Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules

Solubilization of glycosyl-phosphatidylinositol-anchored proteins in quiescent and stimulated neutrophils

AbstractIn human neutrophils, alkaline phosphatase (AlkPase), a low-affinity receptor for IgG (FcRIIIB), and complement decay accelerating factor (DAF) are glycosyl-phosphatidylinositol (GPI)-anchored proteins. Varying greatly in biological function these three integral membrane proteins exhibit regulated cell surface expression in neutrophils. Defined by their common membrane-linkage motif, AlkPase, FcRIIIB, and DAF can be released from the lipid bilayer by the action of phosphatidylinositol-specific phospholipase C and are relatively resistant to low temperature extraction with Triton X-100 (TX-100). In this study we show that neutrophil AlkPase, FcRIII, and DAF display differential extractibility; they are relatively insensitive to TX-100 solubilization at 4° C, but are readily extracted with TX-100 at 37° C or by the detergent octyl glucoside at 4° C. The differential extractibility of these GPI-anchored proteins is the same in unstimulated cells, where these proteins exist primarily in an intracellular pool, and stimulated cells, where they are expressed principally at the cell surface. However, no differential extraction effect is observed with two neutrophil transmembrane proteins, complement receptor 1 (CD35, CRl) and MHC Class I in either stimulated or unstimulated cells

FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy

S U M M A R Y We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for highresolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen

Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC

ホウシャセン チリョウ ガ コウカテキ デ アッタ チョクチョウナイ ブンピツ サイボウ ガン ノ 1 レイ

A 54-year-old woman was seen at the hospital because of anal bleeding. Colonoscopy examination showed a type 2 tumor in the lower rectum and the histological diagnosis from the biopsy specimen was undifferenciated adenocarcinoma. Abdomino-perineal amputation of the rectum were performed. The resected specimen was positive for synaptophysin stain immunohistochemically and we diagnosed it as an endocrine cell carcinoma. We underwent adjuvant chemotherapy by bolus CPT-11, and the post operative course was uneventful. Intra-pelvic recurrence was observed 15 months after operation, and further 4 months later, metastasis developed around the pancreas. And we noticed the tumor in supraclavicular region 21 months after operation. The tumor was sensitive to chemotherapy, but continued to progress. So we underwent radiotherapy for each lesion. The lesion treated with radiotherapy were controlled until she died of cancer, 31 months after the operation. As a result of autopsy, an exellent effect of radiotherapy was confirmed

Transdisciplinary Joint Seminar Studies: Conflits et Interprétations

This report details the research activity carried out from 2009 to date under the Transdisciplinary Joint Seminar Studies of the universities of Aoyama Gakuin, Franche-Comté and Tsukuba. Through study sessions and conferences as well as individual research papers, the issue of conflict and interpretation is addressed across the specific fields of study from the three distinct considerations of circulation, representation and language. 要旨本報告は2009年から現在まで「青山学院大学・フランシュコンテ大学・筑波大学異分野共同セミナー」において行われた研究活動をまとめたものである。分科会、講演、ならびに個々の論文において論じられた「循環」「表象」「言語」の考察を通じて、「衝突」と「解釈」をめぐる諸問題が検証される

Aclarubicin in the treatment of elderly patients with acute nonlymphocytic leukemia.

Thirteen previously untreated patients aged 70 and above with acute nonlymphocytic leukemia were treated with aclarubicin (ACR) alone. Among 10 cases (3, acute myelocytic leukemia; 4, acute myelomonocytic leukemia; 2, acute monocytic leukemia; and one, acute erythroleukemia) in which an evaluation was possible, 5 cases (3, acute myelomonocytic leukemia; and 2, acute monocytic leukemia) obtained complete remission (CR). The CR rate was 83% in 6 patients with acute myelomonocytic leukemia or acute monocytic leukemia. The median CR duration and survival was 7.5 and 10 + months, respectively. Although side effects of the drug on digestive system such as nausea, vomiting and anorexia were observed in all patients, they were controllable by conventional treatments. The results suggest that ACR is effective for the clinical management of elderly patients with acute nonlymphocytic leukemia, especially those with acute myelomonocytic leukemia or acute monocytic leukemia.</p

Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype

Low-Dose Intravenous Alteplase in Wake-Up Stroke

Background and Purpose—We assessed whether lower-dose alteplase at 0.6 mg/kg is efficacious and safe for acute fluid-attenuated inversion recovery-negative stroke with unknown time of onset. Methods—This was an investigator-initiated, multicenter, randomized, open-label, blinded-end point trial. Patients met the standard indication criteria for intravenous thrombolysis other than a time last-known-well >4.5 hours (eg, wake-up stroke). Patients were randomly assigned (1:1) to receive alteplase at 0.6 mg/kg or standard medical treatment if magnetic resonance imaging showed acute ischemic lesion on diffusion-weighted imaging and no marked corresponding hyperintensity on fluid-attenuated inversion recovery. The primary outcome was a favorable outcome (90-day modified Rankin Scale score of 0–1). Results—Following the early stop and positive results of the WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke), this trial was prematurely terminated with 131 of the anticipated 300 patients (55 women; mean age, 74.4±12.2 years). Favorable outcome was comparable between the alteplase group (32/68, 47.1%) and the control group (28/58, 48.3%; relative risk [RR], 0.97 [95% CI, 0.68–1.41]; P=0.892). Symptomatic intracranial hemorrhage within 22 to 36 hours occurred in 1/71 and 0/60 (RR, infinity [95% CI, 0.06 to infinity]; P>0.999), respectively. Death at 90 days occurred in 2/71 and 2/60 (RR, 0.85 [95% CI, 0.06–12.58]; P>0.999), respectively. Conclusions—No difference in favorable outcome was seen between alteplase and control groups among patients with ischemic stroke with unknown time of onset. The safety of alteplase at 0.6 mg/kg was comparable to that of standard treatment. Early study termination precludes any definitive conclusions