Gain-of-function mutations and copy number increases of Notch2 in diffuse large B-cell lymphoma

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T-cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T-cell acute lymphoblastic leukemias. Notch2 , another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B-cell generation. Here, we report that 5 of 63 (~8%) diffuse large B-cell lymphomas, a subtype of mature B-cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain, or a single amino acid substitution at the C-terminus of Notch2 protein. Furthermore, high-density oligonucleotide microarray analysis revealed that some diffuse large B-cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation-sensitive luciferase reporter assay in vitro , mutant Notch2 receptors show increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers. ( Cancer Sci 2009; 100: 920–926)     

Dual antitumor mechanisms of Notch signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model

Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T-cell acute lymphoblastic leukemias (T-ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T-ALL. To further examine this possibility, we applied a γ-secretase inhibitor (GSI) to T-ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T-ALL cell lines that were also sensitive to GSI in vitro . To elucidate the mechanism of action, we transduced DND-41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1-transduced DND-41 cells, whereas it induced complete regression of tumors that developed from mock-transduced DND-41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND-41 cells through the inhibition of cell-autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non-cell-autonomous Notch signaling. ( Cancer Sci 2009; 100: 2444–2450)     

Effects of long-term ethanol administration on the kidneys, bones and muscles of mice]

Reports on effects of ethanol intake on the kidneys and bones are few. Circulation of blood through the kidneys is large in amount; kidney takes water-soluble exogenous substances and their metabolites in from the blood and accumulates them in the cells and interstitial tissues; in addition kidney plays important roles in producing substances that activate a living body and associating with their functions. The author let male and female ICR mice take 16% ethanol (Sake) ad lib for 472 days beginning at the age of 38 days. Observations were carried out on renal tissues and other organs which have some connection with the renal function. These included bones (femur, knee joint, tibia), muscle (gastrocnemius), 1 alpha, 25(OH)2D3 and erythropoietin (EPO). The results were as follows: Renal tissue observations: Significantly more cases of appearances of basophilic renal tubular, swelling of tubular epithelial cells and urinary casts in tubular lumens, PAS positive deposits in glomerulus, and atrophy of glomerulus were seen in the ethanol groups than in a group used as the control. These occurrences were significantly clear and intensive relative to those in the control. Findings in gastrocnemius: Significantly large number of muscle atrophy, random variation of fiber size and multinucleated fibers were seen. Observations on bones based upon soft X-ray pictures: Bone atrophy, thinning of bone trabeculae, thinning of bone cortex, and porosity of bone cortex were disclosed significantly more than the control. Values of 1 alpha, 25(OH)2D3 and EPO were significantly high increased in the ethanol groups relative to the control. Through the above-mentioned results, it has been supposed that ethanol intake affects kidney tissues and consequently affects bones and muscles.     

Tc-99m labeled tissue-type plasminogen activator: Preparation,stability and preliminary imaging of thrombus-bearing rats

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent that directly binds to fibrin formed in clots. In terms of radiolabeling and nuclear imaging, t-PA has several advantages in Tc-99m labeling: it is stable in acidic solution at pH 3, which is suitable for labeling Tc-99m by a method of stannous reduction and blood disappearance after administration is rapid, which is desirable for imaging targets using short-lived radionuclides. Recombinant t-PA was labeled with Tc-99m by a method of stannous reduction without significant degradation of biochemical activity, over 95% of which was retained after the labeling procedure. Labeling efficiency in paper chromatography was over 98%. The moiety of hydrolyzed Tc-99m that was not eluted through the Sephadex column was estimated to be less than 10%. Tc-99m labeled t-PA, however, appeared to become unstable when diluted with normal saline. Nevertheless, in in vitro fibrin binding, Tc-99m labeled t-PA showed high affinity with fibrin: 80% of 100 ng/ml of Tc-99m t-PA bound to 10(-5) mol of the fibrinogen. Preliminary animal studies also showed a concentration of Tc-99m labeled t-PA at fresh thrombi formed in the inferior vena cava. Tc-99m labeled t-PA appears to have potential for thrombus imaging and the preparation of an instant kit.     

Pancreatic Islets after Repeated Injection of Fe3+-NTA An Ultrastructural Study of Diabetic Rats

Pancreatic islet cells were examined ultrastructurally in rats after repeated intraperitoneal injections of ferric nitrilotriacetate (Fe³⁺- NTA) to produce a model of bronze diabetes. Despite diabetic signs such as glycosuria and ketouria, no ultrastructural alterations were found in islet cells up to 90 days after the beginning of the Fe³⁺-NTA injections. After 120 days, however, degenerative changes appeared, with most B cells of the islets of Langerhans showing clumped nuclear chromatin, a dilated nuclear envelope, vacuolated and dilated endoplasmic reticulum, and a loss of cell polarization toward the capillary lumen. The cells contained a number of light secretory granules with an electron-lucent core and a narrow halo. Numerous electron-dense ferritin-like particles were also found in the cytoplasmic matrix, and A and D cells were almost intact. Repeated venesection therapy of rats injected with Fe³⁺-NTA for 120 days resulted in an increase of morphologically normal B cells with a smaller number of necrotizing cells. This process was accompanied by recovery from diabetic symptoms. The toxic effect of injected iron on B cells was thus clarified.     

Reduced midbrain and pons size in children with autism

Recent reports suggested functional abnormalities of the brain stem in autistic children. Moreover, structural brain abnormalities have also been reported, so we analyzed magnetic resonance imaging (MRI) scan in autistic children. MRI scans for 29 autistic children were compared with 15 control MRI scans. The midbrain and pons were measured. The midbrain and pons size were found to be significantly smaller in the autistic group. This suggests that the structure of the brain stem is anatomically altered in autistic children.     

Temporary and partial inhibition of platelets by SM-20302 prevents coronary artery thrombosis in a chronic canine model

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G₁₆ was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

Association between serum gamma-glutamyltransferase and oxidative stress related factors

BACKGROUND/AIMS: To investigate the main factors correlated with the serum gamma-glutamyltransferase activity. METHODOLOGY: We measured serum gamma-glutamyltransferase activity in 248 healthy Japanese people and determined its correlations with serum antioxidants, other plasma or serum factors, urinary 8-hydroxydeoxyguanine, and lifestyle factors. RESULTS: The mean serum gamma-glutamyltransferase activity was 29 IU/L. Gamma-glutamyltransferase activities of males and persons older than 45 years were significantly higher than each counterpart. Gamma-glutamyltransferase levels increased significantly with the number of cigarettes smoked per day and the frequency of alcohol consumption except for the persons who did not take alcohol. Additionally, gamma-glutamyltransferase significantly correlated with urinary 8-hydroxydeoxyguanine, and with more blood factors including serum tocopherols, carotenoids, antioxidative enzymes, lipid peroxide, and free fatty acids than urinary 8-hydroxydeoxyguanine did. In multiple regression analyses, gamma-glutamyltransferase had significant associations with retinol, 8-hydroxydeoxyguanine, docosahexaenoic acid, and cigarette smoking. CONCLUSIONS: Our present findings support the hypothesis that gamma-glutamyltransferase can be used as a marker related with oxidative stress.     

Prediction of acute graft-versus-host disease and detection of distinct end-organ targets by enumeration of peripheral blood cytokine spot-forming cells

BACKGROUND: Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. It was hypothesized that type 1 cytokines promoted aGVHD and type 2 cytokines inhibited it. However, recent publications demonstrated contradictory results in murine models. Type 1/2 paradigm in aGVHD remains to be determined in human. METHODS: Using enzyme-linked immunospot assay that reflects ongoing immune status in vivo, we measured spot-forming cells (SFCs) for interferon (IFN)-gamma, interleukin (IL)-12, IL-4, and IL-10 in peripheral blood from 56 patients with hematological disorders who underwent allogeneic hematopoietic stem cell transplantation. RESULTS: The numbers of IFN-gamma and IL-4 SFCs in patients with grade II approximately IV aGVHD were significantly higher than those in patients with grade 0 approximately I aGVHD. The enumeration of cytokine SFCs predicted aGVHD approximately 4 days before it became clinically evident, since IFN-gamma SFCs in asymptomatic phase that later progressed into grade II approximately IV aGVHD were elevated in 8 out of 8 evaluable patients. Similarly, IL-4 SFCs were elevated in 6 of 8 patients. In addition, Type 1 cytokine SFCs contributed to the intestinal, but not skin and hepatic aGVHD. CONCLUSIONS: Enzyme-linked immunospot assay is clinically useful for predicting aGVHD and detecting distinct end-organ targets following allogeneic hematopoietic stem cell transplantation.