Kaijiro Anzai

Identification of the 23 kDa subunit of tau protein kinase II as a putative activator of cdk5 in bovine brain

Tau protein kinase II (TPKII) was reported previously to be composed of a neuron‐rich cdc2‐related kinase (PSSALRE/cdk5) and 23 kDa subunit. Here we show that the 23 kDa subunit is a putative activator for the kinase activity. Amino acid sequence analysis revealed that the protein was novel and included a partial similarity of amino acids to a cyclin box important for the interaction with cdc2‐related kinase. These results suggest that the 23 kDa subunit, but not cyclin, activates cdk5 in neuronal cells, which no longer exhibit cell cycling but are terminally differentiated cells.

γ-hydroxybutyric acid increases intracellular Ca2+ concentration and nuclear cyclic AMP-responsive element- and activator protein 1 DNA-binding activities through GABAB receptor in cultured cerebellar granule cells

Abstract: In primary cultures of mouse cerebellar granule cells, a brief stimulation by γ‐hydroxybutyric acid (GHB, 0.1–3 mM) significantly increased the intracellular Ca2+ concentration ([Ca2+]i) in a concentration‐dependent manner. In addition, gel mobility assay showed that exposure of the cells to GHB also increased nuclear DNA‐binding activity specific for the cyclic AMP‐responsive element (CRE) and activator protein 1 (AP‐1) transcriptional element in a concentration‐dependent manner. The concentration range of GHB that increased the DNA‐binding activity was essentially the same as the concentration range that elicited the increase in [Ca2+]i. The GHB‐induced increases in [Ca2+]i and nuclear DNA‐binding activity were antagonized by specific GABAB antagonists such as p‐[3‐aminopropyl]‐p‐diethoxymethylphosphinic acid (CGP 35 348) and 3‐N‐[1‐(S)‐(3,4‐dichlorobenzyl)ethanol‐2‐(S)‐hydroxy‐P‐benzylphosphinic acid (CGP 55 845). In addition, the GHB‐induced increase in [Ca2+]i was abolished by pretreatment of the cells with islet‐activating protein. Furthermore, treatment of the cells with 1,2‐bis(2′‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (BAPTA‐AM) and thapsigargin blocked the GHB‐induced increase in nuclear DNA‐binding activity. GHB inhibited [3H]baclofen binding to cultured cerebellar granule cells and mouse cerebellar membranes. These results suggest that stimulation of GABAB receptors by GHB activates intracellular Ca2+ stores and that the increased [Ca2+]i resulting from release of stored Ca2+ plays an important role in increasing the CRE‐ and AP‐1 DNA‐binding activities in cultured cerebellar granule cells.

mRNA population in the liver, kidney and brain of young and senescent mice: Analysis of in vitro translation products

Possible alterations in the population of poly(A)(+)mRNA during ageing were investigated by translation in vitro of poly(A)(+)mRNA from the liver, kidney and brain of male ddY mice of different ages. [35S]Methionine-labeled translation products were analysed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography. A protein product with a molecular weight of 30 000 and isoelectric point of 6.5 was reproducibly observed only in the fluorograms of translation products of poly(A)(+)mRNA derived from the livers of senescent mice (24.5 months old). However, no age-related change was detected in the translation products of the kidney and brain. These results suggest that gene expression in liver cells changes at the level of the population of cytoplasmic poly(A)(+)mRNA during ageing.

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

Abstract: We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82‐nucleotide “identifier” core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12‐nucleotide direct repeat (ATAAATAATTTA).

Isolation and characterization of brain Y-box protein: developmentally regulated expression, polyribosomal association and dendritic localization.

Y-box proteins are DNA- and RNA-binding proteins and control specific gene expression at both transcriptional and translational levels. Particularly in germ cells, it has been reported that Y-box proteins bind to paternal or maternal mRNAs to form mRNPs, mask them from translation and control cell maturation. In this study, we cloned cDNA for a Y-box protein from rat brain. A deduced amino acid sequence of the protein was very similar to that of several other Y-box proteins, and we termed the protein rBYB1 (rat brain Y-box protein 1). rBYB1 was found to be considerably expressed in the cytoplasm of pre- and early postnatal brains, and then decreased to adult levels with brain development. Further, we found rBYB1 to be distributed in both polyribosomal and nonpolyribosomal (mRNP) fractions on a sucrose density gradient, and to be associated with polyribosomes via RNA in the higher-density fractions. Moreover, rBYB1 was localized in dendrites of the primary hippocampal neurons. We compared these sucrose gradient and intracellular rBYB1 localization results with those for fragile X mental retardation protein (FMRP), which is known to be an mRNA-binding and polyribosome-associating translational regulator distributed in neuronal dendrites. Our results suggest that in the brain of prenatal and newborn animals, rBYB1 may function in storage and/or translational regulation of mRNAs involved in the rapid progress of the postnatal brain, and in mature neurons, it may also participate in the control of protein synthesis in dendrites.

Identification of a negative regulatory DNA element for neuronal BC1 RNA expression by RNA polymerase III

BC1 RNA is a neuronal cell-specific RNA polymerase III (Pol III) transcript. The BC1 RNA gene has plural types of Pol III promoters, in addition to which an E-box sequence (E2 site) acts as a transcriptional activator, which is recognized by a brain-specific protein(s). Using an in vitro transcription system, we found that the upstream region of the BC1 RNA gene contained a sequence that interfered with the activity of the E-box element in a distance-independent manner. A tandem repeat within this sequence, which was weakly homologous with the neuron-restrictive silencer element (NRSE) found in the Pol II system, was recognized by a brain nuclear protein. Consistently, the transcriptional activity increased by deleting the tandem repeat sequence. We called this BC1 RNA-repressing element BCRE. The DNA-binding specificities of BCRE-binding protein differed from that of NRSE-binding protein (NRSF). A similar protein with an ability to bind to BCRE was also found in liver and kidney. Furthermore, the glutamate analog kainic acid increased the DNA-binding of both E2 site-binding protein and BCRE-binding protein, and then the levels of BC1 RNA also increased transiently. Our results suggested that both positive and negative regulatory elements contribute to neuronal BC1 RNA expression.

Change with donor age in the degradation rate of endogenous proteins of mouse hepatocytes in primary culture

The degradation rate of [(3)H]leucine labeled intracellular proteins was compared in hepatocytes derived from young (4-7-month-old) and aged (23-month-old) mice. Average half-lives of long-lived proteins were 40 h and 65 h in the cells from young and old animals, respectively. This observation confirms our earlier results that half-lives of foreign proteins introduced into the cells from old mice are 50-60% longer than those of younger counterparts (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133; Ishigami and Goto, 1990, Arch. Biochem. Biophys., 277, 189-195).

Regional distribution of metabotropic glutamate response in the rat brain using Xenopus oocytes

The regional distribution of metabotropic L-glutamate responses was investigated in Xenopus oocytes injected with poly(A)+-RNA from a rat brain which was separated into 3 parts: cerebrum, cerebellum and brainstem. Under voltage-clamp, oscillatory current responses were induced more in cerebellum or brainstem poly (A)+-RNA-injected oocytes, and less in cerebrum poly(A)+-RNA-injected oocytes. These results suggest that the metabotropic glutamate receptor is distributed mostly in cerebellum and brainstem.

Conservation of the ID sequence and its expression as small RNA in rodent brains: analysis with cDNA for mouse brain-specific small RNA

Two cDNA clones for mouse brain-specific small RNA were isolated, whose sequences turn out to be highly homologous to that of a genomic mouse ID-like clone isolated previously. By use of one of these cDNA clones as a probe, the copy numbers of ID-related sequences in several mammalian species were determined. The ID sequence was found to be conserved in mice, rats, hamsters and guinea pigs, but not in non-rodent species. There were about 3 orders of magnitude of difference in the copy numbers of the gene, i.e. rat 4.0 X 10(4), mouse 4.5 X 10(2), hamster 1.5 X 10(2), and guinea pig less than 50. The gene was transcribed as small RNA in the brain of rodent species. The relative concentration of small RNA in the brain was roughly proportional to the copy number of the ID sequence in mice, hamsters, and probably in guinea pigs also. On the other hand, the amount of the RNA in rat brain was much less than that expected from the large copy number of the gene. On the basis of these observations, the possibility is discussed that most of the ID sequences in the rat are pseudogenes.

Brain-specific small RNA during development and ageing of mice

The ID sequence is a repetitive sequence family of rodent species that is transcribed specifically as small RNA in the brain. The amount of brain-specific small RNA in mice was shown to increase greatly in the first month after birth and then to remain unchanged until senescence. This RNA was suggested to be involved in regulation of brain-specific gene expression during postnatal development.