Differential subcellular distribution of Ca2+/calmodulin-dependent protein kinase II isoforms in the striatum and NG108-15 cells

Four subunits of Ca2+/calmodulin-dependent protein kinase II (CaM KII) have several isoforms, which differ in the variable domain. We previously reported that all subunits were highly expressed in rat striatal neurons. To examine intracellular distributions of CaM KII subunits in the rat striatal neurons, we performed immunoblot analysis with antibodies specific to each subunit in cell extracts from the rat striatum after continuous sucrose density gradient fractionation. The alpha subunit, but not the beta, gamma, or delta subunits, was colocalized with synapsin I, and each subunit showed a distinct distribution pattern in the fractions. To examine further the intracellular distributions of CaM KII isoforms in the same subunit, we established NG108-15 cells stably expressing delta1, delta3, and delta4 isoforms and examined distributions of the delta and gamma isoforms in these cell lines after fractionation. Each of the overexpressed exogenous delta isoforms showed a distinct distribution pattern. The endogenous delta2 was colocalized with the overexpressed delta1, delta3, and delta4 isoforms. However, the endogenous gammaB/gammaC isoforms were not colocalized with the overexpressed delta isoforms. Furthermore, the endogenous delta1 was concentrated in the microsomal fraction from the rat striatum. With the results taken together, it is suggested that CaM KII forms oligomers between isoforms in the same subunit but not in different subunits. The variable domain of CaM KII isoforms might possibly be responsible for targeting to certain intracellular compartments.     

Abnormalities of rate-corrected QT intervals in Parkinson's disease-a comparison with multiple system atrophy and progressive supranuclear palsy

A number of patients with Parkinson's disease (PD) and multiple system atrophy (MSA), in whom sudden death does occur occasionally, have QT or rate-corrected QT (QTc) interval prolongation on electrocardiogram (ECG). Although these QT or QTc interval abnormalities are likely related to autonomic dysfunction, the pathophysiology remains unknown. The aim of this study was to compare the degree of QTc interval prolongation among akinetic-rigid syndromes, namely PD and related disorders, and to evaluate the relationship between QTc prolongation and severity of autonomic dysfunction. Thirty-four patients with PD, 22 with MSA, 11 with progressive supranuclear palsy (PSP) and 30 healthy controls underwent standard autonomic function tests, and electrocardiography variables (RR, QT and QTc intervals) were measured by an ECG recorder with an automated analyzer. The relationship between QTc interval and cardiovascular reflex tests were also analyzed. Orthostatic hypotension and decreased heart rate in response to respiratory stimuli were prominent in MSA, while these were relatively mild in PD. Unlike the RR and QT intervals, the QTc interval significantly differed among all groups (p<0.01). The QTc interval was significantly prolonged in PD (409+/-17 ms; p<0.001) and MSA (404+/-14 ms; p<0.05) compared with healthy controls (394+/-19 ms). Neither autonomic dysfunction nor QTc interval prolongation was evident in PSP. QTc intervals and cardiovascular reflexes did not correlate, except for Valsalva ratio. The QTc interval was obviously prolonged in PD patients to an extent that could not be accounted for simply by autonomic dysfunction levels. MSA patients showed slightly prolonged QTc intervals in spite of marked cardiovascular autonomic dysfunction. Abnormalities of the QTc may reflect the degeneration of cardioselective sympathetic and parasympathetic neurons that cannot be fully captured by cardiovascular autonomic function tests.     

Topostin, a novel inhibitor of mammalian DNA topoisomerase I from Flexibacter topostinus sp. nov. II. Purification and some properties of topostin

We describe the isolation of inhibitors of mammalian DNA topoisomerase I, named topostins, from a culture broth of Flexibacter topostinus sp. nov. and some properties of the inhibitors. Topostins A1, A2 and B were isolated by differential solubility in solvents, adsorption chromatography on silica gel and gel filtration on a Sephadex LH-20 column. Topostins A1, A2 and B had specific activities of 4,700, 16,000 and 22,000 U/mg, respectively. The most active metabolite topostin B comprised two components with MW of 567 and 553 in an equimolar ratio.     

Difference of blood cell counts with reference blood cell counters in three different makers]

To determine the difference of CBC values in different blood cell counters, we have measured the values of CBC by using reference cell counters of three major makers; Coulter, Sysmex and Technicon. Thirty ml of whole blood was taken from 2 healthy individuals and mixed with 45 mg of EDTA -2K. The blood samples were then sent to reference laboratory of each maker under identical conditions. The determination of cell counts was performed 4 hours after taking blood. Ten consecutive analysis were then carried out and the mean of 10 values was calculated. The apparent difference was displayed in leucocyte and platelet counts. In both of two samples, the highest values of either leucocyte or platelet count was obtained with Coulter instrument. And second high values were noted with Sysmex counter and the lowest values were observed by Technicon instrument. The extent of difference between each instrument was over 10% in case of platelet count and was 5 to 10% in leucocyte count. The difference of erythrocyte count, Hgb level, hematocrit value and MCV also existed, but the extent of difference was within 5% in all of these examinations. Since CV of each examination was sufficiently low level, this difference might be due to the difference in accuracy of reference cell counter of each maker. These results suggest that distinct differences of CBC count, particularly platelet and leucocyte count can be produced by the different procedures of calibration for reference counter in each maker of automated blood cell counter.     

Role of CD4 molecule in intrathymic T-cell development.

We have investigated the role of CD4 molecules in intrathymic T-cell repertoire selection. The administration of monoclonal antibody (mAb) to CD4 in organ culture of murine foetal thymus (FTOC) completely inhibited the development of CD4+8- cells, and additional treatment with anti-class II MHC (Ia) mAb caused no further effects on this inhibition. On the other hand, when the potentially autoreactive cells in Mls-1a mice were monitored by expression of the Mls-1a-reactive V beta 6 gene product of T-cell receptor alpha beta (TcR alpha beta), the treatment with anti-CD4 resulted in the appearance of V beta 6-bearing cells to some extent, but this effect was considerably reinforced by the combinatory use of anti-Ia mAb with anti-CD4. In a model system where the bacterial superantigen staphylococcal enterotoxin B serves as self-antigen to deplete V beta 8-bearing cells in FTOC, the depletion of V beta 8+ cells was restored partially by anti-CD4 alone but completely by the combination with anti-Ia. These results suggest that CD4 is indispensable for positive selection of all CD4+8- thymocytes, whereas participation of CD4 in negative selection is only partial. It was also observed that the development of TcR alpha beta-bearing cells in the CD4-8- population was inhibited by the treatment with anti-CD4 mAb. In Mls-1a mice, V beta 6-bearing cells were developed in CD4-8+, CD4+8+, and also in CD4-8- populations after anti-CD4 mAb treatment. It is suggested that TcR alpha beta-bearing CD4-8- cells are possibly originated from CD4+ cells and undergo CD4-mediated thymic selection.