山田 晃司

The influence of nicotine on the expression of egr-1 and nur77 genes by nicotine treatment and withdrawal was assessed using PC12 cells. Nicotine treatment significantly increased the amount of mRNA for egr-1 and nur77 genes at 0.5 h post-nicotine treatment in the PC12 cells. In addition, nicotine withdrawal also elevated transcriptional levels of egr-1 and nur77 genes in Northern blot analyses. Nicotine treatment (200 muM) was also found to significantly increase expressional levels of Egr-1 and Nur77 proteins at 0.5 h post-nicotine treatment. In contrast, Egr-1 and Nur77 protein levels were dramatically decreased by nicotine withdrawal. These results suggest that expressional levels of Egr-1 and Nur77 proteins in neural cells may affect the transcriptional activity of late-response genes after nicotine withdrawal.

The ADAM family of membrane-anchored proteins has a unique domain structure, with each containing a disintegrin and metalloprotease, (ADAM) domain. We have isolated mouse and human cDNAs encoding a novel member of the ADAM family. The mouse and human predicted proteins consisted of 797 and 813 amino acids, respectively, and they shared 70% homology of the entire amino acid sequence. The mouse ADAM gene exists at a single gene locus. The human gene was ubiquitously expressed in tissues other than liver, was mapped to human chromosome 20p13, and was found to consist of 22 exerts. Both proteins have domain organization identical to that of previously reported members of the ADAM family, and contain the typical zinc-binding consensus sequence (HEXGHXXGXXHD) in their metalloprotease domain and a pattern of cysteine localization (C(x)(3)C(x)(5)C(x)(5)CxC(x)(8)C) in their EGF-like domain that is typical of an EGF-like motif. The human protein shows homology with Xenopus ADAM13 (44%), human ADAM19 (40%), and human ADAM12 (39%). From the results of phylogenic analysis based on primary amino acid sequence and distribution of the mRNA, these novel ADAM genes were thus named ADAM33. (C) 2002 Elsevier Science B.V. All rights reserved.

Huntington's disease (HD) is a neurodegenerative disorder characterized by the expansion of CAG repeats in axon 1 of the HD gene. To clarify the instability of expanded CAG repeats in HD patients, an HD model mouse has been generated by gene replacement with human axon 1 of the HD gene with expansion to 77 CAG repeats. Chimeric proteins composed of human mutated axon 1 and mouse huntingtin are expressed ubiquitously in brain and peripheral tissues. One or two CAG repeat expansion was found in litters from paternal transmission, whereas contraction of CAG repeat in litters was observed through maternal transmission. Elderly mice show greater CAG repeat instability than younger mice, and a unique case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat instability is particularly pronounced in the liver, kidney, stomach, and brain but not in the cerebellum of 100-week-old mice. The same results of expanded CAG repeat instability as observed in this HD model mouse were confirmed in the human brain of HD patients. Glial fibrillary acidic protein (GFAP)-positive cells have been found to be increased in the substantia nigra (SN), globes pallidus (GP), and striatum (St) in the brains of 40-week-old affected mice, although without neuronal cell death. The CAG repeat instability and increase in GFAP-positive cells in this mouse model appear to mirror the abnormalities in HD patients. The HD model mouse may therefore have advantages for investigations of molecular mechanisms underlying instability of CAG repeats. (C) 2001 Wiley-hiss, Inc.

NTAK (neural- and thymus-derived activator for the ErbB kinase, neuregulin-2) is a novel member of the epidermal growth factor (EGF) family. We have isolated and characterized the human NTAK gene, comprising 12 exons spanning in excess of 55 kilobases (kb). The 7.0 kb long mRNA of the human NTAK gene was expressed in the human neuroblastoma SK-N-SH cell line with two alternative isoforms detected. Furthermore, six isoforms have been identified from rat brain and PC-12 cells. Although the α isoform of the NTAK gene was found to be expressed in all tissues including brain, the β isoform was expressed only in rat brain tissues. Potential regulatory regions included consensus binding sites for AP-2, TF-IIIA, Sp-1, and YY-1 located in the 5'- flanking region of the NTAK gene. (C) 2000 Elsevier Science B.V.

Neural- and thymus-derived activator for ErbB kinases (NTAK) is a recently described member of the neuregulin family that binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Rat NTAK has at least five alternative-spliced isoforms: alpha 1, alpha 2a, alpha 2b, beta, and gamma. In order to understand their biological properties, this study focused on the NTAK alpha 2a and beta isoforms, which have different EGF-like domains. The effect of the se isoforms on cell growth and tyrosine phosphorylation in human breast cancer cells, MDA-MB-453 and T47D, was examined using the recombinant proteins. In terms of cell growth, NTAK alpha 2a and NTAK beta preferentially stimulate T47D cells and MDA-MB-453 cells, respectively, in a dose-dependent manner. Although both NTAKs induce the highest level of tyrosine phosphorylation of ErbB2, NTAK alpha 2a and NTAR beta preferentially induce ErbB3 and ErbB4 phosphorylation, respectively. Thus, NTAK alpha 2a and NTAK beta stimulate cell growth in different ways, by means of different combinations of receptors.

The influence of nicotine on the expression of Fos family proteins, which specifically formed complexes with the AP-I sequence, was assessed, mRNA for c-Fos, c-jun and jun-B were up-regulated at 0.5 h after nicotine treatment, elevated c-Fos also being apparent after withdrawal. Although nicotine failed to up-regulate the mRNA level of the fra-1 gene, the Fra-1 protein was highly expressed after both treatment and withdrawal. These results indicate that nicotine treatment may affect the transcriptional activity of many genes through c-Fos and c-Jun protein expression in neural cells, and that Fra-1 protein may make a contribution. These changes in immediately early genes (IEGs) may be associated with nicotine withdrawal symptoms. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.

Changes in number and the genomic organization of Hox:genes have played an important role in metazoan body-plan evolution. They make cluster(s), and in vertebrates, each cluster contains different number of Hox genes that have been classified into 13 groups. There are 39 Hox genes in four clusters on different chromosomes in the mammalian genome. In the fish, while 31 Hox genes in four clusters have been identified in pufferfish Fugu. rubripes, 47 Hox genes in seven clusters exist in the zebrafish Danio rerio. To estimate the evolutionary origin of Aox organization in ray-finned fishes, we searched for Hox genes in the medaka fish Oryzias latipes, with a taxon thought to be widely separated from those of pufferfish and zebrafish. We synthesized various mixed oligonucleotides that can work as group-specific primers for PCR, then cloned and sequenced amplified fragments. Numbers of Hox genes identified in the present study were 2 for group 1, 2 for group 2, 1 for group 3, 3 for group 4, 6 for groups 5-7, 2 for group 8, 4 for group 9, 3 for group 10, 1 for group 12, and 3 for group 13. The primers specific for group 11 did not function in this study. Thus, at least 27 Box genes are present in medaka genome, suggesting that the Hox gene complexity of the medaka genome is similar to that of the puffer-fish rather than the zebrafish. (C) 1999 Academic Press.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), enhances transcription of many eukaryotic genes, including that for dopamine P-hydroxylase (DBH). In the present study, we report identification and characterization of a novel sequence motif residing in the 5'-flanking region of the human DBH gene, which mediates transcriptional induction by TPA. Deletional analyses indicated the promoter region between -223 and -187 base pairs to be critical. Whereas this region does not contain any putative regulatory motifs with significant sequence homology to the AP-1 motif, extensive deletional and site-directed mutational analyses indicated that a sequence between -210 and -199 base pairs, B'-ATCCGCCTGTCT-3', may represent a novel TPA-response element (TRE), In addition, alteration of the YY1-binding site decreased TPA-mediated induction of the DBH promoter activity, suggesting that contiguous cis-regulatory element(s) cooperate with this novel sequence motif, Furthermore, insertional mutation analyses between the YY1-binding site and the cyclic AMP-responsive element indicated that the stereospecificity of these motifs is important for intact transcriptional induction by TPA, Taken together, these data suggest that transcriptional up-regulation of the human DBH gene in response to TPA requires coordination of a novel TRE (human DBH TRE, hDTRE), cyclic AMP-responsive element, and the YY1-binding site.

A novel member of the epidermal growth factor (EGF) family, the neural-and thymus-derived activator for ErbB kinases (NTAK), has been purified and cloned. Five alternative spliced isoforms have been detected in the rat adrenal pheochromocytoma cell line, PC-12 cells. The rat NTAK alpha 2a isoform exhibits 94% identity in its primary sequence with the human NTAK alpha isoform. In vivo, NTAK is only expressed in the brain of rat E11.5 embryos, and in the brain and thymus of adult rats. The soluble 46 kDa form binds directly to ErbB3 and B4, but not to ErbB1 or B2. NTAK, however, transactivates ErbB1 and B2 via heterodimerization with ErbB3 or E4. NTAK stimulates the differentiation of MDA-MB-453 cells and competitively inhibits the binding of [I-125] neuregulin to these cells. In addition to these neuregulin-like properties, NTAK exhibits limited structural homology to neuregulins in the immunoglobulin (Ig)-like, EGF-like, and hydrophobic domains. Thus, NTAK appears to be a new member of the EGF family displaying neuregulin properties.

To understand the molecular mechanism of nicotine addiction, we examined the mRNA level of the tyrosine hydroxylase (TH) gene and that of the nicotinic acetylcholine receptor (nAChR) genes by long-term nicotine treatment. The transcript levels of the four subunit genes of the nAChR (alpha 3, alpha 5, alpha 7, and beta 4) were down-regulated by the treatment with forskolin, whereas the mRNA levels of the TH gene was increased in PC12 cells. By long-term nicotine treatment, the mRNA level of the nAChR genes did not change, but transcript levels of alpha 3, alpha 5, alpha 7, and beta 4 nAChR genes were still negatively regulated by forskolin. However, the mRNA level of TH gene did not change by forskolin under long-term nicotine treatment. The TH gene may be regulated by a nicotine-related signaling pathway, whereas alpha 3, alpha 5, alpha 7, and beta 4 nAChR genes may be further regulated by a protein kinase A (PKA) pathway under long-term nicotine treatment. (C) 1997 Elsevier Science Ireland Ltd.