Kenji Amano

Differential Activation of Brain-derived Neurotrophic Factor Gene Promoters I and III by Ca2+ Signals Evoked vial-type Voltage-dependent andN-Methyl-d-aspartate Receptor Ca2+Channels

Although the brain-derived neurotrophic factor (BDNF) gene is activated by the intracellular Ca2+signals evoked via Ca2+ influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca2+ signals evoked viaN-methyl-d-aspartate receptors (NMDA-R) and L-type voltage-dependent Ca2+ channels (L-VDCC). There is a critical range in the membrane depolarization caused by high K+ concentrations (25–50 mm KCl) for effective BDNF mRNA expression and transcriptional activation of BDNF gene promoters I and III (BDNF-PI and -PIII, respectively) in rat cortical culture. The increase in BDNF mRNA expression induced at high K+ was repressed not only by nicardipine, an antagonist for L-VDCC, but also by dl-amino-5-phosphonovalerate, an antagonist for NMDA-R, which was supported by the effects of antagonists on the Ca2+ influx. Although the promoter activations at 25 and 50 mm KCl were different, BDNF-PIII was activated by either the Ca2+ influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca2+influx through L-VDCC. Direct stimulation of NMDA-R supported the activation of BDNF-PIII but not that of BDNF-PI. Thus, the alternative BDNF gene promoters responded differently to the intracellular Ca2+ signals evoked via NMDA-R and L-VDCC.

Mutational analysis of the MECP2 gene in Japanese patients with Rett syndrome

AbstractRett syndrome is a neurodevelopmental disorder observed almost exclusively in girls, and is characterized by autistic tendency, severe mental retardation, stereotyped hand movements, seizures, and acquired microcephaly. Recently, the MECP2 (methyl-CpG-binding protein 2) gene, mapped on chromosome Xq28, was reported to be responsible for Rett syndrome. We performed mutational analysis of the MECP2 gene in 26 Japanese patients with Rett syndrome (who were sporadic cases), and identified disease alleles in 19 patients. The mutations consisted of 12 different types including 3 missense, 3 nonsense, and 6 frameshift mutations. Of these, 8 mutations are novel. Most of these mutations affect the functional domains, methyl-CpG binding domain (MBD), and transcriptional repression domain (TRD), and therefore may critically affect the function of MeCP2. The disease phenotype of patients with mutations in the MBD tended to be more severe than the phenotype of those with mutations in the TRD. We also identified 2 types of silent mutations and 4 types of missense mutations as benign variants, and these are all novel ones. Most of the nucleotide substitutions involve C → T transitions at CpG hotspots. The novel disease alleles and benign variants of the MECP2 gene found in this study should contribute to the establishment of a reliable diagnosis of Rett syndrome.

An Alternative Splicing Form of Phosphatidylserine-specific Phospholipase A1 That Exhibits Lysophosphatidylserine-specific Lysophospholipase Activity in Humans

Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192–2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon–intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5′-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon–intron junction) and encodes a 376-amino acid product (PS-PLA1ΔC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1ΔC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1ΔC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA 1 gene was mapped to chromosome 3q13.13–13.2 and was unexpectedly identical to the nmdgene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237–6243).

T-STAR gene: fine mapping in the candidate region for childhood absence epilepsy on 8q24 and mutational analysis in patients

Childhood absence epilepsy (CAE) is one of the most common epilepsies in children. At least four phenotypic subcategories of CAE have been proposed. Among them, a subtype persisting with tonic-clonic seizures has been mapped to 8q24 (ECA1 MIM 600131). By constructing a physical map for the 8q24 region, we recently narrowed the ECA1 locus to a 1.5-Mb region. In the present communication, we show that T-STAR gene is located within the ECA1 region. T-STAR is a novel member of STAR (for signal transduction and activation of RNA) family, and is predicted to encode a spermatogenesis related RNA-binding protein. T-STAR is located within the markers D8S2049 and D8S1753 and its complete coding region spans nine exons. In addition to its known expression in testis, moderate level of transcripts for T-STAR gene was detected in brain, heart and is highly abundant in skeletal muscle. Mutational analysis for the T-SATR gene in CAE families did not show any sequence variation in the coding region, and this suggests that the T-STAR gene is not involved in the pathogenesis of persisting CAE. However, genomic organization of T-STAR gene characterized in the present report might help in understanding the biological functions of T-STAR as well as its suspected involvement in other disorders mapped on this region.

R133C and R168X mutations in Japanese Rett syndrome patients: a caution for misdiagnosis.

Rett syndrome is a neurodevelopmental disorder characterized by regression of motor and mental abilities in females after a period of normal development. The gene, MECP2, has been reported to be responsible for Rett syndrome. Here, we report the cases who were at first misdiagnosed as having homozygous mutations, and later corrected as heterozygous ones. We analyzed the MECP2 gene in three sporadic Japanese patients with Rett syndrome. Direct sequencing by using a primer set that was originally used in the first report of MECP2 mutation suggested two types of homozygous mutations (R133C and R168X). Previous reports of these mutations with heterozygous status, as well as the general nature of dominant inheritance in Rett syndrome females and lethality in hemizygous males, urged us to confirm the homozygosity of these mutations. By using a newly designed PCR primer, we found that these mutations actually occurred heterozygously in these patients. Sequence analyses of PCR products suggested that a C/T polymorphism found upstream of these mutations caused the preferential PCR amplification of the mutated alleles. These results recommend paying attention to biased PCR amplification that may lead to misjudgment of the result for mutational analysis of the MECP2 gene.