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Biomedical subjects

Akemi Tanaka

Publications and source records attributed to Akemi Tanaka.

11 recordsLinked to original sources
PubMed2006

Targeted gene therapy toward astrocytoma using a Cre/loxP-based adenovirus system.

The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.

Adenoviridae↗
PubMed2006

[Case of Sanfilippo syndrome type B and Wilson disease born to unrelated parents].

A 5-year-old boy visited a hospital because of macrocephalus, mental retardation and hepatic dysfunction, and was suspected to have Wilson's disease since his father had this disease. The serum level of ceruloplasmin was low, but urinary copper excretion was not increased markedly. He was treated with D-penicillamine. He was then reffered to our hospital because of his facial features suggesting mucopolysaccharidosis. Based on mucopolysacchariduria and the deficiency of N-acetylglucosaminidase, the diagnosis of Sanfilippo syndrome type B was made. Molecular analyses identified him as a compound heterozygote for both the ATP7B (A844V/2659delG) and alpha-N-acetylglucosaminidase (V241M/R482W) genes, responsible for Wilson's disease and Sanfilippo syndrome type B, respectively. Although born to non-consanguineous parents, he had two rare autosomal recessive diseases. In this case, liver dysfunction was attributed to Wilson's disease, and mental retardation to Sanfilippo syndrome.

Acetylglucosaminidase↗
PubMed2005

Non-invasive screening method for Fabry disease by measuring globotriaosylceramide in whole urine samples using tandem mass spectrometry.

Fabry disease is an X-linked sphingolipidosis due to a deficiency of alpha-galactosidase A, which leads to the accumulation of globotriaosylceramide (GL-3) in several organs. When recombinant human alpha-galactosidase A is intravenously administered repeatedly before the patient develops permanent tissue damage, there is evidence that the accumulation of GL-3 is decreased in some organs and that the clinical symptoms are alleviated in some patients. However, Fabry disease is rare and many patients are not diagnosed until adulthood after irreversible tissue damage has occurred. Our group has developed a simple and non-invasive screening method for Fabry disease that measures total GL-3 in whole urine samples by tandem mass spectrometry. Using this method, we found that the concentration of GL-3 in whole urine sample from hemizygous patients, including pre-symptomatic young children with classic type Fabry disease, was significantly higher than that in controls. The mean concentration of GL-3 in urine from heterozygotes with symptoms was significantly higher than control concentrations, but GL-3 levels in the urine from 2 out of 8 heterozygotes of classic type Fabry disease were within control levels. An asymptomatic 14-year old hemizygote in the family of a cardiac variant did not have elevated urinary GL-3. Therefore, screening for the classic type and probably renal variant of Fabry disease is possible by measuring urinary GL-3, using our method. The early diagnosis of cardiac variant hemizygotes and some heterozygotes with all types of Fabry disease will not be possible using our method. We propose that this procedure can be used as a reliable, non-invasive, simple method for general and high-risk population screening for hemizygotic patients with the classic type and probably renal variant of Fabry disease.

Adolescent↗
PubMed2004

Vesicular acetylcholine transporter can be a morphological marker for the reinnervation to muscle of regenerating motor axons.

This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), which packages acetylcholine into synaptic vesicles, can be used as a marker for regenerating motor axon terminal. We examined motor axon regeneration in the tongue after hypoglossal nerve axotomy, using an anterograde tracer biotin-dextran (BD), retrograde tracer Fluoro-Gold (FG), electron microscopic (EM) observation, and VAChT immunocytochemistry. BD study demonstrated that outgrowth of thin regenerating axons into the frontal area of the tongue was firstly observed at 14 post-operative days, and presynaptic formation of neuromuscular junction (NMJ) was observed from 21 post-operative days. Under electron microscopic observation, reconstruction of new NMJs was observed within the interval between 21 and 28 days. VAChT-immunoreactive nerve terminals disappeared by 3 days after axotomy, slightly appeared at 14 post-operative days, and thereafter gradually increased in number from 21 to 28 post-operative days. The re-expression of VAChT positive presynaptic terminal was almost the same as those obtained in BD, FG and EM studies. Regenerating axons tip in the crush model of the hypoglossal nerve exhibited prominent VAChT immunoreactivity in growing tip of regenerating axons. These indicate that VAChT is an excellent morphological indicator for regenerating nerve terminals of motor neurons.

Animals↗
PubMed2004

Fabry disease female proband with clinical manifestations similar to hypertrophic cardiomyopathy.

Fabry's disease is an X-linked inborn error of glycosphingolipid catabolism, resulting from a deficiency in alpha-galactosidase A (alpha-Gal A). A 56-year-old Japanese woman was at first suspected of having hypertrophic cardiomyopathy. The patient and her son had alpha-Gal A activity in leukocytes that was remarkably below the limit of controls. DNA analysis of the alpha-Gal A gene revealed a novel missense mutation at codon 19 in exon 1, resulting in leucine-to-proline substitution. As a result she was confirmed as a classic Fabry heterozygote. Recent advances in enzyme replacement therapy can reverse the storage of glycosphingolipids in Fabry's disease. Thus, in patients with cardiac hypertrophy, it is important to differentiate Fabry's disease from other causes of hypertrophy. Therefore, it is necessary to measure alpha-Gal A activity in all suspected cases and to analyze genetic abnormalities in heterozygotes.

Cardiomegaly↗
PubMed2003

Structural basis of the GM2 gangliosidosis B variant.

To study the structural basis of the GM2 gangliosidosis B variant, we constructed the three-dimensional structures of the human beta-hexosaminidase alpha-subunit and the heterodimer of the alpha- and beta-subunits, Hex A, by homology modeling. The alpha-subunit is composed of two domains, domains I and II. Nine mutant models due to specific missense mutations were constructed as well and compared with the wild type to determine structural defects. These nine mutations were divided into five groups according to structural defects. R178H is deduced to affect the active site directly, because R178 is important for binding to the substrate. C458Y and W420C are predicted to cause drastic structural changes in the barrel structure carrying the active site pocket. R504C/H is deduced to introduce a disruption of an essential binding with D494 in the beta-subunit for dimerization. R499C/H, located in an extra-helix, is deduced to disrupt hydrogen bonds with domain I and the barrel. R170W and L484P are deduced to affect the interface between domains I and II, causing destabilization. The structural defects reflect the biochemical abnormalities of the disease.

Amino Acid Substitution↗
PubMed2003

Different attenuated phenotypes of GM2 gangliosidosis variant B in Japanese patients with HEXA mutations at codon 499, and five novel mutations responsible for infantile acute form.

Eight mutations of the alpha subunit of beta-hexosaminidase A gene ( HEXA) were identified in eight patients with GM2 gangliosidosis variant B. They were five missense mutations, two splice-site mutations, and one two-base deletion. Five of them, R252L (CGT-->CTT), N295S (AAT-->AAC), W420C (TGG-->TGT), IVS 13, +2A-->C, and del 265-266AC (exon 2), were novel mutations responsible for infantile acute form of GM2 gangliosidosis. Two missense mutations, R499H and R499C, were found in one allele of two patients with attenuated phenotypes. The patient with R499C showed a late infantile form, and the other patient with R499H showed a juvenile form. These two mutations have been reported previously in the patients of other ethnic groups, and they have been known to cause attenuated phenotypes. The milder phenotypes of GM2 gangliosidosis variant B, different from the infantile acute form, have not been reported so far in Japan, and this is the first report of Japanese patients with attenuated phenotypes and their molecular analysis.

Acute Disease↗
PubMed2002

Molecular and structural studies of the GM2 gangliosidosis 0 variant.

To determine the molecular basis of the GM2 gangliosidosis 0 variant, we constructed a three-dimensional structure of the human beta-hexosaminidase beta-subunit by homology modeling. It is composed of two domains, domains I and II, and has three disulfide bonds. C534 is located on an extra helix in domain II and forms a disulfide bond with C551. The extra helix is structurally located near domain I. C534Y, identified in a patient with the infantile form of the disease, was deduced to cause disruption of the disulfide bond, which results in a large conformational change of the extra helix, stabilizing the two domains. The drastic change in the protein structure results in a deficiency of the mature beta-subunit, and deficient activities of beta-hexosaminidases A (abeta) and B (betabeta), followed by abundant accumulation of GM2 ganglioside in the patient's cells. R505 is located on the eighth helix of domain II. R505Q, found in a patient with the chronic form of the disease, is predicted to influence the surface structure of the beta-subunit, although it does not affect the active site. The amino acid substitution causes a partial processing defect and decreased enzyme activities, which result in moderate accumulation of GM2 ganglioside in the patient's cells. The structural defects well reflect biochemical and phenotypic abnormalities of the disease.

Adult↗
PubMed2002

Molecular analysis of the alpha-N-acetylglucosaminidase gene in seven Japanese patients from six unrelated families with mucopolysaccharidosis IIIB (Sanfilippo type B), including two novel mutations.

Molecular analysis of the alpha-N-acetylglucosaminidase gene in seven Japanese patients with Sanfilippo syndrome type B from six unrelated families was carried out, and six disease-causing mutations were found. The parents of Patient 2 had a consanguinous marriage, but other families did not have any record of consanguinity. Two families were from Okinawa Island, where more patients with Sanfilippo syndrome were found than in other areas in Japan. Patients 1 and 6 showed the most severe phenotype with rapid progression. Patients 2, 5, and 7 were moderate. Patients 3 and 4 (sib cases) showed an attenuated form compared with other patients. Patients 1, 2, and 6 were homozygous for R482W, R565W, and R565P, respectively. Patients 3 and 4 were compound heterozygous for F314L and R565P. Patient 5 had delTG2171-2172 in exon 6 in one allele, and the other allele was unknown. Patient 7 was compound heterozygous for V241M and R482W. The family of Patients 3 and 4 and that of Patient 6 are unrelated, although both families are from Okinawa Island, and the patients have the same mutation, R565P; thus, R565P might be a common mutation in the Okinawa district. F314L and V241M are novel mutations.

Acetylglucosaminidase↗