Unexpectedly mild phenotype in an ataxic family with a two-base deletion in the APTX gene.

INTRODUCTION: Early onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia 1 (AOA1) is an autosomal recessive disorder caused by mutations in the APTX gene. In contrast to the recent progress on the molecular mechanism of aprataxin in DNA repair, the genotype and phenotype correlation has not been fully established. A previous study demonstrated that patients with truncation mutations had earlier onset of disease than those with missense mutations METHODS: Genomic DNA analysis was performed in a consanguineous family with relatively late-onset EAOH/AOA1. In addition, mRNA and protein analyses were performed. RESULTS: The proband of the family had a homozygous two-base deletion in the middle of exon 3. Reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays of mRNA revealed an aberrantly spliced mRNA with a cryptic splice site located four bases upstream of the deletion site. The newly identified mRNA retained a frameshift mutation and encoded a truncated protein. Immunoblot analysis did not detect the truncated protein in the patient's fibroblasts, possibly because it was unstable. CONCLUSIONS: Although patients with truncation mutations had an earlier onset of disease, our findings suggest that patients with a truncation mutation resulting in an undetectable protein level can also have a later onset of disease.

Effect of quercetin on the transport of N-acetyl 5-aminosalicylic acid.

OBJECTIVES: The aim of this study was to investigate the transporter-mediated transport of N-acetyl 5-aminosalicylic acid (Ac-5-ASA) and the effect of quercetin on Ac-5-ASA transport. METHODS: Caco-2 cell monolayers grown in Transwells were used to study the transport of Ac-5-ASA in the absence or presence of quercetin, and apical-to-basolateral and basolateral-to-apical apparent permeability (PappAB and PappBA values, respectively) was determined. The effect of transporter inhibitors, such as MK571, quinidine and mitoxantrone, on the transport of Ac-5-ASA was investigated. KEY FINDINGS: In the absence of transporter mediators, the transport of Ac-5-ASA was much higher in the basolateral-to-apical direction than in the opposite direction. The PappBA/PappAB ratio of Ac-5-ASA was 4.89. Quercetin inhibited the apical efflux of Ac-5-ASA and decreased the PappBA/PappAB ratio to 1.05. Of the transporter inhibitors, MK571 decreased the PappBA/PappAB ratio to 1.07; however, neither quinidine nor mitoxantrone had an effect on Ac-5-ASA transport. CONCLUSIONS: Ac-5-ASA was excreted by multidrug resistance-associated protein 2 from Caco-2 cells, and its transport was inhibited by quercetin. Our findings suggest that dose levels of sulfasalazine or 5-aminosalicylic acid can be decreased by coadministration of quercetin, leading to improved pharmaceutical care for inflammatory bowel diseases.

Inhibitory effect of flavonoids on the efflux of N-acetyl 5-aminosalicylic acid intracellularly formed in Caco-2 cells.

N-acetyl 5-aminosalicylic acid (5-AcASA) that was intracellularly formed from 5-aminosalicylic acid (5-ASA) at 200 microM was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 microM) such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 microM) was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of N-acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the N-acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3' or C4' position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells.

Alpha-1-antichymotrypsin gene polymorphism and susceptibility to multiple system atrophy (MSA).

We investigated three genotypes (AA, AT, and TT) produced by signal peptide polymorphism of the alpha-1-antichymotrypsin (ACT) gene in 105 patients with multiple system atrophy (MSA) and age-matched controls. The frequency of ACT-AA genotype was significantly higher in patients with MSA (20.0%) than in controls (10.5%). The onset of MSA was significantly earlier and the disease progressed significantly faster in patients with ACT-AA genotype than in those with non-ACT-AA genotypes. The ACT concentration in cerebrospinal fluid was increased in patients with ACT-AA. To our knowledge, this is the first study to show that the ACT-AA genotype is a risk factor and modulating factor for MSA. Our findings suggest the involvement of ACT-relating inflammatory process in the pathogenesis of MSA.

Sequence-dependent and independent inhibition specific for mutant ataxin-3 by small interfering RNA.

In Machado-Joseph disease (MJD) gene, there is a C/G polymorphism immediately after the CAG repeat; the expanded CAG repeat tract is exclusively followed by C, whereas about half of wild-type alleles are followed by G. Using this C/G polymorphism, we have engineered the small interfering RNA (siRNA) which decreased the expression of mutant ataxin-3, Q79C, by 96.0%, whereas there was minimal reduction on that of the wild type, Q22G (5.9%). Furthermore, unexpectedly, the expression of another wild-type allele, Q22C, was also much less suppressed (22.5%) by this siRNA possibly due to difference of the secondary structure of the target RNA. This is the first report of sequence-independent discrimination of mutant and wild-type alleles by siRNA.

siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme.

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.

Efficient production of human FVIII in hemophilic mice using lentiviral vectors.

Lentiviral vectors (LV) have the ability to integrate their proviral DNA containing a therapeutic gene into the host cell's genome. Therefore, these vectors have a great potential for gene therapy especially in the treatment of hereditary diseases like hemophilia A, which require lifelong expression of the transgene. We constructed an HIV-1-based LV containing human B-domain-deleted factor VIII (FVIII) cDNA under the control of a promoter consisting of the chicken beta-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which is a robust transcription promoter. High levels of FVIII expression from this vector could be demonstrated in vitro in 293T cells, primary liver cells, and hematopoietic progenitor cells. To test whether this viral vector was able to correct the bleeding disorder of C57BL/6 FVIII knockout mice, we transduced these mice with the FVIII LV either by intraperitoneal injection or by transplantation with transduced syngeneic bone marrow. FVIII production was analyzed in the blood plasma for a period of 3 months; however, only low levels of FVIII (<50 mU), which were below 5% of normal FVIII levels of 1000 mU, could be detected. Further analysis revealed that the low levels of FVIII activity present in the blood plasma were due to the presence of neutralizing antibodies to FVIII and not due to lack of expression of FVIII from the viral vector. FVIII expression could be detected in the tissues of the transduced mice by Western blot analysis and in ex vivo cultures. These data demonstrate that LVs are able to produce therapeutic levels of FVIII in knockout mice when administered by ip infection or by transduced hematopoietic cells. The challenge is to overcome the immune barriers to the therapeutic gene product.