Developmental changes and localization of mouse brain serine proteinase mRNA and protein in mouse brain Takashi Yokoi a,b, * , Naoki Yamamoto b , Toyohiro Tada c , Masataka Fujita d , Akihiko Moriyama e , Hitoshi Matsui f , Takayuki Takahashi f , Hajime Togari a , Taiji Kato b , Kiyofumi Asai b a Department of Pediatrics, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan b Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan c Nagoya City University School of Nursing, Mizuho-ku, Nagoya 467-8601, Japan d Department of Neurosurgery, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan e Division of Biomolecular Science, Institute of Natural Science, Nagoya City University, Mizuho-ku, Nagoya 467-8501, Japan f Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Received 7 January 2002; received in revised form 21 January 2002; accepted 25 January 2002 Abstract Serine proteases are known to be involved in neural development and various functions in the central nervous system. Mouse brain serine proteinase (mBSP) is expressed almost exclusively in the mouse brain and it has been characterized at the molecular and biochemical levels. In this study, we analyzed the developmental changes and localization of mBSP mRNA and protein in the mouse brain, using reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Expression of mBSP was strong in the white matter and the nerve tracts after postnatal day 30, especially in the cerebellum and the medulla oblongata. These results suggest that mBSP contributes to development and sustaining the functions in the mouse brain. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serine protease; Oligodendrocyte; Astrocyte; Regional difference; Developmental change; Reverse transcription-polymerase chain reaction; Western blotting; Immunohistochemistry Serine proteases are widely distributed in tissues and organs [12], and take part in various biological functions. In the central nervous system, serine proteases and their inhibitors are considered to play important roles in neuronal migration during development, neurite outgrowth, synaptic plasticity, and neuronal degeneration and death [6,11]. One of the serine proteases, mouse brain serine proteinase (mBSP), expressed predominantly in brain, was identified recently [4], and recognized to be the same enzyme reported as brain and skin serine protease [5]. In the present study, we investigated regional differences and developmental changes of mBSP mRNA and protein in the mouse brain. For the analysis of developmental changes in mBSP gene expression, total RNA was isolated from embryonic day 7 (E7) whole embryos and E14, E17, postnatal day 0 (P0), P7, P30, and adult brains of ICR mice with TRIZOL reagent (Gibco BRL) according to the manufacturer’s protocol. Reverse transcription (RT) was conducted by SUPER- SCRIPT II RNase H - reverse transcriptase (Gibco BRL) with oligo (dT) 12–18 primer and 20 ml of RT product was generated from 1 mg of total RNA. Oligonucleotide primers for polymerase chain reaction (PCR) were synthesized based on the published cDNA sequences. The PCR primers for mBSP, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and b-actin are shown in Table 1. MBP mRNA consists of five isoforms and the primers for MBP yielded two bands corresponding to 318 and 396 base pair cDNA fragments [2]. The PCR reaction contained 2 ml of RT product as template DNA and was carried out for 30 cycles, using a denaturing step at 94 8C for 30 s, an annealing step at 58 8C for 30 s, and an extension step at 72 8C for 45 s. The concentrations of primers for mBSP, GFAP, MBP, and b- actin were 2, 0.5, 0.4 and 0.12 mM, respectively. The PCR Neuroscience Letters 323 (2002) 133–136 0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(02)00122-2 www.elsevier.com/locate/neulet * Corresponding author. Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: 181-52-853-8200; fax: 181-52- 842-3316. E-mail address: tyokoi@med.nagoya-cu.ac.jp (T. Yokoi).