Generation of Rat Monoclonal Antibody for Human IQGAP1 by Immunization of Three-Dimensional-Cultured Cancer Cells.

The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.

Isolation and characterization of bone marrow-derived mesenchymal stem cells in Xenopus laevis.

Mesenchymal stem cells (MSCs) are multipotent cells that exist in mesenchymal tissues such as bone marrow and are able to differentiate into osteocytes, chondrocytes, and adipocytes. MSCs are generally collected as adherent cells on a plastic dish, and are positive for markers such as CD44, CD73, CD90, CD105 and CD166, and negative for CD11b, CD14, CD19, CD31, CD34, CD45, CD79a and HLA-DR. MSCs have been established from many kinds of mammals, but MSCs from amphibians have not yet been reported. We cultured adherent cells from the bone marrow of Xenopus laevis by modifying the protocol for culturing mammalian MSCs. The morphology of these cells was similar to that of mammalian MSCs. The amphibian MSCs were positive for cd44, cd73, cd90 and cd166, and negative for cd11b, cd14, cd19, cd31, cd34, cd45, cd79a and hla-dra. Moreover, they could be induced to differentiate into osteocyte-, chondrocyte-, and adipocyte-lineage cells by cytokine induction systems that were similar to those used for mammalian MSC differentiation. Thus, they are considered to be similar to mammalian MSCs. Unlike mammals, amphibians have high regenerative capacity. The findings from the present study will allow for future research to reveal how Xenopus MSCs are involved in the amphibian regenerative capacity and to elucidate the differences in the regenerative capacity between mammals and amphibians.

Probabilistic exposure assessment of aggregate rates of dermal exposure of Japanese women and children to parabens in personal care products.

Parabens (p-hydroxybenzoic acids) are commonly used as preservatives in personal care products. Although the rate of exposure to a single product may be small, it is possible for an individual to have marked exposure to parabens through the use of multiple personal care products (aggregate exposure). To assess the risks associated with aggregate exposure to parabens, we estimated the dermal exposure rate distributions of four major parabens (methylparaben, MP; ethylparaben, EP; propylparaben, PP; butylparaben, BP) in various products for women (>20 years old) and children (1-3 years old) by using the probabilistic exposure assessment tool ConsExpo. Integrated exposure rates were then calculated as the sum of exposure rates for individual products. Aggregate exposure rates for women were 1.2 (median) (0.13 [5%ile], 6.9 [95%ile]), 0.43 (0.029, 3.0), 0.35 (0.032, 1.9), and 0.25 (0.027, 1.2) mg kg-bw-1 day-1 for MP, EP, PP, and BP, respectively. Those for children were 0.47 (0.054, 2.2), 0.11 (0.012, 0.60), 0.13 (0.012, 0.78), and 0.13 (0.0065, 0.85) mg kg-bw-1 day-1 for MP, EP, PP, and BP, respectively. Integrated exposure rates for women were several times those for children. In both cases, personal care products that were applied to larger areas of skin and were used more frequently were more likely to be associated with higher exposure rates. According to the results of a risk assessment using a margin of exposure approach, aggregate rates of exposure to PP and BP, but not MP or EP, were high enough to warrant concern about disruption of the reproductive system.

Serum Nardilysin, a Surrogate Marker for Epithelial-Mesenchymal Transition, Predicts Prognosis of Intrahepatic Cholangiocarcinoma after Surgical Resection.

PURPOSE: Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC.Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing. RESULTS: High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial-mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC. CONCLUSIONS: Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

A phenomenological reaction kinetic model for Cu removal from aqueous solutions by zero-valent iron (ZVI).

Zero-valent iron (ZVI) being an inexpensive and eco-friendly catalyst has drawn great attention in removal of heavy metals from wastewaters. However, quantitative understandings of ZVI processes are significantly deficient. To compensate for the lack of quantitative analyses of removal of heavy metals by ZVI, a phenomenological reaction kinetic model was newly developed for removal of Cu chosen as a typical heavy metal from acidic aqueous solutions by ZVI. The novel kinetic model is based on the adsorption of Cu2+ and H+ onto ZVI surface and subsequent Cu2+ reduction on ZVI surface and Fe2+ elution from ZVI. Batch experiments were conducted to elucidate effects of pH and Cu loading on Cu removal by ZVI in acidic aqueous solutions and to validate the proposed phenomenological reaction kinetic model. The quick and complete removals of 1.57 mM Cu were established in the rage of pH 2-5. Although the maximum Cu removal rate was obtained at pH 4, effects of pH were insignificant. In the range of Cu loading from 0.393 to 4.72 mM, almost complete Cu removals were obtained at pH 4 within 35 min. The changes in concentrations of Cu2+, Fe2+, H+ and dissolved oxygen were strongly linked with each other. They could be successfully simulated by the proposed model with the average correlation coefficient of 0.979. The capability of the phenomenological reaction kinetic model for dynamic simulation of Cu removal by ZVI under acidic conditions was confirmed.

Removal of antibiotic sulfamethoxazole by zero-valent iron under oxic and anoxic conditions: Removal mechanisms in acidic, neutral and alkaline solutions.

Removal of antibiotic sulfamethoxazole (SMX) by zero-valent iron (ZVI) was examined in the range of pH from 3.0 to 11.0 under oxic and anoxic conditions to clarify mechanisms of SMX removal in acidic, neutral and alkaline solutions. SMX removal was affected by solution pH and related to the speciation of SMX. Under the oxic condition, the maximums of SMX removal efficiency and rate were obtained at pH 3.0. The SMX removal efficiency decreased from 100 to 32% with increasing pH in the acidic solutions (3 â‰¦ pH â‰¦ 5) and increased to 88% in neutral and moderately alkaline solutions (6 â‰¦ pH â‰¦ 10). In highly alkaline solution (pH = 11), the SMX removal was significantly suppressed due to the formation of passive layer on ZVI surface. The removal rate of SMX under the oxic condition significantly declined with increasing pH. Under the anoxic condition, SMX removal was completed within 300 min in the acidic solutions and remained to less than 70% after 300 min in neutral and moderately alkaline solutions. For pH â‰§ 10, no SMX removal practically occurred. The removal rate of SMX under the anoxic condition approximately remained constant in the acidic solution and largely decreased in neutral and moderately alkaline solutions. SMX removal by ZVI was found to be dominated by the reductive degradation and adsorption under both the oxic and anoxic conditions. It was concluded that ZVI has the potential for effective removal of antibiotic SMX under the oxic and anoxic conditions. A kinetic model could reasonably simulate the dynamic profiles of SMX removal.

Cells from subcutaneous tissues contribute to scarless skin regeneration in Xenopus laevis froglets.

BACKGROUND: Mammals cannot regenerate the dermis and other skin structures after an injury and instead form a scar. However, a Xenopus laevis froglet can regenerate scarless skin, including the dermis and secretion glands, on the limbs and trunk after skin excision. Subcutaneous tissues in the limbs and trunk consist mostly of muscles. Although subcutaneous tissues beneath a skin injury appear disorganized, the cellular contribution of these underlying tissues to skin regeneration remains unclear. RESULTS: We crossed the inbred J strain with a green fluorescent protein (GFP)-labeled transgenic Xenopus line to obtain chimeric froglets that have GFP-negative skin and GFP-labeled subcutaneous tissues and are not affected by immune rejection after metamorphosis. We found that GFP-positive cells from subcutaneous tissues contributed to regenerating the skin, especially the dermis, after an excision injury. We also showed that the skin on the head, which is over bone rather than muscle, can also completely regenerate skin structures. CONCLUSIONS: Cells derived from subcutaneous tissues, at least in the trunk region, contribute to and may be essential for skin regeneration. Characterizing the subcutaneous tissue-derived cells that contribute to skin regeneration in amphibians may lead to the induction of cells that can regenerate complete skin structures without scarring in mammals. Developmental Dynamics 246:585-597, 2017. © 2017 Wiley Periodicals, Inc.