Identification of P2X7 (P2Z) receptor in N18TG-2 cells and NG108-15 cells.

ATP activates a nonselective cation current by stimulating the P2Z receptor in NG108-15 cells--a hybrid cell line of the mouse neuroblastoma N18TG-2 cells and the rat glioma C6Bu-1 cells. Recently, the P2X7 receptor was cloned from the rat brain and was found to have electrophysiological properties similar to those of the P2Z receptor. We examined the expression of P2X7 receptor mRNA in NG108-15 cells as well as in their parent cell lines, N18TG-2 and C6Bu-1 cells, by reverse transcription-polymerase chain reaction (RT-PCR). The cDNA templates from these cell lines were amplified with primers specific to the P2X7 receptor sequence. Positive signals were detected in the RT-PCR products from NG108-15 and N18TG-2 cells but not from C6Bu-1 cells. We next examined the effect of ATP on the membrane current in N18TG-2 cells and C6Bu-1 cells by whole-cell voltage clamp. In N18TG-2 cells, ATP induced a sustained current with a reversal potential of 9.3 +/- 1.2 mV (n = 22) in a concentration-dependent manner with an EC50 of 1.76 +/- 0.18 mM (n = 36). In contrast, ATP (1 mM) did not induce any current in C6Bu-1 cells. The ATP-induced current in N18TG-2 cells resembled that in NG108-15 cells in the following points: (a) The currents did not desensitize significantly. (b) EC50 values of ATP are of millimolar order. (c) Benzoylbenzoyl-ATP was a more potent agonist than ATP. (d) The current was larger in methanesulfonate than in Cl- external solution. (e) The current was larger at lower external Mg2+ concentrations. These results suggest that the hybrid NG108-15 cells possess a P2X7 receptor like the P2Z receptor and that the ability of expressing this channel originates from N18TG-2 cells but not from C6Bu-1 cells.

Effects of anions on ATP-activated nonselective cation current in NG108-15 cells.

Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS-) than in Cl- external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density (Imax) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent Imax decreased in the order of aspartic acid (Asp-) > MS- > F- > Cl- > Br- > or = I-. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp- < F- < MS- < Br- < Cl- < I-. Thus both Imax and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl- was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.

ATP-activated nonselective cation current in NG108-15 cells.

ATP (1 mM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca2+. We examined the mechanism of the ATP-elicited sustained [Ca2+]i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 +/- 0.8 mV (n = 10) in Tyrode external solution. The EC50 of ATP was approximately 0.75 mM. The permeability ratio of various cations carrying this current was Na+ (defined as 1) > Li+ (0.92 +/- 0.01; n = 5) > K+ (0.89 +/- 0.03; n = 6) > Rb+ (0.55 +/- 0.02; n = 6) > Cs+ (0.51 +/- 0.01; n = 5) > Ca2+ (0.22 +/- 0.03; n = 3) > N-methyl-D-glucamine (0.13 +/- 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg2+. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5'-thiotriphosphate (ATPgammaS), and adenosine 5'-O-(2-thiodiphosphate), but not adenosine, ADP, alpha,beta-methylene-ATP (AMPCPP), beta,gamma-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPgammaS, but not AMPCPP or AMPPCP, increased the sustained [Ca2+]i increase. We conclude that ATP activates a class of Ca2+-permeable nonselective cation channels via the P2z receptor in NG108-15 cells.

[Robustness of the analytic/holistic processing strategies in various cognitive tasks].

Robustness of the analytic/holistic processing strategies was examined in various cognitive tasks. The tasks in Experiment I were to classify facial patterns and verbal materials of different category structures into two categories. Stimuli were presented either with (thematization condition) or without a theme. Three types of strategies were discerned; consistently-analytic, consistently-holistic and optimal shifting. Experiment II was designed to test their performance in the Kagan type perceptual and memory identification tasks. The results from the two experiments showed that about 30% of the subjects adhered to either analytic or holistic strategy across tasks and experiments. They exhibited responses similar to the reflective and impulsive types. Approximately 40% of the subjects adjusted their strategies according to the task demands.

N-terminal amino acid sequence analysis of small subunits of photosystem I reaction center complex from a thermophilic cyanobacterium, Synechococcus elongatus Nägeli.

Four small subunits (14, 13, 10, and 8 kDa) of the photosystem I reaction center complex were isolated from a thermophilic cyanobacterium Synechococcus elongatus and their N-terminal amino acid sequences determined. Sequence analysis of the 10-kDa subunit revealed that the distribution of cysteine residues, Cys-X-X-Cys-X-X-Cys-X-X-X-Cys-Pro, is characteristic of bacterial-type ferredoxins, and that its partial sequence is highly homologous to that deduced from the chloroplast gene frx A of liverwort. This indicates that the 10-kDa polypeptide is an apoprotein carrying two iron-sulfur centers, FA and FB, assigned as [4Fe-4S] clusters, which mediated the light-activated transfer of electrons from P700 in photosystem I reaction center complex to soluble ferredoxin. The amino acid sequence of the 14-kDa polypeptide also showed similarity to that of the 20-kDa polypeptide from spinach chloroplast that can be chemically crosslinked with soluble ferredoxin. Thus, the 14-kDa polypeptide appears to be the ferredoxin 'docking' protein.

[Combination chemotherapy of cyclophosphamide, adriamycin and cisplatin in advanced urothelial cancer].

Sixteen patients with advanced evaluable urothelial cancer were treated with a chemotherapy regimen of cyclophosphamide, adriamycin and cisplatin (CAP). Cisplatin 50 mg/m2 and adriamycin 30 mg/m2 were given on the first day and cyclophosphamide 200 mg/m2 was given from the second to the fifth day. This course was repeated every 3 weeks. The objective response rate was 25% (4 of 16 patients), with 1 patient achieving complete remission. The survival time of responders was longer than that of nonresponders, although the difference was not significant (generalized Wilcoxon method). As side effects, nausea with vomiting (43.8%), renal dysfunction (6.3%), anemia (12.5%), leucopenia (12.5%), thrombocytopenia (25.0%), alopecia (68.8%), heart failure (6.3%) and peripheral neuropathy (6.3%) were noticed. One patient died of sepsis due to agranulocytosis and another died suddenly of heart failure.

[An analysis of Gestalt characteristics of 881 Japanese/Chinese Kanji].

The Gestalt characteristics of 881 Kanji were rated on each of 10 SD-like scales by 554 subjects. High reliability was found for all rating scales. Principal component analysis of the mean rating values of the Kanji for each scale showed eight of the ten scales to be appropriate for the measurement. The appropriate scales were: complexity, compactness, elongation, openness, straightness, roundness, stability, and symmetry.

Levels of carbenicillin in serum and prostatic tissue after intravenous administration in patients with benign prostatic hyperplasia.

Carbenicillin (CB-PC) was administered intravenously (5 gm over one-half hour) to 25 patients suffering form benign prostatic hyperplasia. Prostatic biopsies and sera were obtained one, two, three, four, and five hours later for histology and biochemical analyses of the serum and prostatic tissue levels of CB-PC. The serum and prostatic tissue levels of carbenicillin attained were sufficient to eradicate gram-negative bacteria within the five time intervals studied. The ratio of prostatic tissue level of CB-PC to the serum level of this agent increased in proportion to the grading of round cell infiltration of the prostatic tissue. The results of this study point to the clinical usefulness of CB-PC in the treatment of chronic and acute prostatitis.