Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of K(ATP) channels

BACKGROUND: Phorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic β-cells by using patch-clamp techniques. RESULTS: In current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 μM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized β-cells in a concentration-dependent manner (0.8–240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K(+) channel (K(ATP)) activity. Based on inside-out patch-clamp recordings, PMA had little effect on K(ATP) activity if no ATP was in the bath, while PMA restored K(ATP) activity that was suppressed by 10 μM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 μM), PMA did not increase, but rather reduced insulin secretion. CONCLUSION: In rat pancreatic β-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing β-cell excitability in a PKC-independent manner. The enhancement of K(ATP) activity by reducing sensitivity of K(ATP) to ATP seems to underlie the PMA-induced impairment of β-cells electrical excitation in response to glucose stimulation

当科において過去9年間（2003年～2012年）に治療を行った歯原性腫瘍75例の臨床的検討

2003年から2012年までの9年間に当科で診断・治療を行った歯原性腫瘍75症例について，2005年WHO分類に基づいて臨床的検討を行った．疾患別症例数は角化嚢胞性歯原性腫瘍が22例，エナメル上皮腫16例，歯牙腫16例，骨性異形性症13例，線維性異形性症3例，石灰化嚢胞性歯原性腫瘍2例，歯原性粘液腫，歯原性線維腫，腺様歯原性腫瘍が各1例であった．男女比は1.5対1であった．手術時年齢は，平均39.8歳であった．発生部位別症例数は，角化嚢胞性歯原性腫瘍，エナメル上皮腫は下顎大臼歯部に多く，歯牙腫は，上顎前歯部に多かった．治療方法は，角化嚢胞性歯原性腫瘍に対しては摘出後閉鎖7例，摘出後開放7例，生検後開窓5例，開窓後摘出3例であった．エナメル上皮腫は摘出後開放8例，区域切除3例，辺縁切除3例，開窓1例，開窓後摘出1例であった．歯牙腫16例に対しては摘出術を施行した．治療成績は75例中71例（94.7％）は再発を認めなかったが，一次症例で，エナメル上皮腫1例，角化嚢胞性歯原性腫瘍1例，歯原性粘液腫1例の3症例，他院で治療後のエナメル上皮腫1例に再発が認められた

The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy.

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression

ミトコンドリア DNA ハプロタイプ ブンセキ ニ ヨル グンマケン ツキノワグマ シュウダン ノ イデンテキ タヨウセイ

日本国内でツキノワグマ(Ursus thibetanus)は本州,四国に生息し,現在5地域の個体群が絶滅の恐れのある地域集団とされている。群馬県でもツキノワグマが生息しているが,その捕獲頭数を定めた群馬県ツキノワグマ適正管理計画は,地域集団の構成を考慮しないまま実施されており,このままでは絶滅を招く危険性をはらんでいる。このことから,ツキノワグマの適切な保全を考慮した農林業被害等の防止対策を実施することが,希少野生動物とともに暮らす地域にとって重要な課題といえる。そこで本研究は群馬県ツキノワグマの遺伝的多様性を明らかにするため,群馬県で捕獲されたツキノワグマ30個体のミトコンドリアDNA D-loop領域706bpの配列を決定し,ハプロタイプ分析を行った。その結果,群馬県のツキノワグマから6つのハプロタイプを同定した。これらは先行研究により東日本に生息するツキノワグマで同定された38ハプロタイプのうち,E01, E06, E10, E11, E31, E34に該当した。ハプロタイプの地理的分布および集団構造解析から,群馬県では南西部集団,中之条集団,北東部集団の3集団が存在する可能性が示唆された。群馬県中央部から南東部にかけては平野が広がっており,ツキノワグマの生息は確認されていない。よって群馬県のツキノワグマ3集団は群馬県の西から東へ南西部集団,中之条集団,北東部集団の順に並んで存在していると思われる。つまり,中之条集団の西側で南西部集団と分かれる境界線があり,東側で北東部集団と分かれる境界線が本研究によって想定された。これらは適正管理計画のもとで人為的に設定された地域個体群(越後・三国地域個体群と関東山地個体群)とは異なる境界分布を示しており,今後ツキノワグマの自然集団を繁栄した適切な保全計画を実施するためにも現在の分布境界線を見直していく必要があることを本研究は提唱する。Asiatic black bears living in Gunma Prefecture, Japan, have been divided into two populations (Echigo-Mikuni and Kanto mountain populations) based on the Asiatic Black Bear Population Management Plan. This management plan puts a ceiling on the number of bears captured in each population. However, for convenience, division of the two populations is based on an administrative boundary, and does not represent the natural population structure of this species in Gunma. Accordingly, to clarify the population structure of the species in Gunma, we performed mitochondrial haplotype analysis using 30 individuals captured in the prefecture. We identified six haplotypes, which corresponded to the haplotypes previously discovered in this species in eastern Japan. On the basis of the geographical distribution of the haplotypes and population structure analyses, we suggest that three populations of Asiatic black bear exist in Gunma Prefecture : the Southwestern, Nakanojo, and Northeastern populations. The Nakanojo population appears to be relatively small and genetically unique in Gunma. We identified two borders among these populations. One is situated between the Southwestern and Nakanojo populations, and the second is situated between the Nakanojo and Northeastern populations. These borders clearly differ from the single border drawn between the Echigo-Mikuni and Kanto mountain populations. We conclude that the current management plan does not reflect the natural population structure of these bears and suggest a revision of the plan based on the existence of three discrete populations

Semi-quantitative analyses of metabolic systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG mice

Analyses of energy metabolism in human cancer have been difficult because of rapid turnover of the metabolites and difficulties in reducing time for collecting clinical samples under surgical procedures. Utilization of xenograft transplantation of human-derived colon cancer HCT116 cells in spleens of superimmunodeficient NOD/SCID/IL-2Rγnull (NOG) mice led us to establish an experimental model of hepatic micrometastasis of the solid tumor, whereby analyses of the tissue sections collected by snap-frozen procedures through newly developed microscopic imaging mass spectrometry (MIMS) revealed distinct spatial distribution of a variety of metabolites. To perform intergroup comparison of the signal intensities of metabolites among different tissue sections collected from mice in fed states, we combined matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI–TOF-IMS) and capillary electrophoresis–mass spectrometry (CE–MS), to determine the apparent contents of individual metabolites in serial tissue sections. The results indicated significant elevation of ATP and energy charge in both metastases and the parenchyma of the tumor-bearing livers. To note were significant increases in UDP-N-acetyl hexosamines, and reduced and oxidized forms of glutathione in the metastatic foci versus the liver parenchyma. These findings thus provided a potentially important method for characterizing the properties of metabolic systems of human-derived cancer and the host tissues in vivo

ゼツヘンペイジョウヒガン ニ オケル センチネル リンパセツ セイケン ニ カン スル ケンキュウ : OSNA ホウ ニ ヨル イデンシ カイセキ ノ ユウヨウ セイ ニ ツ イテ

癌においてリンパ節転移は,重要な予後因子であることより,正確な診断が必要となる.そこで,より正確なリンパ節転移診断を行う方法として,センチネルリンパ節(sentinel lymph node:SLN) 生検が導入され,また,さらなる診断精度の向上を図るために遺伝子解析による転移診断が応用され,その有用性が報告されている.しかしながら,口腔癌のSLN 生検における遺伝子解析は,従来の病理組織学的診断に比べ精度が高いとする報告がある一方,ほぼ同等とする報告もあり,なお一定の見解が得られていない.本研究では,舌扁平上皮癌に対するSLN 生検における遺伝子解析の有用性について検討を行った.対象は,SLN 生検を施行した舌扁平上皮癌StageI,II症例28 例で,病理組織学的診断ならびにone-step nucleic acid amplification(OSNA) 法による遺伝子解析を用いてSLN における転移診断を行い,それぞれを比較検討し,さらに治療成績について,SLN 生検導入前のStage I,II症例23 例と比較検討した.その結果,病理組織学的診断にてリンパ節転移陽性と診断された症例は28 例中5 例(17.9%)で,そのうちの1 例は,当初病理組織学的診断では転移陰性であったが,遺伝子解析にて転移陽性と診断されたため,亜連続切片にて再度検索を行い,病理学的にも転移陽性と診断されたものであった.OSNA 法による遺伝子解析では28 例中6 例(21.4%) が転移陽性で,病理組織診断との一致率は,27 例(96.4%)であった.5 年累積生存率は,SLN 生検施行群94.4%,SLN 生検未施行群86.7%で,有意差はないもののSLN 生検施行群に高い傾向を示した.後発転移は,SLN 生検施行群にはなかったが,SLN 生検未施行群では23 例中3 例(13.0%) に認められ,有意に多かった.以上の結果より,舌扁平上皮癌においてもSLN 生検は有用で,OSNA 法による遺伝子解析は,その診断精度を高めると思われた.Background:Metastasis of cervical lymph node is the most important prognostic factor in oral cancer. In this study, we evaluated the efficacy of one-step nucleic acid amplification (OSNA) assay for intraoperative diagnosis of sentinel lymph node (SLN) metastasis compared with conventional examination in patients with carcinoma of the tongue. The aim of this study was to investigate availability of the genetic diagnosis in carcinoma of the tongue.Patients and Methods:We investigated patients with T1 and T2 squamous cell carcinoma of the tongue with a clinical diagnosis of N0 who underwent SLN biopsy (SLN group;n=28) in comparison with those who did not undergo SLN biopsy (non-SLN group;n=23). SLN biopsy consisted of not only intraoperative pathological examination, but also genetic analysis using the OSNA method.Results:Lymph node metastasis was detected in five of 28 patients in the SLN group( 17.9%). One discordant case was observed between pathological examination and OSNA assay. This case was positive metastatic lymph node according OSNA assay, but negative lymph node at pathological examination. Subsequent cervical lymph node metastasis was not seen in the SLN group, it was seen three patients in the non-SLN group( 13.0%). Five-year survival rate was higher in the SLN group (94.4 %) than the non-SLN group( 86.7%), but this difference was not significant.Conclusion:These results suggest that in early oral cancer, SLN biopsy is highly useful for detecting lymph node metastasis. The high accurate OSNA method can be used in intraoperative diagnosis as a tool for detecting metastasis in the sentinel lymph nodes of tongue carcinoma

Chaotic electrical activity of living beta-cells in the mouse pancreatic islet

To test for chaotic dynamics of the insulin producing beta-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the beta-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations. (c) 2006 Elsevier B.V All rights reserved

2-Aminoethoxydiphenyl Borate Inhibits Agonist-Induced Ca2+ Signals By Blocking Inositol Trisphosphate Formation In Acutely Dissociated Mouse Pancreatic Acinar Cells

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca2+ signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca2+ signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca2+ oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 μM 2-APB completely abolished ACh-but not inositol trisphosphate (InsP3)-induced Ca 2+ oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP3 receptor activation. In addition, 100 μM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP3 receptors and blocking the store-operated Ca2+ pathway, high concentrations of 2-APB also inhibit agonist-induced Ca2+ signals by reducing InsP3 formation