VDUP1 potentiates Ras-mediated angiogenesis via ROS production in endothelial cells.

Vitamin D3 up-regulated protein 1 (VDUP1) is a tumor suppressor of which expression is reduced in a variety of cancer cells, and enforced expression inhibits the tumor cell proliferation. It inhibits the activity of thioredoxin, thus contributing cellular ROS generation. Since ROS is a critical factor for angiogenesis, we investigated the role of VDUP1 in angiogenesis and endothelial proliferation. The expression of VDUP1 was upregulated by overexpression of an oncogene, Ras. Enforced expression of VDUP1 increases ROS production and proliferation of Ras-overexpressing endothelial cells. Overexpression of VDUP1 increases the resistance to the anchorage-dependent cell death and tube formation of the Ras-overexpressing endothelial cell. In addition, the removal of ROS by ROS scavenger attenuates the effect of VDUP1 on tube formation. These results suggest that VDUP1 is involved in Ras-mediated angiogenesis via ROS generation in endothelial cells.

Reproducibility of aortic valve calcification scoring with computed tomography - An interplatform analysis.

BACKGROUND: To investigate whether aortic valve calcification (AVC) scoring performed with different workstation platforms generates comparable and thus software-independent results. METHODS: In this IRB-approved retrospective study, we included 100 consecutive patients with symptomatic aortic stenosis undergoing CT prior to transcatheter aortic valve implantation. Two independent observers performed AVC scoring on non-enhanced images with commercially available software platforms of four vendors (GE, Philips, Siemens, 3mensio). Gender-specific Agatston score cut-off values were applied according to current recommendations to assign patients to different likelihood categories of aortic stenosis (unlikely to very likely). Comparative analysis of Agatston scores between the four platforms were performed by using Kruskal-Wallis analysis, Spearman rank correlation, linear regression analysis, and Bland-Altman analysis. Differences in category assignment were compared using Fisher's exact test and Cohen's kappa. RESULTS: For both observers, each workstation platform produced slightly different numeric AVC Agatston scores, however, without statistical significance (p = 0.96 and p = 0.98). Excellent correlation was found between platforms, with r = 0.991-0.996 (Spearman) and r2 = 0.981-0.992 (regression analysis) for both observers. Bland-Altman analyses revealed small mean differences with narrow limits of agreement between platforms (mean differences: 6 ±â€¯128 to 100 ±â€¯179), for inter-observer (mean differences: 1 ±â€¯43 to 12 ±â€¯70), and intra-observer variability (mean differences: 9 ±â€¯42 to 20 ±â€¯96). Observer 1 assigned 11 (kappa: 0.85-0.97) and observer 2 assigned 10 patients (kappa: 0.88-0.95) to different likelihood groups of severe aortic stenosis with at least one platform. Overall, there was no significant difference of likelihood assignment between platforms (p = 0.98 and p = 1.0, respectively). CONCLUSION: While absolute values differ slightly, common commercially available software platforms produce comparable results for AVC scoring, which indicates software-independence of the method.

Inhibition of retinoic acid receptor signaling by Ski in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with -7/del7q and 11 with normal karyotype) with 23 CD34+ preparations from healthy bone marrow donors. SKI, a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with -7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RARalpha signaling in AML. Ski was co-immunoprecipitated and colocalized with RARalpha. We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RARalpha signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RARalpha signaling.

Evaluation of pulmonary nodules and infection on chest CT with radiation dose equivalent to chest radiography: Prospective intra-individual comparison study to standard dose CT.

PURPOSE: To compare prospectively, in patients undergoing chest computed tomography (CT) for pulmonary-nodules or infection, image-quality and accuracy of standard dose (SD) and reduced dose (RD) CT with tin-filtration. MATERIAL AND METHODS: This IRB-approved study included 100 consecutive patients (36 female;median age 56 years) referred for follow-up of pulmonary-nodules (n=43) or suspicion of infection (n=57) undergoing single-energy CT with SD and RD using tin-filtration at 100 kVp (CTDIvol 2.47 mGy and 0.07 mGy, respectively). Images were reconstructed with advanced modeled iterative reconstruction (ADMIRE) at strength 3 and 5. Image-noise was measured. Two independent readers evaluated nodules and pulmonary-infection. SD CT served as reference standard. RESULTS: No significant difference was found in noise between RD with ADMIRE5 and SD with ADMIRE3 (118HU ± 14 vs. 120HU ± 17; p=0.08). Sensitivity for detection of atelectasis and interstitial lung changes was higher in images reconstructed with ADMIRE5 (93% and 88%; respectively) than in those reconstructed with ADIMRE3 (77% and 78%; respectively). Sensitivity for detection of consolidations was 90% for ADMIRE3 and 89% for ADMIRE5. Sensitivity for nodule detection was 71% for ADMIRE3 and 81% for ADMIRE5. Specificity for detection of atelectasis and interstitial lung changes was 99% and 96% with ADMIRE5 and 99% and 96% with ADMIRE3. Specificity for detection of consolidations was 99% for ADMIRE3 and 5. Specificity for detection of nodules was 87% for both ADMIRE3 and 5. CONCLUSION: Chest CT with a radiation dose equivalent to conventional radiography is feasible and allows for detection of pulmonary infection with high sensitivity, whereas the accuracy for detecting nodules is only moderate.

European LeukemiaNet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia.

Most reports on chronic myeloid leukaemia (CML) treatment with tyrosine kinase inhibitors (TKIs) focus on efficacy, particularly on molecular response and outcome. In contrast, adverse events (AEs) are often reported as infrequent, minor, tolerable and manageable, but they are increasingly important as therapy is potentially lifelong and multiple TKIs are available. For this reason, the European LeukemiaNet panel for CML management recommendations presents an exhaustive and critical summary of AEs emerging during CML treatment, to assist their understanding, management and prevention. There are five major conclusions. First, the main purpose of CML treatment is the antileukemic effect. Suboptimal management of AEs must not compromise this first objective. Second, most patients will have AEs, usually early, mostly mild to moderate, and which will resolve spontaneously or are easily controlled by simple means. Third, reduction or interruption of treatment must only be done if optimal management of the AE cannot be accomplished in other ways, and frequent monitoring is needed to detect resolution of the AE as early as possible. Fourth, attention must be given to comorbidities and drug interactions, and to new events unrelated to TKIs that are inevitable during such a prolonged treatment. Fifth, some TKI-related AEs have emerged which were not predicted or detected in earlier studies, maybe because of suboptimal attention to or absence from the preclinical data. Overall, imatinib has demonstrated a good long-term safety profile, though recent findings suggest underestimation of symptom severity by physicians. Second and third generation TKIs have shown higher response rates, but have been associated with unexpected problems, some of which could be irreversible. We hope these recommendations will help to minimise adverse events, and we believe that an optimal management of them will be rewarded by better TKI compliance and thus better CML outcomes, together with better quality of life.

Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography.

Non-specific lipid-transfer proteins (nsLTPs) are involved in the movement of phospholipids, glycolipids, fatty acids, and steroids between membranes. Several structures of plant nsLTPs have been determined both by X-ray crystallography and nuclear magnetic resonance. However, the detailed structural basis of the non-specific binding of hydrophobic ligands by nsLTPs is still poorly understood. In order to gain a better understanding of the structural basis of the non-specific binding of hydrophobic ligands by nsLTPs and to investigate the plasticity of the fatty acid binding cavity in nsLTPs, seven high-resolution (between 1.3 A and 1.9 A) crystal structures have been determined. These depict the nsLTP from maize seedlings in complex with an array of fatty acids.A detailed comparison of the structures of maize nsLTP in complex with various ligands reveals a new binding mode in an nsLTP-oleate complex which has not been seen before. Furthermore, in the caprate complex, the ligand binds to the protein cavity in two orientations with equal occupancy. The volume of the hydrophobic cavity in the nsLTP from maize shows some variation depending on the size of the bound ligands. The structural plasticity of the ligand binding cavity and the predominant involvement of non-specific van der Waals interactions with the hydrophobic tail of the ligands provide a structural explanation for the non-specificity of maize nsLTP. The hydrophobic cavity accommodates various ligands from C10 to C18. The C18:1 ricinoleate with its hydroxyl group hydrogen bonding to Ala68 possibly mimics cutin monomer binding which is of biological importance. Some of the myristate binding sites in human serum albumin resemble the maize nsLTP, implying the importance of a helical bundle in accommodating the non-specific binding of fatty acids.

Structural changes in alpha-synuclein affect its chaperone-like activity in vitro.

Alpha-synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of alpha-synuclein has not yet been established. We here present biochemical evidence that recombinant alpha-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, alpha-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and alpha-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of alpha-synuclein, which is believed to reorganize the molecular surface of alpha-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, alpha-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, alpha-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that alpha-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that alpha-synuclein could act as a molecular chaperone, and that the conformational change of the alpha-synuclein could explain the aggregation kinetics of alpha-synuclein, which may be related to the abolishment of the chaperonic-like activity.

Differential regulation of P2Y(11) receptor-mediated signalling to phospholipase C and adenylyl cyclase by protein kinase C in HL-60 promyelocytes.

The regulatory mode of the P2Y(11) purinoceptor-mediated signalling cascades towards phospholipase C and adenylyl cyclase was studied in HL-60 promyelocytes. Treatment with the potent P2Y(11) receptor activator dATP evoked an elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)) production that was sustained for longer than 30 min. However, the dATP-induced responses were significantly inhibited by the activation of protein kinase C after a short exposure to phorbol 12-myristate 13-acetate (PMA). dATP also potently stimulated cyclic AMP production with half maximum effect seen at 23+/-7 microM dATP. In addition, a 5-min pretreatment with PMA enhanced the dATP-stimulated cyclic AMP accumulation. PMA potentiated the cyclic AMP production when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment. dATP also enhanced the forskolin-mediated cyclic AMP generation. Treatment of the cells with 10 microM U-73122, which almost completely blocked the dATP-stimulated IP(3) production and [Ca(2+)](i) rise, had no effect on cyclic AMP accumulation, while 10 microM 9-(tetrahydro-2-furyl)adenine (SQ 22536), which inhibited the adenylyl cyclase activation, did not effect the dATP-stimulated phosphoinositide turnover. Taken together, the results indicate that P2Y(11) receptor-mediated activation of phospholipase C and adenylyl cyclase occurs through independent pathways and is differentially regulated by protein kinase C in HL-60 cells.

Pharmacological characterization of adenosine receptors in PGT-beta mouse pineal gland tumour cells.

1. The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches. 2. Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT-beta cells by polymerase chain reaction and nucleotide sequencing. 3. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2)-selective agonist 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarboxamideadenosine (CGS 21680) and the A1-specific agonists R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and N(6)-cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA-mediated cyclic AMP accumulation. 4. Treatment of cells with the A3-selective agonist N(6)-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration-dependent manner with maximal inhibition of 40 - 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB-MECA-induced responses, suggesting that this effect occurred via the pertussis toxin-sensitive inhibitory G proteins. 5. IB-MECA also caused a concentration-dependent elevation in [Ca(2+)]i and IP3 content. Both the responses induced by IB-MECA were attenuated by treatment with U73122 or phorbol 12-myristate 13-acetate. 6. These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.

Studies on liposome-encapsulated heparin.

In order to prolong the anticoagulant activity of heparin in vivo, attempts were made to encapsulate heparin into liposomes. Liposome-encapsulated heparin (lipo-heparin) prepared was large multilamellar vesicles (0.5-4.0 micron in diameter). The activity of lipo-heparin was 1.6-5.2 X 10(3) U/g lipid with recovery rate ranged between 0.4 to 1.3% and stable in saline at 4 degrees C for at least two weeks. When intravenously administered into rats, the anticoagulant activity of lipo-heparin was significantly prolonged (approximately three times), as compared with that of untreated heparin. Furthermore, the activity of lipo-heparin could be neutralized by protamine sulfate. From these observations, it was concluded that liposome-encapsulation of heparin results in the prolonged anticoagulant effect in vivo and lipo-heparin may be applicable for clinical use, after further studies on side effects of liposomes are completed.

Metabolism of liposome-encapsulated heparin.

In order to elucidate the metabolism of liposome encapsulated heparin (LIPO-HEP), LIPO-HEP containing 3H-heparin (3H-HEP) and/or 14C-phosphatidylcholine (14C-PC) was intravenously administered into rats and the radioactivity as well as the biological activity in plasma and certain organs was investigated. The amount of 3H-radioactivity in plasma was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin. The amount of 14C-radioactivity in plasma of rats receiving LIPO-HEP, however, was not proportional to the amount of 3H-radioactivity in the same rats, indicating the dissociation of liposome and heparin in plasma. Incorporation of 3H-radioactivity into various organs examined, i.e., liver, spleen, lung, was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin, e.g. 4.7 and 11.8 times higher in the liver and the spleen, respectively at 150 min after the injection. Thereby, in contrast to the untreated heparin, LIPO-HEP was selectively incorporated into the reticuloendothelial system (RES) and it may be suggested that prolonged biological activity in LIPO-HEP is due to a gradual release of heparin from the liposomes entrapped in RES, and that it is not due to prolonged circulation in blood.

[Lympho- and hemodynamic reactions during the contraction of the skeletal musculature of the hindlimb in dogs]. / Limfo- i gemodinamicheskie reaktsii pri sokrashchenii skeletnoi muskulatury zadnikh konechnostei sobaki.

Alterations in arterial pressure (AP) values, blood and perfusate flow from the thoracic duct, lymph and perfusate flow from the femoral lymphatic vessel during femoral muscle contractions induced by electrical stimulation applied for 1.5 and 3.0 min, were studied: the AP values were decreased by 10-15 mm Hg, the blood flow increased to 293.5 +/- 21.7%, the lymph flow increased to 525.4 +/- 29.8%, the perfusate flow from the thoracic duct raised to 81.8 +/- 4.7%, and the perfusate flow from the lymphatic vessel increased to 650.0 +/- 28.2%. The increase in the lymph flow during 1.5 min stimulation seems to be due to squeezing of lymph out of the lymphatic channel, whereas during 3 min stimulation it was due to enhanced lymph production.

[Development of the contractile activity of the thoracic duct in dogs in postnatal ontogenesis]. / Razvitie sokratitel'noi aktivnosti grudnogo limfaticheskogo protoka u sobak v postnatal'nom ontogeneze.

It has been demonstrated that the thoracic lymph duct in newborn puppies exhibits only tonic (spontaneous) contractile activity. Rhythmic activity in the duct appears at the age of 2 1/2-3 months.

Peripheral artery occlusive disease in chronic phase chronic myeloid leukemia patients treated with nilotinib or imatinib.

Several retrospective studies have described the clinical manifestation of peripheral artery occlusive disease (PAOD) in patients receiving nilotinib. We thus prospectively screened for PAOD in patients with chronic phase chronic myeloid leukemia (CP CML) being treated with tyrosine kinase inhibitors (TKI), including imatinib and nilotinib. One hundred and fifty-nine consecutive patients were evaluated for clinical and biochemical risk factors for cardiovascular disease. Non-invasive assessment for PAOD included determination of the ankle-brachial index (ABI) and duplex ultrasonography. A second cohort consisted of patients with clinically manifest PAOD recruited from additional collaborating centers. Pathological ABI were significantly more frequent in patients on first-line nilotinib (7 of 27; 26%) and in patients on second-line nilotinib (10 of 28; 35.7%) as compared with patients on first-line imatinib (3 of 48; 6.3%). Clinically manifest PAOD was identified in five patients, all with current or previous nilotinib exposure only. Relative risk for PAOD determined by a pathological ABI in first-line nilotinib-treated patients as compared with first-line imatinib-treated patients was 10.3. PAOD is more frequently observed in patients receiving nilotinib as compared with imatinib. Owing to the severe nature of clinically manifest PAOD, longitudinal non-invasive monitoring and careful assessment of risk factors is warranted.