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Biomedical subjects

I Kanazawa

Publications and source records attributed to I Kanazawa.

At least 145 records · Page 8Linked to original sources
PubMed1997

Instability of regions containing expanded CAG repeats during replication in Escherichia coli probed by labeled oligonucleotides.

The instability of plasmid DNAs that contained various numbers of repeated CAG units in Escherichia coli was examined. Synthetic [5'-32P](CAG)15 and [5'-32P]-(CTG)15 oligonucleotides were incubated with plasmid DNA. A minor fraction of the plasmid DNA formed a stable complex with either the repeated CAG or the repeated CTG oligonucleotide when the number of CAG repeats in the plasmid DNA was more than 30, regardless of the polarity of replication. The formation of the complexes appeared to occur by the normal hybridization process. The DNA that formed stable complexes could be purified with a biotinylated fragments of CAG repeats and avidin-coated beads. In addition, in the case of the DNA containing CAG repeats derived from the IT15 gene of a patient with Huntington's disease, there seemed to be an unusual product that contained a partially replicated product that extended only from the origin of replication to the CAG-repeated region. The formation of this product was observed when replication started downstream of IT15 gene.

Base Sequence↗
PubMed1997

Presenilin 1 binds to amyloid precursor protein directly.

Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to accumulation of beta-amyloid peptide in the brain of patients, suggesting that presenilin abnormalities induce pathological processing of amyloid precursor protein (APP) in Alzheimer's disease. For the understanding of pathogenesis in this type of familal Alzheimer disease, it is important to know whether presenilins are directly involved in the metabolism of beta-amyloid or not. To test whether presenilin 1 (PS1) directly binds to APP, we performed two-hybrid interaction assays between these proteins in yeast cells by using bait plasmids for normal and mutant PS1 and prey plasmids for APP fragments corresponding to the different molecular portions. Positive interaction was observed in any combination between PS1 bait plasmids and APP prey plasmids. Therefore, our results show that PS1 binds to APP directly and suggest that the PS1 protein itself is involved in the metabolism of beta-amyloid peptide.

Alzheimer Disease↗
PubMed1997

Screening for imprinted genes by allelic message display: identification of a paternally expressed gene impact on mouse chromosome 18.

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Alleles↗
PubMed1997

A role of dystroglycan in schwannoma cell adhesion to laminin.

Dystroglycan is encoded by a single gene and cleaved into two proteins alpha- and beta-dystroglycan by posttranslational processing. Recently, alpha-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein beta-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of alpha- and beta-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of alpha-dystroglycan, but not beta-dystroglycan, in the culture medium, suggesting secretion of alpha-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against alpha-dystroglycan was shown previously to inhibit the binding of laminin-1 to alpha-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of alpha-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells.

Animals↗
PubMed1997

Machado-Joseph disease gene product identified in lymphocytes and brain.

Machado-Joseph disease (MJD) is associated with the expansion of an unstable CAG repeat. The existence of an abnormal gene product in MJD was previously suggested with the expanded polyglutamine stretch-specific antibody. However, the product of the normal allele has not previously been identified. We generated monoclonal antibodies against the fusion protein (codon 225-310) of the MJD gene product and then identified the MJD1 gene product in normal lymphoblastoid cells as a approximately 50-kDa protein by immunoblot analysis. The electrophoretic mobility differences among the normal allele products corresponds to the molecular size difference produced by the polyglutamine stretch and the polymorphism at the C-terminus. Moreover, abnormal immunoreactive bands which were larger than the normal ones were found as a approximately 60-kDa protein exclusively in the MJD samples. The cytoplasm and the nuclei of neurons and glial cells were stained by these antibodies with immunocytochemistry. As in other CAG repeat diseases, the abnormal and normal allele products were almost equally expressed in lymphoblastoid cells and the brain of MJD patients.

Alleles↗
PubMed1997

Expression of dentatorubral-pallidoluysian atrophy (DRPLA) proteins in patients.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a CAG trinucleotide repeat. The mutant gene is translated into protein whose electrophoretic mobility correlates to the number of expanded CAG trinucleotide repeats, indicating that the protein carries an expanded glutamine repeat. Using two polyclonal antibodies raised against the DRPLA gene product in immunoblotting, we determined the untruncated DRPLA proteins, and showed that the amounts of mutant and wild-type DRPLA proteins were similar in DRPLA brain tissues and lymphoblastoid cells, suggesting that regulation of the level of translation of the DRPLA gene is not central to the development of the disease.

Adult↗
PubMed1997

YAC and cosmid contigs encompassing the Fukuyama-type congenital muscular dystrophy (FCMD) candidate region on 9q31.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of childhood muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with an anomaly of the brain. We had mapped the FCMD gene to an approximately 5-cM interval between D9S127 and D9S2111 on 9q31-q33 and had also found evidence for linkage disequilibrium between FCMD and D9S306 in this candidate region. Through further analysis, we have defined another marker, D9S172, which showed stronger linkage disequilibrium than D9S306. A yeast artificial chromosome (YAC) contig spanning 3,5 Mb, which includes this D9S306-D9S172 interval on 9q31, has been constructed by a combination of sequence-tagged site, Alu-PCR, and restriction mapping. Also, cosmid clones subcloned from the YAC were assembled into three contigs, one of which contains D9S2107, which showed the strongest linkage disequilibrium with FCMD. These contigs also allowed us to order the markers as follows: cen-D9S127-(approximately 800 kb)-D9S306 (identical to D9S53)-(approximately 700 kb)-A107XF9-(approximately 500 kb)-D9S172-(approximately 30 kb)-D9S299 (identical to D9S774)-(approximately 120 kb)-WI2269-tel. Thus, we have constructed the first high-resolution physical map of the FCMD candidate region. The YAC and cosmid contigs established here will be a crucial resource for identification of the FCMD gene and other genes in this region.

Chromosomes, Artificial, Yeast↗
PubMed1997

Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin.

alpha-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein beta-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve alpha-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the alpha-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the alpha-dystroglycan with laminin.

Animals↗
PubMed1997

Ganglioside composition of the human cranial nerves, with special reference to pathophysiology of Miller Fisher syndrome.

Total ganglioside fractions from the human cranial nerves purified on a Phenyl Sepharose column, were given mild alkaline treatment, after which their composition and amounts of lipid-bound sialic acid were determined by HPTLC-densitometry with resorcinol as the coloring reagent. The total amounts of lipid-bound sialic acid were 156.5 ng/mg of wet tissue in the Ist cranial nerve (olfactory tract) and 131.9 ng/mg in the IInd nerve, greater than the amounts in the other nerves (99.1-120.0 ng/mg). The Ist, IInd, and VIIIth nerves had GM4, but not LM1. It may reflect their histological feature of the central nervous system. The IIIrd, IVth, and VIth nerves, as well as the IInd, had significantly higher percentages of GQ1b (11.6-13.2%) than the other nerves (5.2-8.4%). The high proportion of GQ1b specific to these three cranial nerves involved in the ocular movement lends support to the role of serum anti-GQ1b antibody in the pathogenetic mechanisms of ophthalmoplegia in Miller Fisher syndrome and Guillain-Barré syndrome.

Aged↗
PubMed1997

Binding of antibodies against GM1 and GD1b in human peripheral nerve.

Human dorsal root ganglia (DRG), and ventral and dorsal roots were immunostained with rabbit antibodies recognizing GM1, GD1b, or both. Sera from rabbits immunized with GM1 or GD1b were separated in affinity columns into three fractions: Rab1, Rab2, and Rab3. Rab1 recognized only GM1, and Rab2 only GD1b; whereas Rab3 recognized both GM1 and GD1b, presumably by binding to the terminal galactosyl beta 1-3N-acetylgalactosaminyl residue. Rab2 and Rab3 immunostained most of the nerve cell bodies in the DRG and paranodal myelin of the ventral and dorsal roots, whereas Rab1 produced no significant immunostaining. These results show that GD1b is localized on the DRG neurons and the paranodal myelin of human peripheral nerve. These places may be the binding sites for anti-GD1b antibodies, including those cross-reactive with GM1, in the sera from patients with autoimmune neuropathies. GM1 may be dispersed in human DRG and dorsal and ventral roots.

Animals↗
PubMed1997

A Japanese family carrying a novel mutation in the Emery-Dreifuss muscular dystrophy gene.

We report on a Japanese family affected by Emery-Dreifuss muscular dystrophy carrying a novel mutation of the emerin (STA) gene. The cardinal clinical feature of the family was cardiac conduction block and mild myopathy. A deletion of 11 bp with a frameshift was identified in exon 6, causing truncation of the predicted protein. The relationship between mutation and phenotype is discussed.

Adolescent↗
PubMed1997

Brain regional differences in the expansion of a CAG repeat in the spinocerebellar ataxias: dentatorubral-pallidoluysian atrophy, Machado-Joseph disease, and spinocerebellar ataxia type 1.

Three autosomal dominant spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy (DRPLA), Machado-Joseph disease (MJD), and spinocerebellar ataxia type 1 (SCA1), are associated with the expansion of a CAG repeat in the respective genes. To investigate the association between CAG repeat expansion and neuropathological findings, we analyzed several brain regions from 9 cases of DRPLA, 3 cases of MJD, and 1 case of SCA1. We found that the expanded alleles were smaller in the cerebellar cortex than in other brain regions, such as the frontal cortex, in these three diseases. The discrepancy in the expanded CAG repeat length between cerebellar cortex and other tissues was most prominent in DRPLA, and especially in cases of adult-onset DRPLA. A significant correlation was found between the age at onset of DRPLA and the size of the CAG repeat expansion. Cerebella of DRPLA patients were microscopically dissected into three layers, the molecular and granular layers and the white matter, which were analyzed separately. The lower level of CAG repeat expansion in DRPLA cerebella was representative of CAG repeat expansion in the granule cells. The microdissected samples of the granular layer of the hippocampal formation, which is densely packed with neuronal cells, revealed that the degree of CAG repeat expansion in this layer was similar to that in the cerebellum. These observations suggest that granule cells in the cerebellum and hippocampus have low levels of CAG repeat expansion, and that other types of cells exhibit a higher level of CAG repeat expansion, in spinocerebellar ataxias.

Adolescent↗
PubMed1997

Expression of the copper-zinc superoxide dismutase gene in amyotrophic lateral sclerosis.

The demonstration of a genetic linkage between the copper-zinc superoxide dismutase (SOD1) gene and familial amyotrophic lateral sclerosis has aroused interest in the role of SOD1 in motoneuronal death. We investigated the expression of the human SOD1 gene at a cellular level in the motoneurons of patients with sporadic amyotrophic lateral sclerosis, patients with familial amyotrophic lateral sclerosis, and normal control subjects, using a quantitative in situ hybridization technique. There were no significant differences between the amounts of SOD1 messenger RNA observed in patients with sporadic disease, patients with familial disease, and normal control subjects. However, many of the atrophic motoneurons from patients with sporadic or familial disease had significantly lower levels of SOD1 messenger RNA, compared to morphologically intact motoneurons. Moreover, motoneurons in the normal spinal ventral horn and precentral motor cortex exhibited significantly higher levels of SOD1 messenger RNA than did other neurons. Our study indicated that vulnerable neurons in amyotrophic lateral sclerosis exhibit high levels of SOD1 messenger RNA, suggesting a close relationship between the SOD1 gene and the pathogenesis of amyotrophic lateral sclerosis.

Aged↗
PubMed1997

Regional and cellular expression of the dentatorubral-pallidoluysian atrophy gene in brains of normal and affected individuals.

Dentatorubral-pallidoluysian atrophy is an autosomal dominant neurodegenerative disorder characterized by dementia and spinocerebellar degeneration. Recently, part of the gene responsible for this disorder was cloned containing a CAG repeat, and predominant neuronal expression of the gene was proved only through Northern blot analysis in rats. In this study, we investigated the regional and cellular expression of the dentatorubral-pallidoluysian atrophy gene in the central nervous system of normal and affected humans, as well as in rat brains. In normal control human subjects, the gene messenger RNA was present in all brain regions examined, with the highest levels seen in the cerebellum, hippocampus, substantia nigra, and pontine nuclei. Its expression in the striatum, globus pallidus, and cerebral cortex was intermediate. The gene was expressed predominantly in neurons; a low but significant level of expression was also seen in glial cells. Neurons susceptible to degeneration in dentatorubral-pallidoluysian atrophy did not selectively express high or low levels of its gene messenger RNA. In brains affected by the disorder, the distribution and levels of gene messenger RNA were comparable to those of the normal controls in all the areas. In the rat brains, gene messenger RNA expression was very similar to that in human brain. It was also expressed predominantly in neurons, while low-level expression was observed in glial cells. It is apparent from these results that the presence of expanded trinucleotide repeats in dentatorubral-pallidoluysian atrophy does not result in the absence of its gene messenger RNA expression or in altered patterns or levels of expression.

Adult↗
PubMed1997

Effects of tolcapone, a catechol-O-methyltransferase inhibitor, on motor symptoms and pharmacokinetics of levodopa in patients with Parkinson's disease.

The effects of tolcapone, a catechol-O-methyltransferase inhibitor, on the bioavailability and efficacy of levodopa were evaluated in 12 patients with Parkinson's disease (PD), 8 of whom showed signs of daily motor fluctuations (wearing-off phenomenon). Motor disabilities were assessed in 12 patients at 7 time points before and after the chronic administration of tolcapone using the Unified Parkinson's Disease Rating Scale (UPDRS). The UPDRS score was improved at all points of determination. Eight patients with wearing-off phenomenon on levodopa showed symptomatic improvement on the combination. The area under the curve (AUC) for levodopa increased by 34% (p = 0.0059) after the administration of tolcapone. The elimination half-life (T1/2) of levodopa was significantly prolonged by 81% (p = 0.0001) after the treatment. The AUC of 3-O-methyldopa, a metabolite of levodopa, was decreased by 79% (p = 0.0001) and the Cmax (maximum concentration) was also decreased by 80%d after the administration (p = 0.0001) of tolcapone. The combination of tolcapone and levodopa was well tolerated. Our findings suggest that tolcapone improves the pharmacokinetics of levodopa in plasma and motor symptoms of fluctuating PD patients. It is suggested that tolcapone may be useful drug adjunct to levodopa in treating patients with PD with wearing-off phenomena.

Aged↗
PubMed1997

Preferential activation of different I waves by transcranial magnetic stimulation with a figure-of-eight-shaped coil.

Transcranial magnetic stimulation (TMS) over the human primary motor cortex (MI) evokes motor responses in the contralateral limb muscles. The latencies and amplitudes of those responses depend on the direction of induced current in the brain by the stimuli (Mills et al. 1992, Werhahn et al. 1994). This observation suggests that different neural elements might be activated by the differently directed induced currents. Using a figure-of-eight-shaped coil, which induces current with a certain direction, we analyzed the effect of direction of stimulating current on the latencies of responses to TMS in normal subjects. The latencies were measured from surface electromyographic responses of the first dorsal interosseous muscles and the peaks in the peristimulus time histograms (PSTHs) of single motor units from the same muscles. The coil was placed over the MI, with eight different directions each separated by 45 degrees. Stimulus intensity was adjusted just above the motor threshold while subjects made a weak tonic voluntary contraction, so that we can analyse the most readily elicited descending volley in the pyramidal tracts. In most subjects, TMS with medially and anteriorly directed current in the brain produced responses or a peak that occurred some 1.5 ms later than those to anodal electrical stimulation. In contrast, TMS with laterally and posteriorly directed current produced responses or a peak that occurred about 4.5 ms later. There was a single peak in most of PSTHs under the above stimulation condition, whereas there were occasionally two peaks under the transitional current directions between the above two groups. These results suggest that TMS with medially and anteriorly directed current in the brain readily elicits I1 waves, whereas that with laterally and posteriorly directed current preferentially elicits I3 waves. Functional magnetic resonance imaging studies indicated that this direction was related to the course of the central sulcus. TMS with induced current flowing forward relative to the central sulcus preferentially elicited I1 waves and that flowing backward elicited I3 waves. Our finding of the dependence of preferentially activated I waves on the current direction in the brain suggests that different sets of cortical neurons are responsible for different I waves, and are contrarily oriented. The present method using a figure-of-eight-shaped coil must enable us to study physiological characteristics of each I wave separately and, possibly, analyse different neural elements in MI, since it activates a certain I wave selectively without D waves or other I waves.

Adult↗
PubMed1997

Shortening of simple reaction time by peripheral electrical and submotor-threshold magnetic cortical stimulation.

Subthreshold transcranial magnetic stimulation (TMS) over the motor cortex can shorten the simple reaction time in contralateral arm muscles if the cortical shock is given at about the same time as the reaction stimulus. The present experiments were designed to investigate whether this phenomenon is due to a specific facilitatory effect on cortical circuitry. The simple visual reaction time was shortened by 20-50 ms when subthreshold TMS was given over the contralateral motor cortex. Reaction time was reduced to the same level whether the magnetic stimulus was given over the bilateral motor cortices or over other points on the scalp (Cz, Pz). Indeed, similar effects could be seen with conventional electrical stimulation over the neck, or even when the coil was discharged (giving a click sound) near the head. We conclude that much of the effect of TMS on simple reaction time is due to intersensory facilitation, although part of it may be ascribed to a specific effect on the excitability of motor cortex.

Electric Stimulation↗