The Output Signal of Purkinje Cells of the Cerebellum and Circadian Rhythmicity

Measurement of clock gene expression has recently provided evidence that the cerebellum, like the master clock in the SCN, contains a circadian oscillator. The cerebellar oscillator is involved in anticipation of mealtime and possibly resides in Purkinje cells. However, the rhythmic gene expression is likely transduced into a circadian cerebellar output signal to exert an effective control of neuronal brain circuits that are responsible for feeding behavior. Using electrophysiological recordings from acute and organotypic cerebellar slices, we tested the hypothesis whether Purkinje cells transmit a circadian modulated signal to their targets in the brain. Extracellular recordings from brain slices revealed the typical discharge pattern previously described in vivo in single cell recordings showing basically a tonic or a trimodal-like firing pattern. However, in acute sagittal cerebellar slices the average spike rate of randomly selected Purkinje cells did not exhibit significant circadian variations, irrespective of their specific firing pattern. Also, frequency and amplitude of spontaneous inhibitory postsynaptic currents and the amplitude of GABA- and glutamate-evoked currents did not vary with circadian time. Long-term recordings using multielectrode arrays (MEA) allowed to monitor neuronal activity at multiple sites in organotypic cerebellar slices for several days to weeks. With this recording technique we observed oscillations of the firing rate of cerebellar neurons, presumably of Purkinje cells, with a period of about 24 hours which were stable for periods up to three days. The daily renewal of culture medium could induce circadian oscillations of the firing rate of Purkinje cells, a feature that is compatible with the behavior of slave oscillators. However, from the present results it appears that the circadian expression of cerebellar clock genes exerts only a weak influence on the electrical output of cerebellar neurons.     

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.     

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

Minor structural modifications convert a selective PPARalpha agonist into a potent, highly selective PPARdelta agonist

We report the solid-phase synthesis and pharmacological evaluation of a new series of small-molecule agonists of the human peroxisome proliferator-activated receptor delta (PPARdelta) based on a lead structure from our PPARalpha program. Compound 33 showed good pharmacokinetics.     

Automated cerebrospinal fluid cytology: limitations and reasonable applications

OBJECTIVE: To evaluate the precision and clinical applicability of the Bayer ADVIA 120 cytometer (Bayer Healthcare, Fernwald, Germany) for cerebrospinal fluid (CSF) cell count and differentiation. STUDY DESIGN: One hundred six analyses of CSF from 98 patients by the ADVIA 120 were compared with routine cell count and microscopic differentiation. Correlation coefficients were calculated. RESULTS: In general, the total cell counts of both methods correlated well. The best correlations were seen at higher cell counts, > or = 100 cells per microliter with < 100 erythrocytes per microliter. The best correlations of cell differentiation were seen for lymphocytes and neutrophils, while the results for monocytes and eosinophils were less precise. In some cases, considerable differences between automated and microscopic cell counts and differentiation were seen that were relevant to clinical decision making. The detection of pathologic cell types, such as hemosiderophages, mitoses and neoplastic cells, was not provided by automated cytometry. CONCLUSION: When experienced personnel are not available, a preliminary cell count and differentiation between neutrophilic and lymphocytic reactions by automated cytometry may be valuable in allowing initial therapeutic decision making. Since the detection of pathologic cell types is not provided and the precision at low cell counts is only moderate, a personal microscopic evaluation of each sample is still indispensable to avoid misdiagnoses.