<原著>培養軟骨細胞の分化機能発現と細胞増殖動態に関する実験的研究

The present study was undertaken to investigate the relationship among cell morphology, proliferation, and maturation of chondrocytes in primary cultures. Chondrocytes were isolated from the growth cartilages of the rat ribs and cultured for 6 days. In situ DNA cytofluorometry using an inverted epi-illumination cytofluorometer (Nikon P1-I) and 3H-thymidine autoradiography were carried out for the correlated analysis of cell morphology and proliferation. Cytoskeletal staining with fluorescent phalloidin and 35S-sulphate autoradiography were also performed. In addition, in situ hybridization to c-myc mRNA was carried out using DNA probe. According to the results obtained, the cultured chondrocytes were composed of mixed populations of large, polygonal cells and of small, round cells. The round cells showed a significantly higher 35S uptake than the polygonal cells. The cytoskeletal staining clearly revealed stress fibers in the cytoplasm of the polygonal cells, whereas only a fine filamentous structure was shown in the cytoplasm of the round cells. In situ DNA cytofluorometry clearly demonstrated that cell proliferative activity was high in the polygonal cells and low in the round cells. In addition, 3H-thymidine autoradiography with cumulative labeling method revealed that the polygonal cells were changing into the small, round cells. C myc mRNA signals were detected in the cytoplasm of over a half of the round cells, whereas no evidence of c-myc expression were found in the polygonal cells. From these results, it appears that as the shape of the cultured chondrocytes shifts from polygonal to round, the cell proliferative activity decreases in association with cell differentiation. It was also suggested that c-myc mRNA is amplified in the well differentiated round chondrocytes, and not in the proliferative polygonal cells.従来の培養軟骨細胞を用いた研究から, 軟骨細胞の形態と分化機能発現の聞には, 関連性のあることが示されている. 著者らは, 成長軟骨細胞の培養系において細胞形態, 機能が明らかに異なっている2種類の細胞が存在することを見いだした. 本研究では, この培養系を用い, 軟骨細胞の形態・細胞増殖動態・分化機能発現の3者の関連性を総合的に把握することを目的とした. このための方法論として, 細胞形態別増殖動態解析には, in situ DNA 顕微蛍光測光法と3_H-サイミジンオートラジオグラフィーを行い, 分化機能の検索には35_S オートラジオグラフィーを用いた. また, FITC-ファロイジン染色法により, 軟骨細胞の形態と細胞骨格の関係についても調べた. 更に, 本研究では, 悪性腫瘍以外に, 胎生期の細胞や分化途上の細胞にも出現し, 細胞の分化・増殖に深く関係があると考えられている c-myc 遺伝子の発現の有無を, in situ DNA- mRNA hybridization 法を用いて検索した. 実験には, ラット肋軟骨から分離・培養した成長軟骨細胞を用いた. 培養開始4 - 6日目頃の成長軟骨細胞は, 大型多角形の扁平な胞体を持ち, 大きな核を有する細胞(以下, 多角形細胞と略す)と, 比較的小型で類円形ないし球状の胞体と小さな核を有する細胞(以下, 円形細胞と略す)の2種類の細胞から構成されていた. in situ DNA 顕微蛍光測光法による細胞増殖動態解析の結果, 多角形細胞は, 活発な増殖性を示す2倍体細胞と少数の4倍体から構成されているのに対し, 円形細胞は, ほとんど増殖活性を持たない2倍体細胞から構成されていることが判った. 3_H-サイミジンの30分標識の結果から, 多角形細胞の標準率は11%, 円形細胞の標識率は0. 5%であり, その標識率の経時的変化はほとんど認められなかった. 3_H-サイミジンの持続標識実験の結果から, 多角形細胞が円形細胞に形態的に変化することが示唆された. また, 35_S オートラジオグラフィーより, 多角形細胞は, 軟骨基質の産生能が低く, 他方, 円形細胞では, 基質産生が亢進していることがわかった. FITC-ファロイジン染色によるアクチンの細胞内分布パタンを, 両細胞で比較したところ, 多角形細胞ではストレスファイバーがよく発達しているのに対し, 円形細胞には, 分断された線維性構造のみが観察された. 以上の結果をまとめると, 培養軟骨細胞の形態・増殖・分化の3者の間には, たがいに密接な関連が有り, 多角形細胞から円形細胞への形態変化に伴って, 増殖活性が低下し, 分化機能が発現されることが判明した. 次に, c-myc 遺伝子の発現の有無を in situ hybridization 法を用いて検索したところ, 円形細胞の過半数に, c-myc mRNA のシグナルが検出された. このことから, 軟骨細胞では, 分化機能発現と関連して c-myc 遺伝子が発現される可能性が示唆された

Burkholderia insecticola sp. nov., a gut symbiotic bacterium of the bean bug Riptortus pedestris

A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64(T), was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei Glade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314(T) (100%), Burkholderia turbans LMG 29316(T) (99.52%) and Burkholderia ptereochthonis LMG 29326(T) (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome BLAST Distance Phylogeny confirmed B. peredens LMG 29314(T), B. ptereochthonis LMG 29326(T) and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64(T) represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C-16:0, C-18:1 omega 7c and C-17:0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64(T) from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64(T) is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64(T) (=NCIMB 15023(T)=JCM 31142(T))

Usefulness of measuring hepatic functional volume using technetium-99m galactosyl serum albumin scintigraphy in hilar bile duct carcinoma

This case involved a 75-year-old woman with obstructive jaundice who was diagnosed with hilar bile duct carcinoma. After endoscopic retrograde biliary drainage, the total bilirubin level was normalized. The indocyanine green test retention rate at 15 min (ICGR15) was 26%. The liver uptake ratio (LHL15) by technetium-99m galactosyl human serum albumin (99mTc-GSA) liver scintigraphy was 0.87. Left hepatectomy was scheduled by CT volumetry. However, biliary drainage was insufficient, and the functional liver volume showed functional deterioration of the left liver. After percutaneous transhepatic biliary drainage, future remnant liver volume by 99mTc-GSA liver scintigraphy changed to 52% from 42%, and ICGR15 and LHL15 were improved to 16% and 0.914, respectively. Scheduled left hepatectomy was performed following the results of functional liver volume. The measurement of functional volume by 99mTc-GSA liver scintigraphy provides useful information with respect to segmental liver function for deciding operative indications

Intraductal papillary growth of liver metastasis originating from colon carcinoma in the bile duct: report of a case.

Morphologically, liver metastases from colorectal carcinoma usually form as nodular tumor masses, whereas intraductal papillary growth in the bile duct is rare. A 65-year-old man underwent right hemicolectomy for advanced colon carcinoma, and histology of the primary carcinoma confirmed moderately differentiated adenocarcinoma with subserosal invasion, no vascular infiltration, and no lymph node metastasis. A liver tumor was found in the right paramedian Glisson pedicle and intraductal growth of cholangiocarcinoma was seen on imaging. We performed right hepatectomy and macroscopically, the resected specimen contained a growth in the bile duct lumen similar to cholangiocarcinoma. Histological examination revealed intraductal papillary proliferation of well-differentiated adenocarcinoma without vascular infiltration or lymph node metastasis in the hepatic hilum. Immunohistochemical staining revealed that the tumor cells were negative for cytokeratin-7 and positive for cytokeratin-20. Based on these findings, liver metastasis from colon carcinoma was diagnosed. Liver metastasis from colorectal carcinoma rarely arises as intraductal papillary growth in the bile duct, but the possibility of liver metastases with unusual morphology must be borne in mind for patients with a history of carcinoma in the digestive tract

Relationship of hepatic functional parameters with changes of functional liver volume using technetium-99m galactosyl serum albumin scintigraphy in patients undergoing preoperative portal vein embolization: a follow-up report.

To identify predictors of changes in functional hepatic volumes after portal vein embolization (PVE) before hepatectomy, we examined the relationship between hepatic functional parameters and changes in functional volume of the embolized and non-embolized liver based on a previous volumetric analysis

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 10(4 )cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34(+ )cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Seismic Exploration Using Active Sources at Kuchierabujima Volcano, Southwest Japan

Seismic exploration using artificial sources was conducted at Kuchierabujima volcano, southwest Japan in November 2004 by 40 participants from 9 national universities andJapan Meteorological Agency to investigate the subsurface seismic structure. The exploration was the 11th joint experiment under the National Project for Prediction of Volcanic Eruptions. A total of 183 temporal stations equippedwith a 2 Hz vertical component seismometer (including 75 3component seismometers) and a portable data logger were deployed on Kuchierabu Island. Dynamite shots with charges of 10-115 kg were detonated at 19 locations, and seismic signals were successfully recorded. To reveal the P-wave velocity structure, 2955 arrival times of the first motion were picked from the seismograms, and 2187 were classified into ranks A and B. From the record sections and the arrival time data, characteristics reflecting the geological structure were identified. Refracted waves of 5 km/s were observed at stations＞5km from the shot points. Apparent velocities near the shot points depend on the surface geology around the shots. P-wave arrived earlier at stations near the summits. Strongly scattered waves were observed similarly near the summits