Androgen-dependent neurodegeneration by polyglutamine-expanded human androgen receptor in Drosophila

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset, neurodegenerative disorder affecting only males and is caused by expanded polyglutamine (polyQ) stretches in the N-terminal A/B domain of human androgen receptor (hAR). Although no overt phenotype was detected in adult fly eye photoreceptor neurons expressing mutant hAR (polyQ 52), ingestion of androgen or its known antagonists caused marked neurodegeneration with nuclear localization and structural alteration of the hAR mutant. Ligand-independent toxicity was detected with a truncated polyQ-expanded A/B domain alone, which was attenuated with cytosolic trapping by coexpression of the unliganded hAR E/F ligand binding domain. Thus, our findings suggest that the full binding of androgen to the polyQ-expanded hAR mutants leads to structural alteration with nuclear translocation that eventually results in the onset of SBMA in male patients.     

On a kinetic origin of heredity: minority control in a replicating system with mutually catalytic molecules

As the first step in an investigation of the origin of genetic information, we study how some species of molecules are preserved over cell generations and play an important role in controlling the growth of a cell. We consider a model consisting of protocells. Each protocell contains two mutually catalysing molecule species (X and Y), each of which has catalytically active and inactive types. One of the species Y is assumed to have a slower synthesis speed. Through divisions of the protocells, the system reaches and remains in a state in which there are only a few active Y and almost no inactive Y molecules in most protocells, through the selection of very rare fluctuations. In this state, the active Y molecules are shown to control the behavior of the protocell. The minority molecule species act as the carrier of heredity, due to the relatively discrete nature of its population, in comparison with the majority species which behaves statistically in accordance with the law of large numbers. The minority controlled state may give rise to a selection pressure for mechanisms that ensure the transmission of the minority molecule. Once those mechanisms are in place, the minority molecule becomes the ideal storage device for information to be transmitted across generations, thus giving rise to "genetic information". The relevance of this minority controlled state to evolvability is also discussed.     

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate,a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain-to-blood transport of indoxyl sulfate at the blood-brain barrier (BBB) using the Brain Efflux Index method. [(3)H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 x 10(-2)/min, and the process is saturable with a Km of 298 microm. This process is inhibited by para-aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acid and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. RT-PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [(3)H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3-methoxy-4-hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6-mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain-to-blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain-to-blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.     

Age-related decline of serotonin transporters in living human brain of healthy males

There is growing interest in serotonin transporter (5-HTT) function in the human brain, since alteration in 5-HTT has been suggested in a variety of neurophychiatric disorders. Age-related decline in postsynaptic 5-HT receptors has been demonstrated in postmortem human studies and in vivo imaging studies, and has been assumed to be related to changes in mental function in the normal aging process. However, few studies have investigated the aging effect on 5-HTT in human brain in vivo, since the availability of suitable ligands has been limited. To investigate the aging effect on 5-HTT in living human brain, we performed positron emission tomography (PET) scans with a selective ligand for 5-HTT, [11C](+)McN5652. We examined 28 healthy male volunteers aged between 20 and 79 years. The uptake was quantified in the thalamus and midbrain by graphical analysis with the cerebellum as a reference tissue, and binding potential (BP) was used for the index of 5-HTT binding. There was a significant age-related decline in BP in the thalamus and midbrain. The decline in [11C](+)McN5652 binding was 9.6% per decade in the thalamus and 10.5% per decade in the midbrain.     

Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.     

Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2-cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10-15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.     

CYP2D6.10 present in human liver microsomes shows low catalytic activity and thermal stability

Comparing bufuralol 1'-hydroxylase activity among liver microsomes prepared from individuals whose CYP2D6 genotypes had been determined, we found that the activity tended to decrease depending on the number of the CYP2D6*10 allele. Pre-incubation of liver microsomes from individuals homozygous for the CYP2D6*10 allele resulted in a decrease in the enzyme activity more rapidly than those from individuals homozygous for the CYP2D6*1, suggesting that not only the catalytic activity but also the thermal stability of the enzyme appeared to be affected by the genetic polymorphism. To confirm this hypothesis, the kinetic parameters of CYP2D6.1 and CYP2D6.10 were compared for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation using microsomes prepared from yeast transformed with plasmids carrying CYP2D6 cDNAs (*1A and *10B). Kinetic studies of these CYP2D6 forms indicated clear differences in the metabolic activities between the wild (CYP2D6.1) and the mutant enzymes (CYP2D6.10). Bufuralol 1(')-hydroxylase activity in microsomes of yeast expressing CYP2D6.10 was rapidly decreased by heat treatment, supporting the idea that the thermal stability of the enzyme was reduced by amino acid replacement from Pro (CYP2D6.1) to Ser (CYP2D6.10). These data strongly suggest that the thermal instability together with the reduced intrinsic clearance of CYP2D6.10 is one of the causes responsible for the known fact that Orientals show lower metabolic activities than Caucasians for drugs metabolized mainly by CYP2D6, because of a high frequency of CYP2D6*10 in Orientals.