Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively‐charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)‐labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA‐binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC‐labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy. Microsc. Res. Tech. 79:31–37, 2016. © 2015 Wiley Periodicals, Inc.     

Effect of medium-chain triacylglycerols on anti-obesity effect of fucoxanthin

Dietary effects of medium-chain triacylglycerols (MCT) and fucoxanthin (Fc) on abdominal fat weight were determined using KK-Ay obese mouse. Experimental diet contained MCT(0.9%), Fc (0.1%), or MCT (0.9%) +Fc (0.1%). The abdominal fat weight of mice fed with Fc was significantly lower than that of mice fed with MCT. Uncoupling protein 1 (UCP1), a key molecule for metabolic thermogenesis, was clearly expressed in the white adipose tissue (WAT) of mice fed Fc, but little expression in that of the mice fed MCT. The anti-obesity effect of Fc was increased by mixing Fc with MCT. This increase would be due to the increase in the absorption rate of Fc by MCT.     

Angiotensin-I converting enzyme inhibitory peptide derived from porcine skeletal muscle myosin and its antihypertensive activity in spontaneously hypertensive rats

ABSTRACT:  Crude myosin light chain was extracted from Japanese domestic pork loin and digested with pepsin. Antihypertensive peptide was isolated from this digest as a measure of its inhibitory activity for angiotensin-I converting enzyme (ACE). Through isolation with some chromatographies, a single active fraction was isolated, and it was detected as an octapeptide, Val-Lys-Lys-Val-Leu-Gly-Asn-Pro, from 47th to 54th positions of myosin light chain. The 50% inhibitory concentration of this peptide was 28.5 μM. Kinetic evaluation showed that this peptide was a noncompetitive inhibitor, but it was slowly hydrolyzed by ACE. At the dose of 10 mg/kg, this peptide showed antihypertensive activity after a maximum of 3 h of administration and was estimated as a temporally effective hypotensor.     

Deciphering the Biological Significance of ADAR1–Z-RNA Interactions

Adenosine deaminase acting on RNA 1 (ADAR1) is an enzyme responsible for double-stranded RNA (dsRNA)-specific adenosine-to-inosine RNA editing, which is estimated to occur at over 100 million sites in humans. ADAR1 is composed of two isoforms transcribed from different promoters: p150 and N-terminal truncated p110. Deletion of ADAR1 p150 in mice activates melanoma differentiation-associated protein 5 (MDA5)-sensing pathway, which recognizes endogenous unedited RNA as non-self. In contrast, we have recently demonstrated that ADAR1 p110-mediated RNA editing does not contribute to this function, implying that a unique Z-DNA/RNA-binding domain α (Zα) in the N terminus of ADAR1 p150 provides specific RNA editing, which is critical for preventing MDA5 activation. In addition, a mutation in the Zα domain is identified in patients with Aicardi–Goutières syndrome (AGS), an inherited encephalopathy characterized by overproduction of type I interferon. Accordingly, we and other groups have recently demonstrated that Adar1 Zα-mutated mice show MDA5-dependent type I interferon responses. Furthermore, one such mutant mouse carrying a W197A point mutation in the Zα domain, which inhibits Z-RNA binding, manifests AGS-like encephalopathy. These findings collectively suggest that Z-RNA binding by ADAR1 p150 is essential for proper RNA editing at certain sites, preventing aberrant MDA5 activation.     

Laboratory corrosion tests for simulating fireside wastage of superheater materials in waste incinerators

Laboratory corrosion tests were performed to clarify the effects of relative amounts of fused salts in tube deposits on corrosion rates of superheater materials in WTE plants. All test exposures were at 550 °C and of 100 h duration. The nine synthetic ashes used as corrodents consisted of mixtures of chlorides, sulfates and oxides. The test materials were alloy steel T22, stainless steels TP347H, TP310HCbN, and alloys HR11N and 625. The gas atmosphere consisted of 500 to 3000 ppm HCl‐30 ppm SO₂‐10% O₂‐10% CO₂‐20% H₂O‐bal.N₂. Generally, the relative amount of fused salts in non‐fused ash constituents at 550 °C increased with increasing the chlorine content of the ashes. The corrosion rate of T22 steel did not depend directly on ash chlorine content, but for ashes of 7.7 wt.% Cl, the corrosion rate depended on the calculated amount of fused salt at 500 °C. The corrosion rates of TP347H steel and alloy 625 were maximum for ashes of 6–8 wt.% Cl. For ashes of 7.7 wt.% Cl, the corrosion rates of T22 steel, stainless steels, and alloys increased with ashes having higher amounts of fused salts. Increased HCl content of the gas caused higher corrosion of the stainless steels and high‐nickel alloys, but there was no clear corrosion‐exacerbating effect with T22 steel.     

Mechanical properties of tussah silk fibers treated with methacrylamide

Tussah silk fibers were treated with methacrylamide (MAA). The polymerization of MAA onto tussah silk fibers and the mechanical properties of the MAA-treated tussah silk fibers were investigated. The tanning agent contained in tussah silk fibers acted as an inhibitor to the radical polymerization of MAA. The alkali treatment enhanced the swelling of noncrystalline regions of the tussah silk fibers and promoted the polymerization of MAA onto the tussah silk fibers. The cross-sectional area of the MAA-treated tussah silk fiber was given by the sum of the cross-sectional area of the original silk fiber and that of the MAA polymer. Breaking load of the fibers was almost unchanged by the MAA treatment, while rigidity was markedly increased. Young's modulus of the MAA-treated tussah silk fibers decreased with decreasing volume fractions of the fiber in the MAA-treated tussah silk fibers. Young's modulus of the MAA polymer in the MAA-treated tussah silk fibers was estimated by extrapolating the relation between Young's moduli and the volume fractions of the fiber to zero volume fraction. Young's modulus of the MAA polymer in the MAA-treated tussah silk fibers was significantly larger than the modulus of a MAA polymer plate. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 65: 2051–2057, 1997     

Multifaceted Analysis of IL-23A- and/or EBI3-Including Cytokines Produced by Psoriatic Keratinocytes

Interleukin (IL) 23 (p19/p40) plays a critical role in the pathogenesis of psoriasis and is upregulated in psoriasis skin lesions. In clinical practice, anti-IL-23Ap19 antibodies are highly effective against psoriasis. IL-39 (p19/ Epstein-Barr virus-induced (EBI) 3), a newly discovered cytokine in 2015, shares the p19 subunit with IL-23. Anti-IL-23Ap19 antibodies may bind to IL-39; also, the cytokine may contribute to the pathogenesis of psoriasis. To investigate IL23Ap19- and/or EBI3-including cytokines in psoriatic keratinocytes, we analyzed IL-23Ap19 and EBI3 expressions in psoriasis skin lesions, using immunohistochemistry and normal human epidermal keratinocytes (NHEKs) stimulated with inflammatory cytokines, using quantitative real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-electrospray tandem mass spectrometry (LC-Ms/Ms). Immunohistochemical analysis showed that IL-23Ap19 and EBI3 expressions were upregulated in the psoriasis skin lesions. In vitro, these expressions were synergistically induced by the triple combination of tumor necrosis factor (TNF)-α, IL-17A, and interferon (IFN)-γ, and suppressed by dexamethasone, vitamin D3, and acitretin. In ELISA and LC-Ms/Ms analyses, keratinocyte-derived IL-23Ap19 and EBI3, but not heterodimeric forms, were detected with humanized anti-IL-23Ap19 monoclonal antibodies, tildrakizumab, and anti-EBI3 antibodies, respectively. Psoriatic keratinocytes may express IL-23Ap19 and EBI3 proteins in a monomer or homopolymer, such as homodimer or homotrimer.     

Enhancement of Anti-Tumoral Immunity by β-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer

β-Casomorphin-7 (BCM) is a degradation product of β-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20⁺ B cells, CD4⁺ T cells, and regulatory T cells (Tregs), and increased CD8⁺ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8⁺ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.