Norio Ishida

Humoral signals mediate the circadian expression of rat period homologue (rPer2) mRNA in peripheral tissues

Northern blot analysis revealed the circadian expression of rat period homologue (rPer2) mRNA in peripheral mononuclear leukocytes that have no neuronal connections. The mRNA expression in the suprachiasmatic nucleus (SCN), peripheral mononuclear leukocytes, and other peripheral tissues exhibited prominent, synchronous circadian oscillation with peaks at early night. As we have previously shown the peripheral circadian expression of rPer2 mRNA is abolished in SCN-lesioned rats [Sakamoto, K., Nagase, T. Fukui, H., Horikawa, K., Okada, T., Tanaka, H., Sato, K., Miyake, Y., Ohara, O., Kako, K. and Ishida, N., J. Biol. Chem., 273 (1998) 27039-27042], our findings suggest that some humoral signals, driven by the SCN, mediate the circadian expression of mammalian per homologues in peripheral tissues.

Circadian expression of serotonin N-acetyltransferase mRNA in the rat retina

To investigate the molecular mechanism of the melatonin rhythm in the mammalian retina, we examined the temporal mRNA expression pattern of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; the rate-limiting enzyme in melatonin synthesis) in the rat retina. Northern blot analysis showed that in a daily light-dark cycle retinal AA-NAT mRNA was low during the day and increased more than threefold at night, and this daily rhythm persisted even in constant darkness. These findings suggest that AA-NAT mRNA expression in the rat retina is regulated by an endogenous circadian clock.

Light-induced phase-shifts in the circadian expression rhythm of mammalian Period genes in the mouse heart

To investigate the molecular mechanism that regulates circadian rhythms in mammalian peripheral tissues, we examined the phase shifts evoked by light exposure in the circadian mRNA expression rhythms of mammalian Period genes (mPer1, mPer2 and mPer3) and a clock‐controlled gene Dbp, in the mouse heart, by Northern blot analysis. The light pulse did not induce any acute mRNA expression of mPer in the heart, but the pulse gave rise to phase shifts in the circadian mRNA rhythms. On the first day after the exposure, only mPer1 mRNA showed a phase shift, whereas obvious phase shifts were not observed in the rhythms of mPer2, mPer3 and Dbp mRNAs. On the second day, phase shifts occurred to a similar extent in the mRNA rhythms of all four genes examined. The rhythm of mPer1 mRNA shifted fastest among those of the three mPers. Therefore mPer1 seems to play an important role in phase resetting of mammalian peripheral oscillators. Immediate responses to light pulses in mRNA expression of mPers may not be required for phase shifting of peripheral circadian oscillators. Our findings suggest that mammals require more than one day to have peripheral oscillators entrained to a new daily schedule.

The role of transcription factors in circadian gene expression.

There are three basic components present in all species which are essential for the circadian gene expression; an input pathway which connects the clock to the environment, the clock oscillator, and an output pathway which connects the pacemaker to the resulting biological phenomena. In this review, an attempt to separate the three processes will be made from the molecular biological stand point. In the pineal of birds, Drosophila, and algae, cAMP/PKA pathway is functional in the output, but in mammalian suprachiasmatic nucleus (SCN), this pathway including the CREB/c-fos pathway, is believed to function in the input pathway. We propose here a model that easily explains the controversial results of the CREB/c-fos story in the SCN by considering this signal transduction pathway as an output. Finally, we propose the importance of E-box and bHLH-PAS type transcription factors, in the clock oscillator in the SCN.

Molecular cloning of serotonin N-acetyltransferase gene from the mouse and its daily expression in the retina

The primary structure of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT: the rate-limiting enzyme in melatonin synthesis) in the mouse retina was deduced from the cDNA nucleotide sequence. The deduced protein consisted of 205 amino-acid residues with sequences highly conserved in AA-NATs of vertebrates, and was 96% identical to rat AA-NAT. Northern blot analysis of mouse retinal mRNA showed two obvious bands, of 1.5 kb and 4.5 kb in length. The levels of both transcripts were low at day and high at night, but the night-to-day ratios were <2. These findings suggest that the expression mechanism of AA-NAT transcripts in the mouse retina may be different from those in other mammals, where a single transcript of AA-NAT is normally observed in Northern blots.

The pineal gland is not essential for circadian expression of rat period homologue (rper2) mRNA in the suprachiasmatic nucleus and peripheral tissues

To investigate the functional involvement of the pineal gland in circadian expression of the rat period homolog gene (rPer2) in the suprachiasmatic nucleus (SCN) and peripheral tissues, we performed Northern blot analysis in tissues from pinealectomized rats. The ectomy did not have any significant effects on rPer2 mRNA expression patterns both in a daily light-dark condition and in a constant darkness. These results suggest that the rhythmic secretion of pineal melatonin is not essential for the circadian expression of clock genes in the SCN and other peripheral tissues of rats.

Examination of DNA-binding activity of neuronal transcription factors by electrophoretical mobility shift assay.

Electrophoretical mobility shift assay (EMSA) is a simple, rapid, and highly sensitive technique for detection of single- or double-stranded DNA-binding proteins such as transcription factors in crude nuclear extracts (F.M. Ausubel, R. Brent, R.E. Kingston, D. D. Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, 1989, pp. 12.0.1-12.2.10 [1]; J. Carey, Gel Retardation. Methods Enzymol., 208 (1991) 103-117 [2]). By using this technique, it is possible to quantify the abundance, relative affinity and binding specificity of DNA-binding proteins. Since proteins which bind specifically to radiolabeled DNA probes retard the mobility of the probe during electrophoresis (it also called gel retardation assay), discrete bands correspond to the individual DNA-protein complexes. Furthermore, EMSA allows one to determine which member(s) of a certain protein family are included in the DNA-protein complex by means of specific antibodies raised against the DNA-binding protein (supershift assay).

Circadian regulation of per repeat mRNA in the suprachiasmatic nucleus of rat brain

We have recently reported fluctuations in the expression of the period repeat sequence (pp2.5) during light-dark cycles in the suprachiasmatic nucleus (SCN) of rat [Neurosci. Lett., 122 (1991) 113-116]. Presently, we performed in situ hybridization which shows that the fluctuation of pp2.5 expression continues during constant darkness conditions in the SCN of rat. The light exposure during subjective night but not subjective day triggered its elevated expression in a time-dependent manner which is parallel to that of c-fos expression. The results suggest the involvement of pp2.5 in the entrainment pathway of the circadian rhythm in rodent SCN.

Regulation of cAMP response element binding protein (CREB) binding in the mammalian clock pacemaker by light but not a circadian clock

Mammalian circadian rhythms are considered to be regulated by a clock pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanism of entrainment and oscillation of circadian rhythm are not well understood but photic induction of immediate-early gene (IEG) expression in the SCN is thought to play a role. Here we show that under 12 h light:12 h dark (LD) condition, the cAMP response element binding protein (CREB) binding to cAMP responsive promoter element (CRE) of NMDAR1/zeta1 promoter region in the SCN is higher during the light than the dark by electro-mobility shift assay (EMSA). When animals are placed in constant dark, CREB DNA binding activity in the SCN is low and does not vary with circadian time when compared with cortex nuclear extract as a control. Most significantly, photic induction of CREB binding activity in the SCN occurs at all circadian times tested, indicating that CREB DNA binding in the SCN is not gated by the endogenous clock. These results implicate the role of CREB in photic neuronal signaling in the SCN and suggest that CREB DNA binding activities may not be regulated by a circadian clock.

Molecular cloning of the cone-rod homeobox gene (Crx) from the rat and its temporal expression pattern in the retina under a daily light-dark cycle.

The primary structure of the cone-rod homeobox protein (CRX), a member of the OTX-like homeobox protein family, in the rat was deduced from the cDNA nucleotide sequence. The deduced protein consisted of 299 amino acid residues with motifs conserved in mammalian CRXs, and was 98% identical to mouse CRX. Northern blot analysis showed that Crx mRNA levels in the rat retina were constant under a daily light-dark cycle. These findings suggest that the expression mechanism of rat Crx in the retina was different from that in the pineal, where Crx mRNA exhibits a daily expression rhythm.