Association of p62/SQSTM1 excess and oral carcinogenesis.

p62/SQSTM1 (sequestosome1) has never been evaluated in oral epithelium. In order to clarify the role of p62/SQSTM1 in carcinogenesis in oral epithelium, both p62/SQSTM1 and Nrf2 were immunohistochemically evaluated in 54 carcinomas and 14 low grade dysplasias. p62/SQSTM1 knockdowns were also designed in oral cancer cells, and we analyzed the Nrf2 pathway, GSH contents and ROS accumulation. The association between p62/SQSTM1 excess and prognosis was addressed in a clinical cohort of oral carcinoma cases. p62/SQSTM1 excess was more obvious in carcinomas, but Nrf2 was abundant in almost all samples of the oral epithelium. In oral carcinoma cells, p62/SQSTM1 knockdown did not affect the Nrf2-Keap1 pathway but did significantly reduce GSH content with subsequent ROS accumulation, and caused cell growth inhibition in the irradiated condition. Finally, p62/SQSTM1 excess was associated with poor prognosis in a clinical cohort. In oral epithelial carcinogenesis, p62/SQSTM1 excess played a role in GSH induction rather than Nrf2 accumulation, and may cause resistance to cytotoxic stresses such as radiation or chemotherapy. Immunohistochemical evaluation of p62/SQSTM1 may be a potential significant marker to identify early carcinogenesis, chemo-radiotherapeutic resistance or poor prognosis of oral squamous cell carcinomas.滋賀医科大学平成25年

Preparation of Mouse Monoclonal Antibody for RB1CC1 and Its Clinical Application

RB1-inducible coiled-coil 1 (RB1CC1; also known as FIP200) plays important roles in several biological pathways such as cell proliferation and autophagy. Evaluation of RB1CC1 expression can provide useful clinical information on various cancers and neurodegenerative diseases. In order to realize the clinical applications, it is necessary to establish a stable supply of antibody and reproducible procedures for the laboratory examinations. In the present study, we have generated mouse monoclonal antibodies for RB1CC1, and four kinds of antibodies (N1-8, N1-216, N3-2, and N3-42) were found to be optimal for clinical applications such as ELISA and immunoblots and work as well as the pre-existing polyclonal antibodies. N1-8 monoclonal antibody provided the best recognition of RB1CC1 in the clinico-pathological examination of formalin-fixed paraffin-embedded tissues. These monoclonal antibodies will help to generate new opportunities in scientific examinations in biology and clinical medicine

Using environmental DNA analyses to assess the occurrence and abundance of the endangered amphidromous fish Plecoglossus altivelis ryukyuensis

The Ryukyu ayu Plecoglossus altivelis ryukyuensis is an endangered amphidromous fish that inhabits rivers in the Ryukyu Archipelago (Japan). Populations of the species have declined dramatically. Consequently, the Ryukyu ayu has been registered as a natural monument in Japan and monitoring surveys with direct catching are restricted legally. This restriction, unfortunately, makes monitoring of population abundances difficult and creates a barrier to both advancing understanding of the species’ status and the development of appropriate conservation plans.We developed a non-invasive monitoring methodology using eDNA analyses. We designed a specific quantitative PCR assay for the Ryukyu ayu using the mitochondrial ND4 region. Using this primer/probe set, we conducted eDNA analyses in three rivers on Amami-Ohshima Island. The DNA fragments were amplified from the eDNA extracted from natural water in each river. The numbers of DNA fragments detected were positively correlated with individual counts of fish obtained by visual snorkelling surveys. Our method does not contravene restrictions and facilitates abundance monitoring of this endangered fish species

Immunoblot analysis of four monoclonal antibodies and pre-existing polyclonal antibodies.

<p>Four kinds of monoclonal antibodies (N1-8, N1-216, N3-2, and N3-42) showed strong immunoreactivity for ∼200 kDa full-length human RB1CC1 and little cross-reactivity with other proteins in Western blot analysis. Two kinds of pre-existing polyclonal antibodies (N1-poly and N3-poly) were used to the blotting control. Four µg of protein lysates of HEK293 and the cells overexpressing RB1CC1 were applied to the analysis. An arrowhead shows the position of ∼200 kDa full-length human RB1CC1.</p

CBB staining and immunoblot analysis of four kinds of monoclonal antibodies (N1-8, N1-216, N3-2, and N3-42).

<p>N1-8 did not recognize aa residues 25–254, but recognized aa residues 25–271 of N1. Therefore, N1-8 was considered to recognize an epitope located among aa residues 255–271 of RB1CC1. Similarly, N1-216, N3-2, and N3-42 recognized epitopes located among aa residues 235–254, 715–734, and 715–734, respectively. The recombinant mutant proteins (∼27–56 kDa) of N1 and N3 were confirmed by anti-GST immunoblots. Coomassie Brilliant Blue (CBB) staining of each protein band in polyacrylamide gel electrophoresis is shown in the lowest row. Arrows and arrowheads indicate 47 and 39 kDa, respectively.</p

Comparison of immunoreactivities between N1-8 monoclonal and N1 polyclonal anti-RB1CC1 antibodies in formalin-fixed paraffin-embedded breast cancer tissues.

<p>In non-neoplastic mammary ductal epithelium, cytoplasm and nuclei were similarly stained with these two antibodies. In breast cancer tissue samples, these two antibodies reacted similarly in all types of staining variation (I, negative staining in both cytoplasm and nuclei; II, positive staining only in cytoplasm; III, positive staining only in nuclei or in nuclei and cytoplasm). I–II and III were defined as RB1CC1 -negative and -positive, respectively, in the previous clinical cohorts. Bar, 100 µm.</p