WT1 was first identified as a tumor suppressor involved in the development of Wilms' tumor. Recently, oncogenic properties of WT1 have been demonstrated in various hematological malignancies and solid tumors. Because WT1 has been identified as a molecular target for cancer immunotherapy, immunohistochemical detection of WT1 in tumor cells has become an essential part of routine practice. In the present study, the expression of WT1 was examined in 494 cases of human cancers, including tumors of the gastrointestinal and pancreatobiliary system, urinary tract, male and female genital organs, breast, lung, brain, skin, soft tissues and bone by immunohistochemistry using polyclonal (C-19) and monoclonal (6F-H2) antibodies against WT1 protein. Staining for C-19 and 6F-H2 was found in 35-100 and 5-88% of the cases of each kind of tumor, respectively. WT1-positive tumors included tumor of the stomach, prostate, and biliary and urinary systems, and malignant melanomas. A majority of the positive cases showed diffuse or granular staining in the cytoplasm, whereas ovarian tumors and desmoplastic small round cell tumors frequently showed nuclear staining. Glioblastomas, some of soft tissue sarcomas, osteosarcomas, and malignant melanomas of the skin showed extremely strong cytoplasmic staining as compared with other tumors. Western blot analysis showed that WT1 protein was predominantly expressed in the cytoplasm of the tumor cells in two cases of lung adenocarcinoma, supporting the intracytoplasmic staining for WT1 using immunohistochemistry. Immunohistochemical detection with routinely processed histologic sections could provide meaningful information on the expression of WT1 in cancer cells.

The role of intracellular magnesium ions is of high interest in the fields of pharmacology and cellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg2+, there is a strong desire for the development of optimized Mg2+ fluorescent probes. In this paper we describe the design, synthesis, and cellular application of the three novel Mg2+ fluorescent probes KMG-101, -103, and -104. The compounds of this series feature a charged beta-diketone as a binding site specific for Mg2+ and a fluorescein residue as the fluorophore that can be excited with an Ar+ laser such as is widely used in confocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent response toward Mg2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants (Kd,Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ion concentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg2+ measurements. For intracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfully incorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporated cells, the resulting increase in the free magnesium ion concentration could be followed over time. By using a confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorily observed.

To determine the nature of intracellular Mg2+ stores and Mg2+ release mechanisms in differentiated PC12 cells, Mg2+ and Ca2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg2+ concentration ([Mg2+]i) and the [Ca2+]i in these cells. Possible candidates as intracellular Mg2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg2+]i was induced by caffeine application, intracellular IP3 or Ca2+ liberated by photolysis, it appears that no Mg2+ release mechanism thus exists that is mediated via the action of Ca2+ on membrane-bound receptors in the ER or via the offloading of Mg2+ from binding sites as a result of the increased [Ca2+]i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg2+]i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg2+ store in PC12 cell. Simultaneous measurements of [Ca2+]i and mitochondrial membrane potential, and also of [Ca2+]i and [Mg2+]i, revealed that the initial rise in [Mg2+]i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg2+ in the FCCP-induced [Mg2+]i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg2+ release.

Inverse spin Hall effect (ISHE) allows the conversion of pure spin current into charge current in nonmagnetic materials (NM) due to spin-orbit interaction (SOI). In ferromagnetic materials (FM), SOI is known to contribute to anomalous Hall effect (AHE), anisotropic magnetoresistance (AMR), and other spin-dependent transport phenomena. However, SOI in FM has been ignored in ISHE studies in spintronic devices, and the possibility of "self-induced ISHE" in FM has never been explored until now. In this paper, we demonstrate the experimental verification of ISHE in FM. We found that the spin-pumping-induced spin current in permalloy (Py) film generates a transverse electromotive force (EMF) in the film itself, which results from the coupling of spin current and SOI in Py. The control experiments ruled out spin rectification effect and anomalous Nernst effect as the origin of the EMF.Comment: 28 page, 5 figures, supplemental information (To appear in Physical Review B (Regular Article)

Intracellular signal transduction relies on spatial and temporal signal transmitter dynamics. To clarify the correlations of these transmitter molecules, multicolor-imaging has been widely used. However, in the case of applying multiple indicators in a cell, spectral overlap of the indicators prevents accurate quantitative analysis. Moreover, the invasive (toxic) effect, the localization, the metabolism, as well as photobleaching of these indicators complicate the situation. Here, we show that single-molecular multifluorescent probes can overcome these problems. While intracellular calcium plays a critical role as a signal transmitter and magnesium acts as a cofactor in many situations, the correlations between the two cations are now the main issue. We designed and synthesized a Ca2+-Mg2+ responsive multifluorescent probe, KCM-1. KCM-1 shows a spectral blue shift upon complexation to Ca2+ and a red shift to the presence of Mg2+. With data analyzed at different excitation wavelengths, the concentrations of Ca2+ and Mg2+ are simultaneously quantified. Furthermore, by using the AM-ester method, intracellular Ca2+ and Mg2+ concentrations are simultaneously imaged. Such a type of intracellular multiple analyte imaging by a single-molecular multifluorescent probe is successfully demonstrated for the first time.

Novel Mg2+ fluorescent molecular probes (KMG-20-AM and KMG-27-AM; where AM is an acetoxymethyl group) based on a coumarin possessing a charged beta-diketone structure were designed and synthesized. These fluorescent probes produced a red shift from 425 to 445 nm in the absorption spectra after formation of a complex with Mg2+. The fluorescence spectra of these probes also showed a red shift from 485 to 495 nm and an increasing fluorescence intensity after formation of a complex with Mg2+. The optimum experimental conditions were excitation wavelength of 445 nm and a monitored wavelength of 500 nm, where these probes functioned as an indicator showing an image of increasing fluorescence in the presence of Mg2+. These probes showed a "seesaw-type" fluorescent spectral change with the isosbestic point at 480 nm due to the light excitation at 445 nm, which indicates that ratiometry can be used for the measurement. The molecular probes formed a 1:1 complex with Mg2+ and the dissociation constant (Kd) was 10.0 mM for KMG-20. The association constants of the probes with Mg2- were approximately 3 times higher than that with Ca2+, which showed that the selectivity of Mg2+ versus Ca2+ for these probes was over 200 times higher than that for commercially available Mg2+ fluorescent molecular probes such as mag-fura-2, Magnesium Green. As an application of these probes, intracellular fluorescent imaging of Mg2+ was demonstrated using a fluorescent microscope. After the addition of KMG-20-AM and KMG-27-AM into PC12 cells, a strong fluorescence was observed in the cytoplasm and a weak fluorescence in the nuclei region. After treatment with a high-K+ medium, the fluorescence intensity increased due to increasing intracellular Mg2+. The real image of Mg2+ release from the magnesium store was successfully observed with these Mg2+ fluorescent probes.

Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.

The effects of IAA-94, a chloride channel blocker and/or low chloride perfusate on afferent arteriolar (AA) constriction by angiotensin II (Ang II), norepinephrine (NE) and increasing pressure (80 to 160 mm Hg) were assessed using isolated perfused hydronephrotic kidneys. In the first series of experiments, Ang II (0.3 nM) constricted AAs by 33 +/- 3% (N = 5, P < 0.01). Subsequent addition of diltiazem (10 microM) restored the decrements in the AA diameters. In the presence of diltiazem (10 microM), increasing pressure did not constrict AAs. In the second series of experiments. elevation of pressure constricted AAs by 20 +/- 2% (N = 7. P < 0.01). Subsequent addition of IAA-94 (30 microM) failed to alter the basal AA diameter and myogenic responsiveness. However, Ang II-induced AA constriction was abolished by IAA-94. In the third series of experiments, decreasing extracellular chloride exaggerated AA constriction by 0.1 nM of Ang II (from 13 +/- 2 to 20 +/- 3%, N = 6, P < 0.05). Similarly, low chloride perfusate enhanced NE (0.1 microM)-induced AA constriction (from 14 +/- 2 to 19 +/- 2%, N = 6, P < 0.05). In contrast, myogenic responsiveness was not influenced by reducing chloride concentrations. The present data provide evidence that both Ang II and NE induce AA constriction by opening chloride channels and subsequent activation of voltage-dependent calcium channels, and suggest that the myogenic response is mediated by activating voltage-dependent calcium channels independently of chloride channels.