Continuous correlation between brain activities

In free-behaving cats, bearing chronically implanted electrodes, the cross-correlation coefficients between EEG, EMG and unit activity in the Lateral Geniculate nucleus (LGU) have been computed continuously at 1 sec interval for epochs of several minutes, in the three basic physiological states: slow wave synchronized sleep (S-sleep), REM-sleep and Arousal. The coefficients exhibit continuous and aperiodic oscillations between high positive and high negative values. The means of the coefficients for each pair of variables exhibit significant differences between the three states. The phase times also are varying significantly for each variable pair in different states. During the transition from one state to another the coefficients follow characteristic time patterns. In the stationary phase of the different physiological states the correlation coefficients follow similar waxing and waning patterns. Embedded in the steady waxing and wanings of the coefficients are frequently found r time patterns similar to those of a state transition: the incomplete and sham transitions. All the results support the hypothesis that the diffuse projecting systems are inducing a continuous pressure to change the state of the brain activity, which is revealed by the changing of the correlations between the different brain structures.     

Recognition of cationic species with synthetic receptors

In the past year progress in the study of cationic species has been made, particularly in our understanding of the factors which control the selective recognition of biologically important cations such as ammonium, alkali and alkaline earth metal ions, and of metal ions used in biomedicine such as lanthanides and iron(III). Based on this knowledge, several new hosts with improved transport, photophysical and biological properties have been designed.     

Increase in fluidity in the membrane of MT3 breast cancer cells correlates with enhanced cell adhesion in vitro and increased lung metastasis in NOD/SCID mice

To study whether membrane fluidity of tumor cells have an influence on metastasis, MT3 breast cancer cells harvested during exponential growth and under confluent conditions were compared. Electron paramagnetic resonance (EPR) data revealed that, in comparison to growing cells, confluent cells have a significant higher fluidity in their membrane related to a higher relative portion of disordered domains and a reduced portion of the most ordered domains. Further, sialyl Lewis X and/or A ligand-mediated adhesion of these cells was 2-fold enhanced. Confocal laser scanning microscopy further demonstrated a higher motility of ligands in the membrane of confluent cells, together with an accumulation of these ligands in distinct areas. Both facts are suggested to be responsible for an enhanced cell adhesion observed. Finally, an increased number of large distinct metastatic foci was registered in lungs of mice after i.v. inoculation of confluent cells. The results indicate that domain organization and fluidity of the cell membrane affect tumor cell adhesion and can have in this way also an impact on the malignancy of breast cancer cells.     

Cadmium and copper change root growth and morphology of Pinus pinea and Pinus pinaster seedlings

Heavy metal loads in forest soils have been increasing over time due to atmospheric inputs. Accumulation in the upper soil layers could affect establishment of seedlings and forest regeneration. Mediterranean species show a high initial root development, allowing seedlings to reach the moisture of deeper soil layers. In the present work seedlings of stone pine ( Pinus pinea L.) and maritime pine ( Pinus pinaster Ait.), were grown in culture solution supplied with 0.0, 0.1, 1 or 5 μ M CdSO₄ or with 1 μ M CdSO₄ and 1 μ M CuSO₄ combined. In both species tap-root elongation was drastically reduced in the 5 μ M Cd²⁺ and in the (Cd²⁺+ Cu²⁺) treatments. A supply of 0.1 or 1 μ M Cd²⁺, however, enhanced root elongation in Pinus pinea without significantly influencing root elongation in Pinus pinaster . In both species the root density (weight per unit length) and the width of the cortex increased in response to Cd²⁺ exposure. In Pinus pinaster the mitotic index decreased at the higher Cd²⁺ concentrations and when Cd²⁺ and Cu²⁺ were combined. The data suggest that cell elongation is more sensitive to Cd²⁺ than cell division. The number and length of the lateral roots were also affected by Cd²⁺ treatment to a higher degree in Pinus pinaster than in Pinus pinea, reflecting the different Cd- tolerance of the two species.     

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo–N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.     

Detection of ferryl myoglobin in the isolated ischemic rat heart

Reflectance spectroscopy was utilized to monitor the oxidation states of myoglobin (Mb) in isolated, buffer-perfused rat hearts. Hearts were subjected to 30 min global, no-flow ischemia, followed by reperfusion under anoxic conditions. The addition of Na₂S to the buffer at reperfusion permitted the detection of ferryl myoglobin (MbIV) as its sulfmyoglobin derivative. The accumulation of MbIV was prevented by addition of ascorbic acid (1 mM), ergothioneine (2mM), or desferal (1mM) to the buffer prior to ischemia. Ascorbate and other agents have been previously shown to serve as one-electron reductants of MbIV. We propose that during the early phases of ischemia, deoxymyoglobin is oxidized to MbIV by residual H₂O₂. It also seems reasonable that the peroxidative activity of Mb(IV), during oxygenated reperfusion, might lead to cellular damage if this hypervalent form of Mb is not reduced.     

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3^rCDDP¹⁰⁰⁰ in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.