The human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) in its 3'-untranslated region (UTR). The linkage and association between the VNTR polymorphism of DAT1 and various neuropsychiatric disorders have been reported. We have determined the genomic structure of DAT1 genes containing 7-, 9-, 10-, and 11-repeat alleles and examined the effect of VNTR polymorphism in the 3'-UTR region of DAT1 on gene expression using the luciferase reporter system in COS-7 cells. Luciferase expression was significantly higher when the 3'-UTR of the DAT1 gene contained the 10-repeat allele than when it contained the 7- or 9-repeat alleles. This suggests that VNTR polymorphism affects the expression of the dopamine transporter.

p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M-to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.Proteolysis in cytosol is a key reaction to modulate various intracellular protein functions such as signal transduction, protein turnover, and cell structure. Calpain, Ca 2ϩ -dependent cysteine proteinase (EC 3.4.22.17), is one of the major intracellular proteinases known as interacting various protein kinases, transcription factors, and cytoskeletal proteins (1-5). Ubiquitous -and m-calpains, dimers of a large catalytic (CL 1 (6) and mCL (7), respectively) and small regulatory (30K (8)) subunit. Recently, we found that the ubiquitous calpain large subunit monomer can express full proteolytic activity, and that 30K dissociates from the large subunit upon activation by Ca 2ϩ (9, 10). In other words, 30K together with calpastatin, a specific proteinaceous inhibitor for calpain, play pivotal roles in regulation of calpain activity.p94 is a muscle-specific member of the calpain large subunit family, and distinct not only from the other members but also from other proteases in that it autolyzes very rapidly and extensively leading to almost complete disappearance right after translation even in the presence of EGTA and leupeptin as observed in vitro (11,12). Ubiquitous calpains as well as many other proteases also undergo autolysis at the NH 2 terminus, but only to a limited extent. Quite recently, p94 was identified as responsible for limb girdle muscular dystrophy type 2A (LGMD2A), the first demonstration of the involvement of an enzyme in muscle dystrophy (13). To elucidate physiological meaning of this exhaustive autolysis and the molecular mechanism connecting LGMD2A and p94 function, it is important to clarify the substrates of p94 and the manner to regulate the proteolytic activity of p94.30K is the first candidate to bind to and regulate p94, since the calmodulin-like Ca 2ϩ -binding domains of CL and mCL (see Fig. 2), which are the binding sites for 30K, are highly homologous to p94 (5). Analysis of p94 at the protein level, however, is very difficult because of the extremely rapid autolysis, and, thus, we examined using the yeast two-hybrid system (14). As a result, 30K was revealed not to bind to p94...

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Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid b peptide (Ab), is generated from amyloid precursor protein (APP) by b-and c-secretase-mediated cleavage. Because b-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Ab and grow normally, a b-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel b-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits b-secretase activity in cultured cells in a dosedependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Ab production in vivo in the soluble fraction compared with vehicle, but the level of Ab in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wildtype mice remarkably reduced Ab production in both the soluble and insoluble fractions. Our results indicate that the b-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD. Keywords: Alzheimer's disease, amyloid b peptide, b-secretase inhibitor, beta-site APP cleaving enzyme 1, hydroxymethylcarbonyl isostere, KMI inhibitor. Alzheimer's disease (AD) is a progressive neurodegenerative disorder whose primary pathogenic event is the extracellular accumulation of amyloid b peptide (Ab), followed by oxidative damage to neurons that ultimately results in