Current status of pan-Arctic terrestrial ecosystem and its possible changes

第6回極域科学シンポジウム特別セッション：[S] 北極温暖化とその影響 ―GRENE 北極気候変動プロジェクトと新しい方向性―11月18日（水）　国立極地研究所 2階 大会議

Optical second-harmonic spectroscopy of Au(887) and Au(443) surfaces

In order to investigate the electronic states of step sites on Au surfaces, we have observed the reflected optical second-harmonic (SH) intensity from Au(887) and Au(443) surfaces in ultrahigh vacuum with a normal incident excitation light beam, as a function of the photon energy and the incident and SH light polarizations. The second-order surface nonlinear susceptibility elements observed in this measurement configuration were X^_, X^_ and X^_, where x and y are defined as the [112^^-] and [1^^-10] directions, respectively. The step edges lie in the y direction. The ratios of the nonlinear susceptibility elements |X^_|/|X^_| and |X^_|/|X^_| were different on the two surfaces in the photon energy range from 2ħω=2.5 to 3.3 eV. The deviation of the SH response from that of an ideal 3m symmetric Au(111) surface was found to be larger for the Au(443) surface than for the Au(887) surface. This deviation is attributed to the atomic steps created by the miscut of the samples. In order to analyze the observed SH spectra, we calculated the electronic states of a Au(554) slab using a density-functional theory. We found that the low-energy onset of the SH intensity caused by the steps can be qualitatively interpreted by referring to the calculated partial density of the d-electronic states of the step and terrace atoms on the Au slab

Short Hairpin RNA Library-Based Functional Screening Identified Ribosomal Protein L31 That Modulates Prostate Cancer Cell Growth via p53 Pathway

<div><p>Androgen receptor is a primary transcription factor involved in the proliferation of prostate cancer cells. Thus, hormone therapy using antiandrogens, such as bicalutamide, is a first-line treatment for the disease. Although hormone therapy initially reduces the tumor burden, many patients eventually relapse, developing tumors with acquired endocrine resistance. Elucidation of the molecular mechanisms underlying endocrine resistance is therefore a fundamental issue for the understanding and development of alternative therapeutics for advanced prostate cancer. In the present study, we performed short hairpin RNA (shRNA)-mediated functional screening to identify genes involved in bicalutamide-mediated effects on LNCaP prostate cancer cells. Among such candidate genes selected by screening using volcano plot analysis, ribosomal protein L31 (RPL31) was found to be essential for cell proliferation and cell-cycle progression in bicalutamide-resistant LNCaP (BicR) cells, based on small interfering RNA (siRNA)-mediated knockdown experiments. Of note, <i>RPL31</i> mRNA is more abundantly expressed in BicR cells than in parental LNCaP cells, and clinical data from ONCOMINE and The Cancer Genome Altas showed that RPL31 is overexpressed in prostate carcinomas compared with benign prostate tissues. Intriguingly, protein levels of the tumor suppressor p53 and its targets, p21 and MDM2, were increased in LNCaP and BicR cells treated with <i>RPL31</i> siRNA. We observed decreased degradation of p53 protein after <i>RPL31</i> knockdown. Moreover, the suppression of growth and cell cycle upon <i>RPL31</i> knockdown was partially recovered with <i>p53</i> siRNA treatment. These results suggest that RPL31 is involved in bicalutamide-resistant growth of prostate cancer cells. The shRNA-mediated functional screen in this study provides new insight into the molecular mechanisms and therapeutic targets of advanced prostate cancer.</p></div

p53 partially mediates the function of RPL31 in BicR cells.

<p>(A) Knockdown effects of RPL31 and p53 on MDM2 and p21 mRNA. BicR cells were transfected with siRPL31, sip53, siRPL31 plus sip53, or siLuc. qRT-PCR for RPL31, p53, MDM2, and p21 mRNAwas performed. Experiments were performed in triplicate; mRNA expression is normalized to GAPDH and shown as mean ± s.d. (n = 3; **, <i>P</i><0.01). (B) sip53 partially cancelled the repression of cell growth induced by siRPL31. BicR cells were transfected with 10 nM each siRPL31, sip53, siRPL31 plus sip53 or siLuc, and cultured with the medium containing 1 µM bicalutamide. WST-8 cell proliferation assay was performed at the indicated time points. The absorbances of the wells in the plates were measured using a microplate reader at a 450 nm. Data are presented as mean ± s.d. (n = 4; **, <i>P</i><0.01).</p

Effects on bicalutamide-resistant BicR cell growth by treatment with selected siRNAs that target genes determined by shRNA screening.

<p>Growth inhibition of BicR cells by siRNA targeting <i>RPL31</i> (siRPL31), <i>HIST1H2BD</i> (siHIST1H2BD), and <i>ADAMTS1</i> (siADAMTS1) was shown. Cells were transfected with 10 nM siRNA in culture medium. Twelve hours after transfection, cells were then further cultured in medium containing 1 µM bicalutamide. WST-8 cell proliferation assays were performed at the indicated time points after transfection. The absorbance of the wells in the plates was measured using a microplate reader at 450 nm. Data are presented as mean ± s.d. (n = 3; *, <i>P</i><0.05; **, <i>P</i><0.01).</p

The list of genes targeted by shRNAs exhibiting bicalutamide-mediated downregulation in lentiviral library-transduced LNCaP cells and the knockdown efficiency of each siRNA chosen for validation.

a)<p>Silencer select pre-designed siRNAs targeting the candidate genes were obtained from Ambion.</p>b)<p>Knockdown efficiency of siRNA was determined by qRT-PCR and normalized to that of siControl.</p><p>The list of genes targeted by shRNAs exhibiting bicalutamide-mediated downregulation in lentiviral library-transduced LNCaP cells and the knockdown efficiency of each siRNA chosen for validation.</p

<i>RPL31</i> is overexpressed in BicR cells and clinical prostate cancer tissues.

<p>(A) Expression levels of <i>RPL31</i>, <i>HIST1H2BD</i>, and <i>ADAMTS1</i> mRNA evaluated by quantitative reverse-transcription PCR analysis (qRT-PCR) with gene-specific primers. Data are normalized to <i>GAPDH</i> and shown as mean ± s.d. (n = 3; **, <i>P</i><0.01). (B) <i>RPL31</i> mRNA is abundantly expressed in clinical prostate carcinoma tissues compared with normal prostate tissues (by > 2-fold), as retrieved from datasets by Tomlins <i>et al.</i> in the ONCOMINE database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108743#pone.0108743-Rhodes1" target="_blank">[30]</a>. Normal: normal prostate tissue, PCa: prostate cancer, PIN: prostatic intraepithelial neoplasia. (C) <i>RPL31</i> mRNA expression is elevated in clinical prostate cancer samples <i>versus</i> normal samples in a study of RNA-sequencing in The Cancer Genome Analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108743#pone.0108743-Chin1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108743#pone.0108743-Chin2" target="_blank">[32]</a>.</p

RPL31 regulates levels of p53 protein expression.

<p>(A) Knockdown of RPL31 increases p53, MDM2, and p21 protein expression. LNCaP and BicR cells were transfected with siRPL31 or siLuc for 48 h. Cell extracts were subjected to SDS-PAGE and western blot analysis using the indicated antibodies. (B) RPL31 regulated the degradation of p53 protein. BicR cells were transfected with siRPL31 or siLuc for 60 h and treated with 50 µg/mL cycloheximide (CHX) for the indicated time. Cell extracts were analyzed by western blotting. (C) p53 protein levels were quantified by densitometry and normalized to the levels of the corresponding β-actin protein and shown as mean ± s.d. (n = 3; *, <i>P</i><0.05).</p