Toru Takumi

A light-independent oscillatory gene mPer3 in mouse SCN and OVLT.

A new member of the mammalian period gene family, mPer3, was isolated and its expression pattern characterized in the mouse brain. Like mPer1, mPer2 and Drosophila period, mPer3 has a dimerization PAS domain and a cytoplasmic localization domain. mPer3 transcripts showed a clear circadian rhythm in the suprachiasmatic nucleus (SCN). Expression of mPer3 was not induced by exposure to light at any phase of the clock, distinguishing this gene from mPer1 and mPer2. Cycling expression of mPer3 was also found outside the SCN in the organum vasculosum lamina terminalis (OVLT), a potentially key region regulating rhythmic gonadotropin production and pyrogen‐induced febrile phenomena. Thus, mPer3 may contribute to pacemaker functions both inside and outside the SCN.

A new mammalian period gene predominantly expressed in the suprachiasmatic nucleus

In mammals, two possible clock genes (Clock, Per1) have very recently been reported. mPer1 (the first identified mouse period gene), in particular, shows a circadian expression in suprachiasmatic nuclei (SCN), the mammalian circadian centre. However, only mPer1 and Clock as clock components may not be sufficient to understand all the events in circadian oscillation and entrainment.

Identification of novel homologues of mouse importin α, the α subunit of the nuclear pore-targeting complex, and their tissue-specific expression

Transport of karyophilic proteins into the nucleus is mediated by nuclear localization signals (NLSs) via a multistep process. The karyophiles are recognized by the importin α subunit in the cytoplasm to form a stable complex, termed the nuclear pore‐targeting complex (PTAC). To date, three different mammalian α subunits (mSRP1/NPI‐1, PTAC58/mPendulin/Rch1 and Qip1) have been identified. In this study, we report the identification of three additional mouse genes homologous to the known α subunits using RT‐PCR methodology and show that the mouse α subunits can be classified into at least three subfamilies, α‐P, α‐Q and α‐S families, each composed of closely related members (more than 80% amino acid sequence identity). These three subfamilies, however, have ∼50% amino acid identity to one another. Northern blot analysis showed that all were differentially expressed in various mouse tissues. These results suggest that the function of these proteins may be controlled in a tissue‐specific manner and that their combinatorial expression may play a role in differentiation and organogenesis.

A mammalian ortholog of Drosophila timeless, highly expressed in SCN and retina, forms a complex with mPER1.

It is now becoming clear that the circadian rhythm of behaviours and hormones arises from a rhythm at the level of gene expression, and that mammals and Drosophila essentially use homologous genes as molecular gears in the control of circadian oscillation. In Drosophila, the period and timeless genes form a functional unit of the clock and its autoregulatory feedback loop for circadian rhythm. However, in mammals, the counterpart of timeless has not been found.

A very small frame-shifting deletion within exon 19 of the Duchenne muscular dystrophy gene

We report the molecular characterization of a Japanese Duchenne muscular dystrophy (DMD) patient. The analysis of genomic gene by polymerase chain reaction indicates that the individuals have a limited deletion within an amplified region, which encompasses exon 19 of DMD gene. The amplified region was sequenced. Comparison of the deletion joint sequence with the normal amplified region sequence indicates that both 5 and 3 deletion end points are present within exon 19 and the deletion removes total 52 bp out of 88 bp of exon 19. Both his mother and sister are carriers of the deletion-containing allele. The mutation introduces a termination codon at residue 791 in exon 20, and is predicted to result in the production of a severely truncated protein. This sort of deletion (designated as DMD-Kobe) might be classified as a new type of DMD gene abnormality.

Inhibition of cardiac delayed rectifier K+ currents by an antisense oligodeoxynucleotide against IsK (minK) and over-expression of IsK mutant D77N in neonatal mouse hearts.

Abstract. The IsK (minK or KCNE1) protein is known to co-assemble with the KvLQT1 (KCNQ1) protein to form a channel underlying the slowly activating delayed rectifier K+ current (IKs). Controversy remains as to whether the IsK protein assembles with ERG (the ether-a-go-go-related gene) products to form or modulate the channel underlying the rapidly activating delayed rectifier K+ current (IKr). We investigated the effects of antisense oligodeoxynucleotides (AS-ODN) against IsK and its mutant D77N [which underlies a form of long QT syndrome (LQT5) in humans] on the delayed rectifier K+ current (IK) of neonatal mouse ventricular myocytes in primary culture. Patch-clamp experiments on these cells showed that IK consists of IKs and IKr. IK was not recorded from ventricular cells transfected with AS-ODN, while it was recorded from cells transfected with the corresponding sense oligodeoxynucleotides (S-ODN). IK was not recorded from cells transfected with the D77N mutant, and the action potential duration was much longer than in cells transfected with wild-type IsK. Furthermore, HERG could not induce currents in COS-1 cells co-expressed with the D77N mutant and HERG (the human form of ERG). These results indicate that the IsK protein associates with both KvLQT1 and ERG products to modulate IKr and IKs in cardiac myocytes.

Restoration of circadian behavioural rhythms in a period null Drosophila mutant (per01) by mammalian period homologues mPer1 and mPer2

Background: Recent molecular studies suggest that mammals and Drosophila utilize similar components to generate circadian (≈ 24 h) rhythms. The first identified circadian clock gene, the period (per) gene, is indispensable for behavioural rhythms in Drosophila and is represented in mammals by three orthologues, the relative roles of which are not known. In this study, we investigated the functional conservation of per by introducing the mouse mPer1 and mPer2 genes, driven by the Drosophila timeless (tim) promoter, into Drosophila melanogaster.

Evidence that TGFβ May Directly Modulate POMC mRNA Expression in the Female Rat Arcuate Nucleus

The purpose of the present study was to determine whether TGFβ, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGFβ1, and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 × 10−10 m TGFβ1 was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGFβ receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGFβ and the presence of type II receptor for TGFβ, respectively, in POMC neurons. The...

Adrenoleukodystrophy without adrenal insufficiency and its magnetic resonance imaging

SummaryFatty acids of plasma and erythrocyte membrane sphingomyelin were determined by gas chromatography-mass spectrometry in adrenoleukodystrophy (ALD) without adrenal insufficiency. Mass chromatogram tracing with the ion at m/z 143 [(CH2)6 COOH3]+ showed increases of saturated very long chain fatty acids in plasma and erythrocyte membrane sphingomyelin in ALD. The C26:0/C22:0 ratios in plasma were 0.121, 0.057 and 0.007 in cases 1 and 2, and a control subject, respectively. The C26:0/C22:0 ratios in erythrocyte membrane sphingomyelin were 0.386, 0.211 and 0.093 in cases 1 and 2 and the control subject, respectively. The demyelinating process of ALD was clearly observed in both the inversion recovery 2100/500 and spin echo 2100/80 scans on magnetic resonance imaging. The magnetic resonance image in case 1 revealed widespread demyelinated lesions, involving almost the entire cerebrum and cerebellum, at 4 years after the onset, while that in case 2 revealed demyelinated lesions mainly limited to parieto-occipital areas at 1 year after the onset.

Transient hyperphenylalaninaemia with a high neopterin to biopterin ratio in urine

A case of transient hyperphenylalaninaemia with a maturational delay of dihydropteridine synthesis is described. With the Guthrie test, the patient showed a blood phenylalanine level of 38 mg dl−1, which had fallen to a normal value without a phenylalanine restricted diet by 3 months of age. The neopterin level and the neopterin to biopterin ratio in the patients urine were very high at 19 days of age. The blood phenylalanine level did not decrease when tetrahydrobiopterin (2.5 mg kg−1) was administered at 19 days of age, while administration of tetrahydrobiopterin (7.5 mg kg−1) at 20 days of age had decreased the blood phenylalanine level to 50% of the preloading level after 24 h. The oral phenylalanine loading test showed the pattern of classic phenylketonuria (PKU) at 15 days of age, but it showed the normal pattern at 1 year 8 months of age.