Tadaomi Morimasa

Proteome analysis of mouse brain: Two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two‐dimensional gel electrophoresis (2‐DE). 2‐DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino‐terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N‐termini and 122 proteins were identified. Twenty‐seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2‐DE map images and the respective spot data files with 15 related references.

Circadian rhythm of [3H]imipramine binding in the rat suprachiasmatic nuclei

High affinity imipramine binding undergoes circadian variations of ca. 35% amplitude in many rat brain nuclei. The suprachiasmatic nuclei of the anterior hypothalamus (considered to be the circadian pacemaker driving many overt rhythms) has highest imipramine binding at the end of the dark and lowest at the end of the light phase. A similar circadian rhythm has previously been observed for serotonin uptake in the suprachiasmatic nuclei. In conjunction with other findings, these data indicate that serotonergic turnover in the suprachiasmatic nuclei decreases at lights on and increases at lights off.

Hypothalamicα2- and β-adrenoceptor rhythms are correlated with circadian feeding: evidence from hronic methamphetamine treatment and withdrawal

Abstract The circadian regulation of food intake in rats is correlated with a bimodal rhythm of β-adrenoceptor binding in the lateral hypothalamus and a unimodal rhythm ofα2-adrenoceptor binding in the medial hypothalamus. Chronic methampheamine treatment provides evidence for a functional correlation: β-adrenoceptor binding in the lateral hypothalamus is reduced at dusk, together with reduction of food intake;α2-adrenoceptor binding in the medial hypothalamus is increased at dawn, together with persistent food intake. Longterm changes in these two adrenergic systems are also correlated with homeostasis of food intakke: 24-h mean β-adrenoceptor binding is reduced andα2-adrenoceptor binding is increased upon methamphetamine withdrawal, when rebound feeding occurs. Corticosterone, although normally coupled to adrenergic mechan isms that regulate feeding, is phase delayed after chronic methamphetamine treatment.

CHANGES IN MUSCARINIC CHOLINERGIC, PCP, GABAA, D1, AND 5-HT2A RECEPTOR BINDING, BUT NOT IN BENZODIAZEPINE RECEPTOR BINDING IN THE BRAINS OF AGED RATS

We used in vitro quantitative autoradiography to investigate changes in neurotransmitter receptor binding, including muscarinic cholinergic, PCP, GABAA, benzodiazepine, D1 and 5-HT2A receptor, in the brains of aged rats, compared with such binding in young rats. Scatchard analysis revealed that the maximal number of binding sites for [3H]quinuclidinyl benzilate (QNB) in the caudate/putamen and accumbens was significantly decreased in aged rats compared with young rats, while its affinity remained unchanged. The specific binding of [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) for the ion channels coupled with N-methyl-D-aspartate receptors in the caudate/putamen and hippocampus was significantly decreased in aged rats compared with young rats. The [3H]muscimol binding in aged rats was decreased in all brain regions examined compared with that in young rats, whereas [3H]flunitrazepam binding was not changed in any brain regions. The [3H]SCH23390 binding for dopamine D1 receptors was significantly increased in the parietal cortex, but decreased in the caudate/putamen and accumbens of aged rats compared with that in young rats. The [3H]ketanserin binding for 5-HT2A receptors in the cortex and accumbens was significantly decreased in aged rats compared with young rats. These results suggest that uneven changes in receptors for various neurotransmitters throughout the brain may be responsible for the decline of brain function in aged rats.

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock‐out mice (p53−/−) were analyzed by two‐dimensional gel electrophoresis (2‐DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18‐week‐old non‐stress‐induced mice were analyzed. The morphology of brains from p53−/− mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2‐D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53−/− specific, whereas 12 of 1008 spots from lung tissue were p53−/− specific. N‐terminal amino acid sequence was determined for p53−/− specific proteins. In all brain tissues from p53−/− mice, a newly identified mouse mitochondrial NADH‐ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain‐type creatine kinase B chain and tubulin β‐5 N‐terminal fragment were increased in the p53−/− cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53−/− mice. The alterations in protein expression identified in this study may imply a p53‐related brain function. This is the first proteomic analysis on the p53−/− mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

Chronic methamphetamine and its withdrawal modify behavioral and neuroendocrine circadian rhythms

Chronic methamphetamine (MA) administration via the drinking water not only induced hyperactivity, but phase delayed the rat rest-activity cycle under entrained conditions. The minimum and/or maximum of the rhythms in eating, drinking, body weight, core body temperature and plasma/urine corticosterone were delayed. The different phase shifts of peak and trough values can also be a result of modulation in the wave form of the rhythms. The fall, but not the rise, of nocturnal pineal melatonin secretion occurred 4 hours earlier in MA-treated rats than in controls: this pattern was still present 1 week after withdrawal, but no longer after 4 weeks withdrawal. Neither chronic MA nor its withdrawal had any effect on plasma thyrotropin. These patterns after chronic MA intake fall into two groups: those rhythms whose peak and/or trough are delayed and those that are not. We thus interpret chronic MA application as modulating the eating rhythm (though not directly through rhythmic MA levels in the CNS), and that this in turn changes all food-dependent rhythms. In contrast, the circadian rhythms of melatonin and thyrotropin remain independent.

The capsid protein of bacteriophage lambda and of its prehead

Abstract Several complex DNA bacteriophages are formed by packing the genome into precapsids. In many of these cases the capsid enlarges during the packaging process (Casjens & King, 1975; Hohn et al., 1976; leading to further references). At least in bacteriophage λ this transition is conservative and main capsid subunits are neither added to, nor removed from the maturing precapsid (Hohn et al., 1974,1976; Kaiser et al., 1975). Thus the enlargement of the head is caused by a rearrangement of the subunits and/or a structural shift within each subunit. There is evidence that the transition from precapsid to capsid is unidirectional (Hohn et al., 1976), and such an irreversible process would be most easily understood if it is caused by a change or modification of primary structure of the subunit. In fact, in bacteriophages T5, P2 and T4, morphogenetic protein cleavage of the main capsid subunit was observed (for a review see Hershko & Fry, 1975). However, it is not yet clear whether this cleavage is the cause or the result of head enlargement and DNA filling. In bacteriophage λ protein cleavage of the major capsid protein (E-protein; pE) is not detectable by standard sodium dodecyl sulfate/acrylamide gel electrophoresis. To detect possible minor cleavages, in this study amino acid composition and end groups of precapsid and capsid protein were determined. Furthermore, it was checked whether glucosylation or phosphorylation of the capsid protein occurs. The study was extended to include pE from a further type of small capsid (Nu3− petit λ), which we suspect to have still another configuration than the prehead and the head (Hohn et al., 1975).

TEMPORAL DISTRIBUTION OF (3H)-IMIPRAMINE BINDING IN RAT BRAIN REGIONS IS NOT CHANGED BY CHRONIC METHAMPHETAMINE

Specific binding of [3H]-imipramine in the rat suprachiasmatic nuclei, occipital cortex and caudate putamen underwent significant and replicable changes throughout 24 hr under a light-dark cycle or under constant conditions. Daily variations were also found in the medial and dorsal raphe nuclei and the lateral hypothalamus. Methamphetamine, a psychoactive drug with marked effect on circadian rhythms in physiological and hormonal parameters and adrenergic receptors, did not have any significant effect on imipramine binding rhythms in eight discrete brain regions. Thus a drug known to reduce serotoninergic neurotransmission did not change characteristics of the modulatory binding site related to serotonin uptake.

Circadian Feeding Rhythms: Central Mechanisms and Methamphetamine Modulation

The circadian rhythm of food intake is driven by a putative central pacemaker in the suprachiasmatic nuclei (SCN). Lesions of the SCN abolish circadian rhythms of feeding and drinking as well as the rest-activity cycle (Moore, 1983). The connections from the SCN to the ventromedial hypothalamus (VMH), the “satiety” centre, and to the lateral hypothalamus (LH), the “feeding” centre, appear indispensable to the circadian organisation of ingestive behaviour (see also Rietveld, this volume). Since anticipatory running activity induced by a restricted food regimen persists in SCIM-lesioned rats (Stephan et al., 1979), and maintains certain circadian characteristics, it is considered that weak circadian oscillators, located in the VMH and LH, may persist in the absence of the SCN (Moore, 1983).

Levodopa induces AP-1 and CREB DNA-binding activities in the rat striatum

Abstract In order to elucidate the effect of levodopa and bromocriptine on the DNA‐binding activities of transcription factors, AP‐1 and CREB DNA‐binding activities were investigated using gel‐shift assay. Intraperitoneal administration of 100 mg/kg levodopa with 50 mg/kg benserazide in rats increased both AP‐1 and CREB DNA‐binding activities in the dorsolateral aspect of the striatum. The major proteins composing the increased AP‐1 were JunB and JunD. Bromocriptine at doses of 2.5 and 5.0 mg/kg, however, did not increase these binding activities. Present results suggest that levodopa but not bromocriptine induces these transcription‐regulating proteins in the striatum with normal dopaminergic functioning.