Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches.

The microfold (M) cell residing in the follicle-associated epithelium is a specialized epithelial cell that initiates mucosal immune responses by sampling luminal antigens. The differentiation process of M cells remains unclear due to limitations of analytical methods. Here we found that M cells were classified into two functionally different subtypes based on the expression of Glycoprotein 2 (GP2) by newly developed image cytometric analysis. GP2-high M cells actively took up luminal microbeads, whereas GP2-negative or low cells scarcely ingested them, even though both subsets equally expressed the other M-cell signature genes, suggesting that GP2-high M cells represent functionally mature M cells. Further, the GP2-high mature M cells were abundant in Peyer's patch but sparse in the cecal patch: this was most likely due to a decrease in the nuclear translocation of RelB, a downstream transcription factor for the receptor activator of nuclear factor-κB signaling. Given that murine cecum contains a protrusion of beneficial commensals, the restriction of M-cell activity might contribute to preventing the onset of any excessive immune response to the commensals through decelerating the M-cell-dependent uptake of microorganisms.

Unique lamina propria stromal cells imprint the functional phenotype of mucosal dendritic cells.

Mucosal dendritic cells (DCs) in the intestine acquire the unique capacity to produce retinoic acid (RA), a vitamin A metabolite that induces gut tropism and regulates the functional differentiation of the T cells they prime. Here, we identified a stromal cell (SC) population in the intestinal lamina propria (LP), which is capable of inducing RA production in DCs in a RA- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent fashion. Unlike DCs, LP SCs constitutively expressed the enzymatic machinery to produce RA even in the absence of dietary vitamin A, but were not able to do so in germ-free mice implying regulation by microbiota. Interestingly, DCs promoted GM-CSF production by the SCs indicating a two-way cross-talk between both cell types. Furthermore, RA-producing LP SCs and intestinal DCs localized closely in vivo suggesting that the interactions between both cell types might have an important role in the functional education of migratory DCs and therefore in the regulation of immune responses toward oral and commensal antigens.

Scattered radiation from dental metallic crowns in head and neck radiotherapy.

We aimed to estimate the scattered radiation from dental metallic crowns during head and neck radiotherapy by irradiating a jaw phantom with external photon beams. The phantom was composed of a dental metallic plate and hydroxyapatite embedded in polymethyl methacrylate. We used radiochromic film measurement and Monte Carlo simulation to calculate the radiation dose and dose distribution inside the phantom. To estimate dose variations in scattered radiation under different clinical situations, we altered the incident energy, field size, plate thickness, plate depth and plate material. The simulation results indicated that the dose at the incident side of the metallic dental plate was approximately 140% of that without the plate. The differences between dose distributions calculated with the radiation treatment-planning system (TPS) algorithms and the data simulation, except around the dental metallic plate, were 3% for a 4 MV photon beam. Therefore, we should carefully consider the dose distribution around dental metallic crowns determined by a TPS.

Glomerulocystic kidney disease in an adult with enlarged kidneys: a case report and review of the literature.

We report the case of a 31-year-old male with enlarged kidneys and glomerulocystic kidney disease (GCKD). The patient had no family history of renal disease or other diseases. On initial presentation he complained of poor eyesight, and hypertensive retinopathy and elevated serum creatinine (5.0 mg/dl) were found at that time. Renal biopsy showed cystic dilatation of Bowman's capsule and atrophy of the glomerular tuft. Thus, an adult case of sporadic GCKD was diagnosed. Based on previous reports, kidney size in patients with adult type GCKD varies from small to large. Our patient's kidneys are the largest ever reported (right kidney was 22 cm×10 cm, left kidney was 19 cm×10 cm). A review of the literature dealing with sporadic adult GCKD suggested that it is difficult to diagnose this disease early in its course.

Serum adiponectin level is not only decreased in metabolic syndrome but also in borderline metabolic abnormalities.

OBJECTIVE: Along with the increasing prevalence of obesity and related diseases, particularly atherosclerotic diseases, metabolic syndrome (MetS) is now a common and major public health issue in many countries around the world. Adiponectin, a protein secreted by the adipose tissue, has become recognized as a key player in the development of MetS. These days, not only MetS but also borderline metabolic/physiological abnormalities, such as impaired fasting glucose, high normal blood pressure and high normal plasma cholesterol, have been reported to be risk factors for atherosclerotic disease. Therefore, we undertook this study to determine the relationship between adiponectin and borderline metabolic/physiological abnormalities, as well as MetS. DESIGN: A cross-sectional study performed from April 2007 to November 2009. SUBJECTS: In 16 892 Japanese adults (10 008 men and 6884 women), we examined the relationship between the serum adiponectin concentration and borderline metabolic/physiological abnormalities or MetS by a questionnaire survey about medical treatment, body size measurement and measurement of laboratory parameters including the serum adiponectin concentration. RESULTS: Adiponectin showed a significant negative correlation with the number of MetS components. In subjects without overt diabetes mellitus, hypertension or dyslipidemia, the adiponectin concentration also showed a significant negative correlation with the number of borderline metabolic abnormalities. CONCLUSION: The decrease of circulating adiponectin may start before the development of diabetes mellitus, hypertension, dyslipidemia or MetS. Adiponectin is an important biomarker for reflecting the adverse influence of visceral fat in persons with MetS, and also in these subclinical states.

Suppression of cell-cycle progression by Jun dimerization protein-2 (JDP2) involves downregulation of cyclin-A2.

We report here a novel role for Jun dimerization protein-2 (JDP2) as a regulator of the progression of normal cells through the cell cycle. To determine the role of JDP2 in vivo, we generated Jdp2-knockout (Jdp2KO) mice by targeting exon-1 to disrupt the site of initiation of transcription. The epidermal thickening of skin from the Jdp2KO mice after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) proceeded more rapidly than that of control mice, and more proliferating cells were found at the epidermis. Fibroblasts derived from embryos of Jdp2KO mice proliferated faster and formed more colonies than fibroblasts from wild-type mice. JDP2 was recruited to the promoter of the gene for cyclin-A2 (ccna2) at the AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin-A2 mRNA. Furthermore, reintroduction of JDP2 resulted in the repression of transcription of ccna2 and of cell-cycle progression. Thus, transcription of the gene for cyclin-A2 appears to be a direct target of JDP2 in the suppression of cell proliferation.

The effect of gantry and collimator angles on leaf limited velocity and position in dynamic multileaf collimator intensity-modulated radiation therapy.

The purpose of the study is to evaluate the limiting velocity (LV) of a multileaf collimator and the leaf position in various collimator and gantry angles. Both leading leaves and trailing leaves began to move with a constant acceleration from 0 to 4 cm s(-1). When the beam hold occurred, the leaf velocity was defined as the leaf LV. Dynamic irradiation was performed at eight gantry angles of every 45 degrees with three different collimator angles. The analysis of the LV and the leaf position was performed with a log file from a leaf motion controller. The mean LVs for Varian Clinac 21EX (21EX) ranged from 2.51 to 3.10 cm s(-1). The mean LVs for Clinac 600C ranged from 2.91 to 3.12 cm s(-1). When only central 5 mm leaves of 21EX moved, LVs were significantly higher than those when all 60 pairs of leaf moved, while the leaf position inconsistencies of the two accelerators were within 1 mm at the leaf velocities from 0.5 to 2.0 cm s(-1). It was recognized that the LV was affected by gravity. This measurement method can be utilized as routine quality assurance for a dynamic multileaf collimator (DMLC) is and easily reproducible.

Four-dimensional quantitative analysis of the gait of mutant mice using coarse-grained motion capture.

To analyze an abnormal gait pattern in mutant mice (Hugger), we conducted coarse-grained motion capture. Using a simple retroreflective marker-based approach, we could detect high-resolution mutant-specific gait patterns. The phenotypic gait patterns are caused by extreme vertical motion of limbs, revealing inefficient motor functions. To elucidate the inefficiency, we developed a musculoskeletal computer model of the mouse hindlimb based on X-ray CT data. By integrating motion data with the model, we determined mutant-specific musculotendon lengths, suggesting that three major muscles were involved in the abnormal gait. This approach worked well on laboratory mice, which were putatively too small to be motion capture subjects. Motion capture technology was originally developed for human study, and our approach may help fill neuroscience gaps between mouse and human behavioral phenotypes.

An experimental attenuation plate to improve the dose distribution in intraoperative electron beam radiotherapy for breast cancer.

Intraoperative electron beam radiotherapy (IOERT) is a technique in which a single-fraction high dose is intraoperatively delivered to subclinical tumour cells using an electron beam after breast-conserving surgery. In IOERT, an attenuation plate consisting of a pair of metal disks is commonly used to protect the normal tissues posterior to the breast. However, the dose in front of the plate is affected by backscatter, resulting in an unpredictable delivered dose to the tumour cells. In this study, an experimental attenuation plate, termed a shielding plate, was designed using Monte Carlo simulation, which significantly diminished the electron beam without introducing any backscatter radiation. The plate's performance was verified by measurements. It was made of two layers, a first layer (source side) of polymethyl methacrylate (PMMA) and a second layer of copper, which was selected from among other metals (aluminium, copper and lead) after testing for shielding capability and the range and magnitude of backscatter. The optimal thicknesses of the PMMA (0.71 cm) and copper (0.3 cm) layers were determined by changing their thicknesses during simulations. On the basis of these results, a shielding plate was prototyped and depth doses with and without the plate were measured by radiophotoluminescence glass dosimeters using a conventional stationary linear accelerator and a mobile linear accelerator dedicated for IOERT. The trial shielding plate functioned as intended, indicating its applicability in clinical practice.

A comparison of prone three-dimensional conformal radiotherapy with supine intensity-modulated radiotherapy for prostate cancer: which technique is more effective for rectal sparing?

The purpose of this study was to assess the potential dose reductions to the rectum with three-dimensional conformal radiotherapy in the prone position (prone 3D-CRT) compared with intensity-modulated radiotherapy in the supine position (supine IMRT) for prostate cancer. 17 prostate cancer patients underwent treatment planning CT scans in the supine and prone positions. Prone 3D-CRT and supine IMRT plans were constructed for each patient and compared in terms of the volume of rectum exposed to the V90 (volume of rectum receiving at least 90% of the prescription dose) as the high dose region. It was confirmed that supine IMRT was significantly superior to prone 3D-CRT (p = 0.023). Although, in some cases, the distance between the seminal vesicles and the rectum could change by more than 20 mm in the transition from supine to prone, the change in distance was approximately 5 mm in many other cases. While prone 3D-CRT resulted in significant improvements in some patients in terms of rectal sparing, the degree of the effect may be dependent on a patient's anatomy and physical condition in prone 3D-CRT compared with supine IMRT. If the cases in which prone 3D-CRT was more effective in rectal dose reduction could be extracted using some anatomical predictor before treatment planning, prone 3D-CRT may be appropriate in such a case. We consider that prone 3D-CRT still warrants further investigation because of its advantages in terms of simplicity, cost-effectiveness and labour saving; continued research to find an appropriate anatomical predictor is required.

JDP2 suppresses adipocyte differentiation by regulating histone acetylation.

Among the events that control cellular differentiation, the acetylation of histones plays a critical role in the regulation of transcription and the modification of chromatin. Jun dimerization protein 2 (JDP2), a member of the AP-1 family, is an inhibitor of such acetylation and contributes to the maintenance of chromatin structure. In an examination of Jdp2 'knock-out' (KO) mice, we observed elevated numbers of white adipocytes and significant accumulation of lipid in the adipose tissue in sections of scapulae. In addition, mouse embryo fibroblasts (MEFs) from Jdp2 KO mice were more susceptible to adipocyte differentiation in response to hormonal induction and members of the CCAAT/enhancer-binding proteins (C/EBP) gene family were expressed at levels higher than MEFs from wild-type mice. Furthermore, JDP2 inhibited both the acetylation of histone H3 in the promoter of the gene for C/EBPdelta and transcription from this promoter. Our data indicate that JDP2 plays a key role as a repressor of adipocyte differentiation by regulating the expression of the gene for C/EBPdelta via inhibition of histone acetylation.

Paternal dual barrier by Ifg2-H19 and Dlk1-Gtl2 to parthenogenesis in mice.

The functional difference between the maternal and paternal genome, which is characterized by epigenetic modifications during gametogenesis, that is genomic imprinting, prevents mammalian embryos from parthenogenesis. Genomic imprinting leads to nonequivalent expression of imprinted genes from the maternal and paternal alleles. However, our research showed that alteration of maternal imprinting by oocyte reconstruction using nongrowing oocytes together with deletion of the H19 gene, provides appropriate expression of maternally imprinted genes. Here we discuss that further alteration of paternally imprinted gene expressions at chromosomes 7 and 12 allows the ng/fg parthenogenetic embryos to develop to term, suggesting that the paternal contribution is obligatory for the descendant.

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide.

In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, approximately 20 mmHg) without glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by complex III inhibitor: antimycin A (24 +/- 3%, P < 0.0001), or stigmatellin (31 +/- 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 +/- 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.

Multivariate spline interpolation as a novel method to optimize pharmaceutical formulations.

One of the difficulties in the quantitative approach for formulation design is the difficulty in understanding the actual relationship between causal factors and individual pharmaceutical responses. In this regard, several techniques were applied to determine the relationship between causal factors and the pharmaceutical responses. The generation of response surfaces using multivariate spline interpolation (MSI) has provided rapid and detailed information. Nevertheless, no application of MSI in the pharmaceutical field has been reported to date, even though it promises potential applications. To overcome the shortcomings of the classical response surface method, we newly developed a multi-objective simultaneous optimization method, in which MSI had been incorporated. The method was applied to the optimization problem of a transdermal hydrogel formulation for ketoprofen containing several chemical enhancers. Results suggested a superior function of the MSI approach.

Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines.

Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.

Vasopressin with delayed combination of nitroglycerin increases survival rate in asphyxia rat model.

Recently, vasopressin has been reported as a more effective drug than epinephrine (adrenaline) for cardiopulmonary resuscitation (CPR). However, vasopressin decreases myocardial blood flow (MBF) because of its strong vasoconstriction, to maintain better coronary perfusion pressure (CPP) compared with epinephrine. Nitroglycerin is well known to be able to maintain MBF and increase survival rate. In a VF model, vasopressin combined with nitroglycerin maintained CPP; however, low survival rates were observed compared with vasopressin alone. We investigated the effectiveness of the delayed use of nitroglycerin combined with vasopressin in a severe asphyxia model. Fourteen Sprague-Dawley male rats were divided into two groups: vasopressin 0.8 U/kg alone (V-Gr.), and nitroglycerin 0.3 microg/kg 45 s after the administration of 0.8 U/kg vasopressin (VN-Gr.). Six min after asphyxia induced by obstructing the tracheal tube, CPR was performed in two ways. Three animals resuscitated in the V-Gr. (42%) and six/seven (84%) in the VN-Gr. (P<0.05). In the 6 min of asphyxia rat model, vasopressin combined with delayed nitroglycerin is more effective than vasopressin alone.

Vasopressin and epinephrine are equally effective for CPR in a rat asphyxia model.

Epinephrine has been administered as a drug essential for cardiopulmonary resuscitation (CPR). Recently, vasopressin has been reported to be more effective than epinephrine for CPR in a ventricular fibrillation (VF) model. As a different myocardial pathology is speculated to exist between the VF model and the asphyxia model, we investigated whether vasopressin is also effective in a rat asphyxia model. Twenty-one Sprague-Dawley male rats were divided into three groups: vasopressin 0.8 U/kg (Vaso-Gr), epinephrine 0.05 mg/kg (Epi-Gr), and saline same volume as the other two drugs (Sal-Gr). Five minutes after suffocation induced by obstruction of the tracheal tube, CPR was performed using each drug. Although only one animal survived (17%) in the Sal-Gr, 6/7 (85%) survived in both Vaso-Gr and Epi-Gr (P<0.01). Vasopressin is as effective as epinephrine for CPR in asphyxia-induced rats.

Limited association between a catechol-O-methyltransferase (COMT) polymorphism and breast cancer risk in Japan.

BACKGROUND: Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy estradiol and 4-hydroxy estradiol. To date, three studies in Caucasians and one study in Chinese have been conducted to determine the association with breast cancer risk of a functional polymorphism (G-to-A, Val158Met) of this enzyme, but the results were inconsistent. In order to examine the impact of this polymorphism on breast cancer risk in Japan, a case-control study was conducted, at Aichi Cancer Center Hospital. METHODS: The cases were 150 patients with histologically confirmed breast cancer who had been diagnosed within 4 years before enrollment at this hospital. The controls were 165 non-cancer patients, mainly from the gastroenterology and breast surgery clinics at the hospital. COMT-H (Val) is the wild-type allele, with high enzyme activity, while the COMT-L (Met) allele has low activity. Genotyping was conducted by a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. RESULTS: The allele frequency of COMT-L was 36.0% for cases and 33.0% for controls. Age-adjusted odds ratios relative to the COMT-HH genotype were 1.46 (95% confidence interval [CI], 0.90-2.36) for COMT-HL, and 0.99 (95% CI, 0.49-2.02) for the COMT-LL genotype. Significant odds ratios were not observed for any subgroup stratified by menopausal status, age at menarche, age at birth of first child, body mass index, and breast cancer history of mother and/or sister(s). CONCLUSION: The present study suggested that any association of the COMT polymorphism with breast cancer risk is limited in Japanese.