Bee Venom Phospholipase A2 Protects against Acetaminophen-Induced Acute Liver Injury by Modulating Regulatory T Cells and IL-10 in Mice

The aim of this study was to investigate the protective effects of phospholipase A2 (PLA2) from bee venom against acetaminophen-induced hepatotoxicity through CD4⁺CD25⁺Foxp3⁺ T cells (Treg) in mice. Acetaminophen (APAP) is a widely used antipyretic and analgesic, but an acute or cumulative overdose of acetaminophen can cause severe hepatic failure. Tregs have been reported to possess protective effects in various liver diseases and kidney toxicity. We previously found that bee venom strongly increased the Treg population in splenocytes and subsequently suppressed immune disorders. More recently, we found that the effective component of bee venom is PLA2. Thus, we hypothesized that PLA2 could protect against liver injury induced by acetaminophen. To evaluate the hepatoprotective effects of PLA2, C57BL/6 mice or interleukin-10-deficient (IL-10^−/−) mice were injected with PLA2 once a day for five days and sacrificed 24 h (h) after acetaminophen injection. The blood sera were collected 0, 6, and 24 h after acetaminophen injection for the analysis of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PLA2-injected mice showed reduced levels of serum AST, ALT, proinflammatory cytokines, and nitric oxide (NO) compared with the PBS-injected control mice. However, IL-10 was significantly increased in the PLA2-injected mice. These hepatic protective effects were abolished in Treg-depleted mice by antibody treatment and in IL-10^−/− mice. Based on these findings, it can be concluded that the protective effects of PLA2 against acetaminophen-induced hepatotoxicity can be mediated by modulating the Treg and IL-10 production.     

Optimal Extent of Prophylactic Irradiation of Paraaortic Lymph Nodes in Patients with Uterine Cervical Cancer

Purpose

The purpose of this study was to determine optimal extent of prophylactic irradiation of paraaortic lymph nodes (PALN) in patients with uterine cervical cancer who had metastatic pelvic LNs.

Methods and Materials

We retrospectively evaluated 103 patients with cervical cancer and pelvic lymph node metastasis who were treated with prophylactic semi-extended field radiotherapy (SEFRT) between 1990 and 2012. The semi-extended field included PALN below the second lumbar spine with prescribed doses of 45 to 50 Gy. Survival outcomes were calculated using the Kaplan-Meier method, and acute and late toxicities were scored using the Radiation Therapy Oncology Group and European Organisation for Research and Treatment of Cancer toxicity criteria.

Results

The median follow-up after SEFRT was 61 (range 5–296) months. Overall, 28 patients (27.2%) experienced treatment failures, which were classified as local in 8 patients (7.8%), regional in 8 patients (7.8%), and distant in 13 patients (12.6%). Of the regional failures, only two involved PALN failure around the renal artery or the renal hilum area at the upper margin of the semi-extended field. At 5 years, the overall survival was 82%. Grade 3 or higher acute gastrointestinal and genitourinary toxicities occurred in one and two patients, respectively. As a late toxicity, one patient developed grade 3 small bowel obstruction.

Conclusion

Prophylactic SEFRT provided favorable outcomes with little acute or late gastrointestinal toxicity. For prophylaxis of PALN recurrences, upper part of PALN might not need to be included in patients with uterine cervical cancer and metastatic pelvic LNs.     

The Subcellular Distribution of an RNA Quality Control Protein, the Ro Autoantigen, Is Regulated by Noncoding Y RNA Binding

The Ro autoantigen is a ring-shaped RNA-binding protein that binds misfolded RNAs in nuclei and is proposed to function in quality control. In the cytoplasm, Ro binds noncoding RNAs, called Y RNAs, that inhibit access of Ro to other RNAs. Ro also assists survival of mammalian cells and at least one bacterium after UV irradiation. In mammals, Ro undergoes dramatic localization changes after UV irradiation, changing from mostly cytoplasmic to predominantly nuclear. Here, we report that a second role of Y RNAs is to regulate the subcellular distribution of Ro. A mutant Ro protein that does not bind Y RNAs accumulates in nuclei. Ro also localizes to nuclei when Y RNAs are depleted. By assaying chimeric proteins in which portions of mouse Ro were replaced with bacterial Ro sequences, we show that nuclear accumulation of Ro after irradiation requires sequences that overlap the Y RNA binding site. Ro also accumulates in nuclei after oxidative stress, and similar sequences are required. Together, these data reveal that Ro contains a signal for nuclear accumulation that is masked by a bound Y RNA and suggest that Y RNA binding may be modulated during cell stress.     

Detection of abnormally high amygdalin content in food by an enzyme immunoassay

Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.     

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane protein-associated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.     

Identification of novel antitubercular compounds through hybrid virtual screening approach

Growing resistance of prevalent antitubercular (antiTB) agents in clinical isolates of Mycobacterium tuberculosis (MTB) provoked an urgent need to discover novel antiTB agents. Enoyl acyl carrier protein (ACP) reductase (InhA) from Mtb is a well known and thoroughly studied as antitubucular therapy target. Here we have reported the discovery of potent antiTB agents through ligand and structure based approaches using computational tools. Initially compounds with more than 0.500 Tanimoto similarity coefficient index using functional class fingerprints (FCFP_4) to the reference chemotype were mined from the chemdiv database. Further, the molecular docking was performed to select the compounds on the basis of their binding energies, binding modes, and tendencies to form reasonable interactions with InhA (PDB ID = 2NSD) protein. Eighty compounds were evaluated for antitubercular activity against H37RV M. tuberculosis strain, out of which one compound showed MIC of 5.70 μM and another showed MIC of 13.85 μM. We believe that these two new scaffolds might be the good starting point from hit to lead optimization for new antitubercular agents.     

High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins

We describe a high-throughput strategy for tuning the expression of recombinant proteins through engineering their early nucleotide sequences. After randomizing the +2 and +3 codons of the target genes, each of the variant genes was isolated in vivo and subsequently expressed using in vitro protein synthesis techniques. When several hundreds of clones were examined in parallel, it was found that expression levels of target genes varied as much as 70-fold depending on the identity of the codons in the randomized region. This broad and continuous distribution of expression levels enabled the selection of specific codon arrangements for the expression of target genes at a desired level. Furthermore, codon-dependent variations in protein expression were reproduced when the same genes were expressed in vivo. Thus, we expect that the methodology reported here could be utilized as a versatile platform for rapid expression of protein molecules at modulated levels either in vitro or in vivo.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.