Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4⁺ T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein–Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3′UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3′UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3′UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3′UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.     

Influence of Plasmonic Nanoparticles on the Performance of Colorimetric Cell Viability Assays

The conventional colorimetric assays based on measurement of the metabolic activity are routinely used to evaluate the cytotoxicity of nanomaterials (NMs). However, due to the varying absorbance properties of plasmonic NMs in the visible region of the spectrum, obtained results can be misleading. In this study, MTT, MTS, and WST-1 colorimetric cell viability assays were evaluated in the presence of gold (AuNPs) or silver nanoparticles (AgNPs). Since a living cell a complex system containing many molecular and ionic species, the plasmonic AuNP and AgNPs may selectively interact with intracellular components possessing thiol, amino, and carboxyl group moieties change the aggregation behavior of the NMs and thus their absorbance. A series of UV/Vis and DLS experiments were conducted to understand the interference possibility of the tested plasmonic NMs. The results show that the AuNPs and AgNPs do not have absorption at the wavelength where MTT formazan is measured while the both NPs may interfere with absorbance of MTS and WST-1 formazan.The overall assessments show that MTT assay is more suitable for the cell viability evaluation of spherical AuNPs and AgNPs with an average diameter of 50 nm. This study also suggests that a preliminary ex situ evaluation of plasmonic nanoparticles can provide valuable information for the suitability of the assay.     

Recovery of bean plants from boron-induced oxidative damage by zinc supply

The effects of zinc on growth, boron uptake, lipid peroxidation, membrane permeability (MP), lypoxygenase (LOX) activity, proline and H₂O₂ accumulation, and the activities of major antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) in bean plants were investigated under greenhouse conditions. Treatments consisted of control, 20 mg/kg B, and 20 mg/kg B plus 20 mg/kg Zn. When the plants were grown with 20 mg/kg Zn, B toxicity was less severe. Zinc supplied to soil counteracted the deleterious effects of B on root and shoot growth. Excess B significantly increased and Zn treatment reduced B concentrations in shoot and root tissues. Applied Zn increased the Zn concentration in the roots and shoots. While the concentrations of H₂O₂ and proline were increased by B toxicity, their concentrations were decreased by Zn supply. Boron toxicity increased the MP, malondialdehyde content, and LOX activity in excised bean leaves. Applied Zn significantly ameliorated the membrane deterioration. Compared with control plants, the activity of SOD was increased while that of CAT was decreased and APX remained unchanged in B-stressed plants. However, application of Zn decreased the SOD and increased the CAT and APX activities under toxic B conditions. It is concluded that Zn supply alleviates B toxicity by preventing oxidative membrane damage. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 555–562. This text was submitted by the authors in English.     

Venous thromboembolism after total joint arthroplasty: results from a Japanese multicenter cohort study

Introduction

Real-world evidence of the effectiveness of pharmacological thromboprophylaxis for venous thromboembolism (VTE) is limited. Our objective was to assess the effectiveness and safety of thromboprophylactic regimens in Japanese patients undergoing joint replacement in a real-world setting.

Method

Overall, 1,294 patients (1,073 females and 221 males) who underwent total knee arthroplasty (TKA) and 868 patients (740 females and 128 males) who underwent total hip arthroplasty (THA) in 34 Japanese national hospital organization (NHO) hospitals were enrolled. The primary efficacy outcome was the incidence of deep vein thrombosis (DVT) detected by mandatory bilateral ultrasonography up to post-operative day (POD) 10 and pulmonary embolism (PE) up to POD28. The main safety outcomes were bleeding (major or minor) and death from any cause up to POD28.

Results

Patients undergoing TKA (n = 1,294) received fondaparinux (n = 360), enoxaparin (n = 223), unfractionated heparin (n = 72), anti-platelet agents (n = 45), or no medication (n = 594). Patients undergoing THA (n = 868) received fondaparinux (n = 261), enoxaparin (n = 148), unfractionated heparin (n = 32), anti-platelet agents (n = 44), or no medication (n = 383). The incidence rates of sonographically diagnosed DVTs up to POD10 were 24.3% in patients undergoing TKA and 12.6% in patients undergoing THA, and the incidence rates of major bleeding up to POD28 were 1.2% and 2.3%, respectively. Neither fatal bleeding nor fatal pulmonary embolism occurred. Significant risk factors for postoperative VTE identified by multivariate analysis included gender (female) in both TKA and THA groups and use of a foot pump in the TKA group. Only prophylaxis with fondaparinux reduced the occurrence of VTE significantly in both groups. Propensity score matching analysis (fondaparinux versus enoxaparin) showed that the incidence of DVT was lower (relative risk 0.70, 95% confidence interval (CI) 0.58 to 0.85, P = 0.002 in TKA and relative risk 0.73, 95% CI 0.53 to 0.99, P = 0.134 in THA) but that the incidence of major bleeding was higher in the fondaparinux than in the enoxaparin group (3.4% versus 0.5%, P = 0.062 in TKA and 4.9% versus 0%, P = 0.022 in THA).

Conclusions

These findings indicate that prophylaxis with fondaparinux, not enoxaparin, reduces the risk of DVT but increases bleeding tendency in patients undergoing TKA and THA.

Trial registration

University Hospital Medical Information Network Clinical Trials Registry: UMIN000001366. Registered 11 September 2008.     

Immunological comparison of native and recombinant egg allergen, ovalbumin, expressed in Escherichia coli

Chicken ovalbumin is one of the major egg white allergens which causes IgE-mediated food hypersensitivity. A gene encoding for chicken ovalbumin (Gad dI) was isolated from chicken oviduct by PCR amplification and was cloned under the control of T5 promoter fused with a six-histidine tag at the N-terminal end. Escherichia coli harbouring this construct expressed high quantities of the recombinant protein in the form of soluble fraction. The protein was purified using affinity chromatography on a Ni(2+)-nitrilotriacetic acid agarose column and was further purified to homogeneity by ion exchange chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot and secondary conformation analysis. The reactivity of the recombinant and native protein was tested against six egg allergic human patient's sera and the IgE and IgG binding activity was tested using both Western blot and ELISA. When compared to native ovalbumin, the recombinant protein had similar binding activity in immunoblotting, but slightly increased activity by ELISA. Circular dichroism revealed that the recombinant protein had a slightly less compact structure than the native form. Both antigens exhibited a similar immunogenicity in mice.     

A peptide derived from the parasite receptor,complement C2 receptor inhibitor trispanning,suppresses immune complex-mediated inflammation in mice

Complement C2 receptor inhibitor trispanning (CRIT) is a Schistosoma protein that binds the human complement protein, C2. We recently showed that peptides based on the ligand binding region of CRIT inhibit the classical pathway (CP) of complement activation in human serum, using hemolytic assays and so speculated that on the parasite surface CRIT has the function of evading human complement. We now show that in vitro the C2-binding 11-aa C terminus of the first extracellular domain of CRIT, a 1.3-kDa peptide termed CRIT-H17, inhibits CP activation in a species-specific manner, inhibiting mouse and rat complement but not that from guinea pig. Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study we show that by inhibiting the CP, CRIT-H17 is able to reduce immune complex-mediated inflammation (dermal reversed passive Arthus reaction) in BALB/c mice. Upon intradermal injection of CRIT-H17, and similarly with recombinant soluble complement receptor type 1, there was a 41% reduction in edema and hemorrhage, a 72% reduction in neutrophil influx, and a reduced C3 deposition. Furthermore, when H17 was administered i.v. at a 1 mg/kg dose, inflammation was reduced by 31%. We propose that CRIT-H17 is a potential therapeutic agent against CP complement-mediated inflammatory tissue destruction.     

Dissolved Organic Nitrogen Hydrolysis Rates in Axenic Cultures of Aureococcus anophagefferens (Pelagophyceae): Comparison with Heterotrophic Bacteria

The marine autotroph Aureococcus anophagefferens (Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.     

PROGRESSION OF SPERMATIAL NUCLEAR DIVISION REQUIRES CALCIUM INFLUX DURING FERTILIZATION OF THE RED ALGA PALMARIA SP.1

During fertilization of the red alga Palmaria sp. (Palmariales), the prophase-arrested nucleus of the uninucleate spermatium resumes its division after direct attachment of the spermatium to the trichogyne of a carpogonium. Treatments that reduce Ca²⁺ influx inhibit the progression of spermatial nuclear division. The ratio of the number of spermatia released from prophase arrest (those in meta-phase to binucleate stages) to the total spermatia attached to trichogynes was significantly reduced by incubating the spermatia in the culture solution having low Ca²⁺ concentration. Similar inhibition was observed by addition of either inorganic (La³⁺ and Co²⁺) or organic (nifedipine and tetramethrin) Ca²⁺ channel inhibitors to the culture solution containing 10 mM Ca²⁺. These results indicate that the prophase/metaphase transition of spermatial nuclear division requires an influx of Ca²⁺ and suggest that Ca²⁺ acts as a second messenger to the mechanical or chemical stimulus that initiates mitotic progression of spermatia in this alga.     

Effects of weak microwave fields amplitude modulated at ELF on EEG of symmetric brain areas in rats

Averaged electroencephalogram (EEG) frequency spectra were studied in eight unanesthetized and unmyorelaxed adult male rats with chronically implanted carbon electrodes in symmetrical somesthetic areas when a weak (0.1–0.2 mW/cm) microwave (MW, 945 MHz) field, amplitude-modulated at extremely low frequency (ELF) (4 Hz), was applied. Intermittent (1 min “On,” 1 min “Off”) field exposure (10-min duration) was used. Hemispheric asymmetry in frequency spectra (averaged data for 10 or 1 min) of an ongoing EEG was characterized by a power decrease in the 1.5–3 Hz range on the left hemisphere and by a power decrease in the 10–14 and 20–30 Hz ranges on the right hemisphere. No differences between control and exposure experiments were shown under these routines of data averaging. Significant elevations of EEG asymmetry in 10–14 Hz range were observed during the first 20 s after four from five onsets of the MW field, when averaged spectra were obtained for every 10 s. Under neither control nor pre- and postexposure conditions was this effect observed. These results are discussed with respect to interaction of MW fields with the EEG generators. Bioelectromagnetics 18:293–298, 1997. © 1997 Wiley-Liss, Inc.