not cleaved by recombinant caspase-1, chymase, or PR3 in vitro, overexpression of IL-38 cDNA produced a soluble form of IL-38 protein. Furthermore, immunohistochemical analysis showed that synovial tissues obtained from RA patients strongly expressed IL-38 protein. To investigate the biological role of IL-38, C57BL/6 IL-38 gene-deficient (^?/?) mice were used in an autoantibody-induced rheumatoid arthritis (RA) mouse model. As compared with control mice, IL-38 ^(?/?) mice showed greater disease severity, accompanied by higher IL-1β and IL-6 gene expression in the joints. Therefore, IL-38 acts as an inhibitor of the pathogenesis of autoantibody-induced arthritis in mice and may have a role in the development or progression of RA in humans.     

Osteodystrophy in Cholestatic Liver Diseases Is Attenuated by Anti-γ-Glutamyl Transpeptidase Antibody

Background

Cholestatic liver diseases exhibit higher levels of serum γ-glutamyl transpeptidase (GGT) and incidence of secondary osteoporosis. GGT has been identified as a novel bone-resorbing factor that stimulates osteoclast formation. The aim of this study was to elucidate the interaction of elevated GGT levels and cholestatic liver disease-induced bone loss.

Methods

Wistar rats were divided into three groups: sham-operated control (SO) rats, bile duct ligation (BDL) rats, and anti-GGT antibody-treated BDL rats (AGT). Serum GGT level was measured. Bone mineral density (BMD) was analyzed by dual-energy X-ray absorptiometry. Bone morphometric parameters and microarchitectural properties were determined by micro-computed tomography and histomorphometry of the distal metaphysis of femurs. Alterations of bone metabolism-related factors were evaluated by cytokine array. Effects of GGT on osteoblasts or stromal cells were evaluated by RT-PCR, enzyme activity, and mineralization ability.

Results

Serum levels of GGT were significantly elevated in the BDL-group. In the BDL group, BMD, bone mass percentage, and osteoblast number were significantly decreased, whereas osteoclast number was significantly increased. These alterations were markedly attenuated in the AGT group. The mRNA levels of vascular endothelial growth factor-A, LPS-induced CXC chemokine, monocyte chemoattractant protein-1, tumor necrosis factor-α interleukin-1β and receptor activator of nuclear factor-kappa B ligand were upregulated, and those of interferon-γ and osteoprotegerin were downregulated in the GGT-treated stromal cells. Furthermore, GGT inhibited mineral nodule formation and expression of alkaline phosphatase and bone sialo-protein in osteoblastic cells.

Conclusion

Our results indicate that elevated GGT level is involved in hepatic osteodystrophy through secretion of bone resorbing factor from GGT-stimulated osteoblasts/bone marrow stromal cells. In addition, GGT also possesses suppressive effects on bone formation. Managing elevated GGT levels by anti-GGT antibody may become a novel therapeutic agent for hepatic osteodystrophy in chronic liver diseases.     

Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K₂ (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg²⁺/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.     

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.     

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.     

Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms

Objective

Intra-arterial stem cell transplantation exerts neuroprotective effects for ischemic stroke. However, the optimal therapeutic time window and mechanisms have not been completely understood. In this study, we investigated the relationship between the timing of intra-arterial transplantation of allogeneic mesenchymal stem cells (MSCs) in ischemic stroke model in rats and its efficacy in acute phase.

Methods

Adult male Wistar rats weighing 200 to 250g received right middle cerebral artery occlusion (MCAO) for 90 minutes. MSCs (1×10⁶cells/ 1ml PBS) were intra-arterially injected at either 1, 6, 24, or 48 hours (1, 6, 24, 48h group) after MCAO. PBS (1ml) was intra-arterially injected to control rats at 1 hour after MCAO. Behavioral test was performed immediately after reperfusion, and at 3, 7 days after MCAO using the Modified Neurological Severity Score (mNSS). Rats were euthanized at 7 days after MCAO for evaluation of infarct volumes and the migration of MSCs. In order to explore potential mechanisms of action, the upregulation of neurotrophic factor and chemotactic cytokine (bFGF, SDF-1α) induced by cell transplantation was examined in another cohort of rats that received intra-arterial transplantation at 24 hours after recanalization then euthanized at 7 days after MCAO for protein assays.

Results

Behavioral test at 3 and 7 days after transplantation revealed that stroke rats in 24h group displayed the most robust significant improvements in mNSS compared to stroke rats in all other groups (p’s<0.05). Similarly, the infarct volumes of stroke rats in 24h group were much significantly decreased compared to those in all other groups (p’s<0.05). These observed behavioral and histological effects were accompanied by MSC survival and migration, with the highest number of integrated MSCs detected in the 24h group. Moreover, bFGF and SDF-1α levels of the infarcted cortex were highly elevated in the 24h group compared to control group (p’s<0.05).

Conclusions

These results suggest that intra-arterial allogeneic transplantation of MSCs provides post-stroke functional recovery and reduction of infarct volumes in ischemic stroke model of rats. The upregulation of bFGF and SDF-1α likely played a key mechanistic role in enabling MSC to afford functional effects in stroke. MSC transplantation at 24 hours after recanalization appears to be the optimal timing for ischemic stroke model, which should guide the design of clinical trials of cell transplantation for stroke patients.     

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3''→5'' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5''-end is deaminated more effectively than one less close to the 5''-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2''s activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3''→5'' polarity. Examination of the effects of different salt concentrations on the 3''→5'' polarity indicated that the higher the salt concentration, the less prominent the 3''→5'' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.