Takashi Mita

Identification of (Na,K)ATPase inhibitor in brine shrimp, Artemia salina, as long-chain fatty acids

SummaryAn inhibitory activity to (Na,K)ATPase was found in cell extracts of the brine shrimp, Artemia salina, irrespective of its developmental stages. Organic solvent extraction together with gas chromatographic analysis reveals that the inhibitory activity is due to long-chain, non-esterified fatty acids and their derivatives. Unsaturated fatty acids, especially with cis-configuration, are more effective in inhibition than saturated ones.

Phenytoin, an antiepileptic drug, competitively blocked non-NMDA receptors produced by Xenopus oocytes

The blocking effect of phenytoin (PHT) and other antiepileptic agents on the glutamate receptors was investigated under voltage-clamp conditions using Xenopus oocytes that translated ddY-stock mouse brain mRNA. PHT shifted the concentration-response curve for kainate to right without significant block on the maximum response obtained, indicating that PHT competitively blocks the non-NMDA receptors. An apparent dissociation constant of PHT was 1.9 x 10(-4) M. The block was voltage-independent. Other antiepileptic agents examined hardly blocked the kainate responses except phenobarbital that blocked the responses. The possibility that the competitive block constitutes a part of the antiepileptic action of PHT was discussed.

Analysis of cis-acting regions upstream of the rat Na+/K+-ATPase α1 subunit gene by in vivo footprinting

By means of in vivo footprinting, we examined the putative cis-acting DNA elements located between -50 and -122 of rat Na+/K(+)-ATPase alpha 1 subunit gene ATP1A1. Proximal and distal GC box sequences and a consensus sequence for the active transcription factor (ATF) were protected for all the tissues examined (kidney, brain and liver). Putative cooperation between two binding factors on the ATF site and the proximal GC box was observed. The overall in vivo footprinting profiles of the three tissues did not exhibit any marked differences that could account for the variation in the extent of tissue-specific transcription. The alpha 1 regulatory element (ARE) found by Suzuki-Yagawa et al. does not appear to be an element responsible for tissue-specific regulation of the gene.

Characterization of a novel promoter structure and its transcriptional regulation of the murine laminin B1 gene

Expression of the laminin B1 gene is known to be induced late during the differentiation of F9 cells by retinoic acid (RA) and dibutyryl cAMP. The involvement of retinoic acid receptors (RARs) has been demonstrated recently in the late induction of laminin B1 gene expression, although the precise regulatory mechanism is not known. In this study, we have reconstituted an efficient in vitro transcription system using F9 nuclear extracts and defined the core promoter structure of the murine laminin B1 gene. The laminin B1 gene was shown to lack a TATA box. The level of the in vitro transcription of the laminin B1 gene was determined by at least three regions between the transcription initiation sites and -100. The most distal region (from -89 to -69) contained three GC boxes. The second region (from -62 to 47) contained a direct repeat of TG(C/A)GCA motif. The proximal region (from -45 to -11) contained another direct repeat of CCTCCCT(C/A)GG motif. A deletion of any one of the three regions respectively decreased the level of transcription to about 20% of wild type DNA. The protein binding analyses revealed that F9 cells contain a factor(s) binding to the TG(C/A)GCA repeat, which was also found in HeLa cells. Together with the observation that the 5 ends of the laminin B1 mRNA from the differentiated F9 cells were identical to those from the undifferentiated F9 cells, it was concluded that the three regions identified here constitute the core promoter of the laminin B1 gene.

The rat peptidylarginine deiminase-encoding gene: structural analysis and the 5′-flanking sequence

Genomic clones of the rat peptidylarginine deiminase (PAD)-encoding gene (PAD) were isolated, and the gene organization was analyzed by restriction mapping and nucleotide sequencing. The PAD spans more than 50 kb and contains 16 exons and 15 introns. The lengths of the introns from 0.5 kb to more than 16.5 kb. A 1.7-kb sequence in the 5-flanking region was determined. S1 nuclease mapping revealed two putative cap sites 79 and 81 bp upstream from the N-terminal ATG codon of PAD, which had been determined by amino acid sequence analysis. This ATG was confirmed to be the translation start site, since no other ATG codon was found in the open reading frame downstream from the cap sites. The 5-flanking sequence contains four potential SP1-binding sites, a putative Pit-1/GHF-1-binding site, four short sequences either identical or homologous to the sequences in the promoter regions of rat or human growth hormone encoding genes, as well as a sequence similar to an estrogen-responsive element. However, neither a typical TATAA box, nor CCAAT box is present. These results provide important clues for elucidating the mechanism of female-specific and/or sex cycle-dependent gene expression.

Properties of isolated extrachromosomal nucleoli fromTetrahymena pyriformis

Extrachromosomal nucleoli were isolated from log phase cells ofTetrahymena pyriformis (amicronucleate strain) in a highly purified state. Nucleoli located at the periphery of the macronucleus were detached from the nucleoplasmic mass of isolated macronuclei with agitation and separated from macronuclei by filtration through a Nuclepore membrane filter (pore size 5 μm). The filtrate constitutes the crude nucleolar preparation, as judged by electron microscopy and DNA analysis. Further purification of the nucleoli was performed by isopycnic centrifugation of the filtrate in a Metrizamide density gradient. After this step, the purity of the nucleoli, as defined by rDNA content and measured by analytical CsCl centrifugation, was almost 100%. Electron microscopy of the purified nucleoli revealed structures that resemble those of in situ nucleoli. Undergraded 35S pre-rRNA, together with 26S and 17S rRNA, could be isolated from purified nucleoli. In vitro RNA synthetic activity was associated with isolated nucleoli. This activity is insensitive to low and high concentrations of α-amanitin, indicating that the form I RNA polymerase is functioning.

254 GABAergic synaptic transmission in the hippocampus of el mice

We have studied if GABAergic inhibition is altered in epileptic El mice compared to control ddY mice with extracellular and intracellular recordings in the hippocampal slices. In El mice, paired-pulse inhibition of the population spike at the short intervals (10 ms) was decreased in the CA1 and the dentate gyrus. Inhibitory postsynaptic potentials in the CA1 decayed faster compared to ddY mice, suggesting decrease in presynaptic GARA release in El mice. In the present study, non-NMDA receptor-mediated discharges and GABAA receptormediated potentials induced by Caminopyridine (4-AP) were analyzed. In El mice, the duration of non-NMDA receptor-mediated discharges was longer than that in ddY mice. GABAA receptor-mediated potentials were not induced by 4-AP. Taken together, our results suggest that GABAergic synaptic transmission in El mice is blocked by interictal epileptiform discharges.