Flow cytometric determination of basophils in whole blood with n-propyl Astra Blue iodide

Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.     

Immunogenicity of Hepatitis B Virus Vaccine (Heptavax-B)—Response of Healthy Adults

We vaccinated 30 healthy adults who were susceptible to hepatitis B virus with 20 μg of Merck Sharp & Dohme vaccine at zero, one and six months and measured their antibody (anti-HBs) response weekly for four weeks and monthly for at least seven months. A month following the first dose 40% had antibodies and 90% were positive for anti-HBs a month following the second dose. The third dose raised the response rate to 93.3% but its major effect was to substantially raise the titer of virtually all of the vaccinees. Two persons had no antibody response to vaccination and four others were hyporesponders. No complications or significant side effects were observed.     

Thrombospondin-4 promotes bladder cancer cell migration and invasion via MMP2 production

Bladder cancer (BC) is the second most common urological tumour in Western countries. Approximately, 80% of patients with BC will present with non-muscle invasive bladder cancer (NMIBC), whereas a quarter will have muscle invasive disease (MIBC) at the time of BC diagnosis. However, patients with NMIBC are at risk of BC recurrence or progression into MIBC, and an MIBC prognosis is determined by the presence of progression and metastasis. Matrix metalloproteinase 2 (MMP2), a type of matrix metalloproteinase (MMP), plays a major role in tumour invasion and is well-characterized in BC prognosis. In BC, the mechanisms regulating MMP2 expression, and, in turn, promote cancer invasion, have hardly been explored. Thrombospondin-4 (THBS4/TSP4) is a matricellular glycoprotein that regulates multiple biological functions, including proliferation, angiogenesis, cell adhesion and extracellular matrix modelling. Based on the results of a meta-analysis in the Gene Expression Profiling Interactive Analysis 2 database, we observed that TSP4 expression levels were consistent with overall survival (OS) rate and BC progression, with the highest expression levels observed in the advanced stages of BC and associated with poor OS rate. In our pilot experiments, incubation with recombinant TSP4 promoted the migration and invasion in BC cells. Furthermore, MMP2 expression levels increased after recombinant TSP4 incubation. TSP4-induced-MMP2 expression and cell motility were regulated via the AKT signalling pathway. Our findings facilitate further investigation into TSP4 silencing-based therapeutic strategies for BC.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1β secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1β expression, it suppressed the secretion of cleaved IL-1β. In contrast, HGF decreased active IL-1β secretion without affecting pro-IL-1β expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1β expression level and active IL-1β secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.     

CFS-1686 Causes Cell Cycle Arrest at Intra-S Phase by Interference of Interaction of Topoisomerase 1 with DNA

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin''s induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.     

Allyl Isothiocyanate Ameliorates Angiogenesis and Inflammation in Dextran Sulfate Sodium-Induced Acute Colitis

Allyl isothiocyanate (AITC) is a phytochemical found in cruciferous vegetables that has known chemopreventive and chemotherapeutic activities. Thus far, the antiangiogenic activity of AITC has not been reported in in vivo studies. Herein, we investigated the effect of AITC on angiogenesis and inflammation in a mouse model of colitis. Experimental colitis was induced in mice by administering 3% dextran sulfate sodium via drinking water. To monitor the activity of AITC in this model, we measured body weight, disease activity indices, histopathological scores, microvascular density, myeloperoxidase activity, F4/80 staining, inducible nitric oxide synthase (iNOS) expression, cyclooxygenase-2 (COX-2) expression, and vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR2) expression in the mice. We found that AITC-treated mice showed less weight loss, fewer clinical signs of colitis, and longer colons than vehicle-treated mice. AITC treatment also significantly lessened the disruption of colonic architecture that is normally associated with colitis and repressed the microvascularization response. Further, AITC treatment reduced both leukocyte recruitment and macrophage infiltration into the inflamed colon, and the mechanism these activities involved repressing iNOS and COX-2 expression. Finally, AITC attenuated the expression of VEGF-A and VEGFR2. Thus, AITC may have potential application in treating conditions marked by inflammatory-driven angiogenesis and mucosal inflammation.     

Antifungal effect of CopA3 monomer peptide via membrane-active mechanism and stability to proteolysis of enantiomeric d-CopA3

In our previous study, coprisin, a 43-mer defensin-like peptide, was derived from the dung beetle, Copris tripartitus, and a 9-mer CopA3 (monomer), truncated coprisin analog peptide, was designed. However, the antifungal effects of CopA3 are not known yet. In this study, the antifungal activity and mechanism of CopA3 were investigated and to develop a more effective antimicrobial peptide under physiological conditions, the enantiomeric d-CopA3 was designed. l- and d-CopA3 had a similar antifungal activity without chiral selectivity, and their activity was more potent than that of melittin used as a positive control. Furthermore, l- and d-CopA3 did not even show any hemolysis against human erythrocytes. Membrane studies using propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)], suggested that the antifungal effect of l- and d-CopA3 was due to the membrane-active mechanism, by contrast with coprisin possessing apoptotic mechanism without membrane permeabilization. Finally, the proteolytic resistance and antifungal activity of l- and d-CopA3 against trypsin was analyzed by HPLC and colony count assay. The results showed that only d-CopA3 maintained a potent antifungal activity despite the proteolytic condition. Therefore, this study suggests that d-CopA3 has potential as a novel antimicrobial agent.