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Biomedical subjects

N Shimizu

Publications and source records attributed to N Shimizu.

At least 253 records · Page 14Linked to original sources
PubMed2000

[Neurology of eye movements].

Eye movements are necessary for clear and stable vision, for which images of the world should be brought to the fovea and be held steady on the retina. The main types of eye movements consist of saccadic eye movements, vergence, vestibular eye movements, smooth pursuit eye movements and eye movements for visual stabilization. Saccades are fast eye movements and have consistent relationship between their peak velocity and the size of the movements. The burst neurons in the brainstem are the generator of saccades and receive projections from the frontal eye field, supplementary eye field, parietal eye field, basal ganglia, superior colliculus and cerebellum. Saccades are evaluated by a speed and accuracy of the size. There are different types of saccade, such as visually triggered saccades, antisaccades, memory-guided saccade and predictive saccades. The specific test paradigms of saccades may show the localization and the type of diseases. Opsoclonus, flatter-like oscillation, ocular myoclonus, square-wave jerks are characteristic involuntary eye movements. Syndromes of the paramedic pontine reticular formation, medial longitudinal fascicules and one-and-a-half syndrome are caused by disease of the pons.

Brain Stem↗
PubMed1999

Chimeric virus-like particle formation of adeno-associated virus.

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2, and VP3. These capsid proteins have a common amino acid sequence, being expressed from different initiation codons on the same open reading frame. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but their functions are poorly understood. To investigate the role(s) of the capsid proteins in capsid formation, we used a baculovirus protein expression system to produce virus-like particles (VLPs). We found that varying the ratios of VP2 and VP3 did not affect VLP formation. Further, their physical properties were equivalent to those of empty wild-type particles. The function of VP3 was studied further by fusing a peptide tag, FLAG, to its N-terminus. This chimeric viral protein, in combination with VP2, could assemble into VLPs, indicating that the chimerism of VP3 did not affect VLP formation. Although the monomeric native form of the FLAG-VP3 chimera could react with anti-FLAG antibody, VLP containing the chimeric VP3 could not, suggesting that the N-terminal region of VP3 is located inside the VLP. These observations indicate that it may be possible to utilize AAV VLP as vectors of a broad range of drugs since fusion of the VP3 N-terminus with defined molecules could impose distinct physical properties onto the internal environment of the VLP.

Animals↗
PubMed1999

A CXC chemokine receptor, CXCR5/BLR1, is a novel and specific coreceptor for human immunodeficiency virus type 2.

G protein-coupled receptors serve as coreceptors in the infection process of human immunodeficiency virus type-1 (HIV-1), type-2 (HIV-2), and simian immunodeficiency virus (SIV). In this study, we showed that a CXC-CKR, CXCR5/BLR1, is a novel coreceptor for HIV-2, but for neither HIV-1 nor SIV. The expression of CXCR5 was detected by polymerase chain reaction after reverse transcription of cellular mRNA from S+L-HOS/CD4 cells and MT-2 human T cells, and the CXCR5 gene was cloned into an expression vector. S+L-HOS/CD4 cells were susceptible to several HIV-2 strains but not most HIV-1 strains. To examine a coreceptor activity of CXCR5, we used NP-2/CD4, which is a human glioma cell line, NP-2, transduced with the CD4 gene that shows strict resistance to infection with HIV-1, HIV-2, SIVmac, SIVagm, or SIVmnd strain. When CXCR5 was transduced into NP-2/CD4 cells, they became highly susceptible to HIV-2ROD and HIV-2CBL23 strains in a CD4-dependent manner but to not to HIV-1 or SIV strains. Anti-CXCR5 monoclonal antibody and a ligand for CXCR5, BCA-1, inhibited HIV-2 infection to NP-2/CD4/CXCR5 cells. Our findings suggest a possibility that CXCR5/BLR1 serves as a coreceptor for HIV-2 strains in vivo.

Animals↗
PubMed1999

A novel glycosyltransferase with a polyglutamine repeat; a new candidate for GD1alpha synthase (ST6GalNAc V)(1).

The fifth type GalNAcalpha2,6-sialyltransferase (mST6GalNAc V) was cloned from a mouse brain cDNA library. mST6GalNAc V exhibited type II transmembrane topology containing a polyglutamine repeat, which showed 42.6% and 44.8% identity to mouse ST6GalNAc III and IV, respectively. Northern blot analysis revealed that the mST6GalNAc V gene was specifically expressed in forebrain and cerebellum. mST6GalNAc V exhibited GD1alpha synthetic activity from GM1b the same as mST6GalNAc III and IV. The activity ratio of GM1b toward fetuin and the expression pattern were completely different among the three ST6GalNAcs. Interestingly, the polyglutamine repeat number was different from that of inbred mice. We report the first glycosyltransferase with a polymorphic polyglutamine repeat.

Amino Acid Sequence↗
PubMed1999

The DNA sequence of human chromosome 22.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

Animals↗
PubMed1999

High-level expression of the Mnb/Dyrk1A gene in brain and heart during rat early development.

We previously isolated human MNB/DYRK1A cDNA from "the Down syndrome critical region" of human chromosome 21 (Shindoh et al., 1996, Biochem. Biophys. Res. Commun. 225: 92-99). As described herein, we prepared a polyclonal anti-MNB/DYRK1A antibody and used it in a Western blot assay to assess the expression of the MNB/DYRK1A protein during rat development. The MNB/DYRK1A protein was expressed strongly not only in the brain but also in other tissues from embryonic rats. At the early postnatal stage, expression of the protein was high in the central nervous system and heart, but low in liver, lung, spleen, and kidney. The level of MNB/DYRK1A protein in all tissues studied gradually decreased with postnatal growth. Similarly, Northern blot analysis revealed that a major 6.0-kb transcript of the Mnb/Dyrk1A gene was expressed at a high level in the brain during the early postnatal period but that its level was low in the adult. The finding that the MNB/DYRK1A protein is expressed strongly in the central nervous system and heart may indicate a significant role for this protein in the development of these organs.

Amino Acid Sequence↗
PubMed1999

A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2.

Human chromosome 11q23.2 has been proposed to contain a tumor suppressor gene(s) whose deletion has been associated with cancer of the lung and breast and with neuroblastoma. To analyze the genomic structure and to isolate a candidate tumor suppressor gene from this region, we constructed a 2-Mb sequence-ready contig map using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The map comprises a contig of 24 overlapping P1, BAC, and PAC clones. To isolate gene fragments from the region, we performed direct cDNA library screening, exon trapping, EST mapping, and genomic sequencing using the P1, BAC, and PAC clones. Sequence analysis of 5 clones, which spans 23% (458,738 bp) of the region, and extensive gene scanning along the entire region revealed that the region is extraordinarily scarce in genes, but we identified one ubiquitously expressed novel gene and one testis-specific gene fragment. The novel gene, which we call IGSF4 (immunoglobulin superfamily 4), is transcribed into a 1.6- or 4.4-kb RNA encoding a 442-amino-acid protein. It shares strong homology with mouse IGSF-B12 and cell adhesion molecules NCAM1 and NCAM2 within their Ig-like C2-type domains. The IGSF4 gene, a novel gene that is shown to be located in the common loss of heterozygosity region, possesses a number of interesting features and may be good candidate for a tumor suppressor gene.

Amino Acid Sequence↗
PubMed1999

rDLG6: a novel homolog of Drosophila DLG expressed in rat brain.

Drosophila DLG (Discs Large Tumor Suppresser Protein) is a component of septate junctions, and disruption of its gene leads to over growth of imaginal discs. Homologs of Drosophila DLG recently isolated from mammalian tissue have been classified as members of the MAGUK (Membrane Associated GUanylate Kinase) superfamily of proteins. Using a modified RT-PCR method applied to rat tissues, we have isolated cDNA clones encoding a novel MAGUK family member that we have named rDLG6. Immunoblot and immunohistochemical analyses revealed that rDLG6 protein is predominantly expressed in brain. GST pull-down assays showed that the PDZ domain of rDLG6 protein binds to the C-terminus of the AMPA (alpha-Amino-3-hydroxy-5-Methyl-isoxazole-4-Propionic Acid) receptor GluR2 subunit.

Amino Acid Sequence↗
PubMed1999

Identification of a putative regulatory subunit of a calcium-activated potassium channel in the dup(3q) syndrome region and a related sequence on 22q11.2.

Duplication of a segment of the long arm of human chromosome 3 (3q26.3-q27) results in a syndrome characterized by multiple congenital abnormalities and neurological anomalies in some patients. We have identified a novel gene (KCNMB3) that maps to this region. KCNMB3 has significant sequence similarity to the regulatory subunit of the large-conductance calcium-activated potassium channel. Due to the significance of potassium channels in neuronal functions, the overexpression of this gene may play a role in the abnormal neurological functions seen in some of these patients. A related sequence corresponding to the second and third exons of this gene resides in the pericentromeric region of 22q11, where a number of other unprocessed pseudogenes are known to map.

Abnormalities, Multiple↗
PubMed1999

Physiological mechanism for enhancement of paracellular drug transport.

We examined the action mechanisms of enhancers that improve paracellular drug transport. For sodium caprate (C10), the increase in the intracellular calcium level was considered to induce the contraction of calmodulin-dependent actin filaments, followed by dilation of the paracellular pathway. Although decanoylcarnitine (DC) also increased the intracellular calcium level, the action was independent of calmodulin and thus, the action mechanism of acylcarnitines was considered to differ from that of C10. Other acylcarnitines, lauroylcarnitine (LC) and palmitoylcarnitine (PC) and organic acids, tartaric acid (TA) and citric acid (CA) decreased the intracellular ATP level and the intracellular pH. From these results, it was considered that one of the action mechanism of acylcarnitines and organic acids is that the intracellular acidosis increases the calcium level through the decrease in ATP levels, followed by opening the tight junction. Membrane dysfunction which was expected from the above mechanism was assessed by the transport function of electrolytes. Membrane conductance, which was increased by C10, LC and PC, returned to the control value during a 3- to 6-h recovery period. On the other hand, Cl(-) ion secretion, which was obtained from short-circuit current (I(sc)), was decreased by these enhancers, but was normalized by C10 but not by LC and PC. Accordingly, C10 can be considered a safer enhancer than acylcarnitines.

Acylation↗
PubMed1999

Human and mouse ISLR (immunoglobulin superfamily containing leucine-rich repeat) genes: genomic structure and tissue expression.

We have previously reported a transcript of the novel gene for human immunoglobulin superfamily containing leucine-rich repeat (ISLR). By additional screening of a human retina cDNA library, we isolated another type of transcript with a 5' UTR different from that of the previously reported type. Genomic sequencing of the ISLR gene revealed that these two types of transcripts, ISLR-1 and ISLR-2, originated from the same gene but are composed of different first exons. Because the entire open reading frame is contained in the second exon, these two transcripts produce the same protein. Radiation hybrid mapping linked the ISLR gene to AFM248yh1, which is in the critical region of Bardet-Biedl syndrome type 4 (BBS4) on chromosome 15. Sequence analysis of the ISLR gene in five BBS4 patients, however, showed no mutations, although a few polymorphic changes were detected. Cloning of the mouse homolog of ISLR (Islr) revealed that the predicted protein consists of 428 amino acids, 86% of which are identical to those of ISLR. The Islr gene was expressed in various mouse tissues, including retina, in which Islr mRNA was detected in the ganglion cell layer, the inner nuclear layer, and the inner segment of the photoreceptor.

Animals↗
PubMed1999

Five different genes, Eif4a1, Cd68, Supl15h, Sox15 and Fxr2h, are clustered in a 40 kb region of mouse chromosome 11.

We characterized a region of the mouse genome disrupted by integration of a gene trap (GT) vector in ES cells. On 5' rapid amplification of cDNA ends analysis of the fusion transcripts containing the GT vector, we identified the eukaryotic protein synthesis initiation factor 4A1 gene (Eif4a1) as a promoter-trapped gene. Plasmid rescue was used to show that the other end of the integrated vector disrupted the murine homolog of the human fragile X mental retardation syndrome-related protein 2 gene (Fxr2h). Structural analysis of P1 clones, isolated from the wild-type mouse genome by PCR with Eif4a1-specific primers, indicated that the integration of the GT vector was accompanied by the deletion of about 35 kb of genomic DNA and that the disrupted region also included three genes, Cd68, Supl15h and Sox15, the latter two of which are transcribed in opposite directions with overlapping 3' ends. These five different genes at least, Eif4a1, Cd68, Supl15h, Sox15 and Fxr2h, are clustered in a 40 kb region. The chromosomal location of this region was mapped by means of interspecific backcross panel DNAs to the central part of mouse chromosome 11, exhibiting a known region of synteny with human chromosome 17.

Animals↗
PubMed1999

Enhanced activity of cyclin A-associated kinase in immortalized human fibroblasts.

Deregulation of cell cycle regulatory mechanisms leads to disruption of normal control of cell growth and may be associated with neoplastic transformation. To determine whether immortalized human cells have alterations in their cell cycle regulatory mechanisms, we analyzed cell cycle regulatory proteins in 2 immortalized human fibroblast cell lines, KMST-6 and OUMS-24/P6X, established by repeated irradiation with (60)Co gamma rays alone or mutant p53 gene transfection plus X-ray irradiation, respectively. Both the immortalized cell lines had markedly enhanced activity of cyclin A-associated kinase as compared with their normal counterparts. The high activity of cyclin A-associated kinase was well correlated with the increased expression of cyclin A mRNA and its protein. In addition, the immortalized cell lines showed significantly reduced amounts of p21(Cip1/Waf1/Sdi1), a potent inhibitor of cyclin dependent kinases. Furthermore, among the pRb family of proteins, p107 and p130, were hyperphosphorylated in both the immortalized cell lines, suggesting possible participation in upregulation of cyclin A associated kinase activity. These changes represent some important characteristics of immortalized cells.

Blood Proteins↗
PubMed1999

Alteration in amino acids in motor neurons of the spinal cord in amyotrophic lateral sclerosis.

Little is known concerning the changes of amino acid composition in different regions of the spinal cord in patients with amyotrophic lateral sclerosis (ALS). We performed quantitative amino acid analyses in the posterior funiculus, the lateral corticospinal tract, and the anterior horn of cervical enlargement of the spinal cord from seven ALS patients, and the results were compared with those of seven patients with other neurologic diseases (control A) and seven patients without neurologic diseases (control B). The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract and the anterior horn of ALS patients than in controls A and B. The contents of the acidic amino acids glutamate and aspartate were also significantly decreased in the lateral corticospinal tract and the anterior horn of ALS patients as compared with those of controls A and B. These data suggest that decreased contents of collagen-associated amino acids and excitatory amino acids are related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS.

Adult↗
PubMed1999

Heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human T-cell lymphotropic virus type I.

Heat shock cognate protein 70 (HSC70) has been shown to bind to the peptide corresponding to amino acids 197 to 216 of human T-cell lymphotropic virus type I (HTLV-I) envelope protein, gp46, and an anti-HSC70 monoclonal antibody (mAb) inhibits HTLV-I-induced syncytium formation. These findings suggest that HSC70 is necessary for the entry of HTLV-I into its target cells. Here we showed that HSC70 directly binds to gp46 by co-immunoprecipitation of HSC70 and gp46 from HTLV-I-producing human T-cell lysate. However, transduction of human HSC70 cDNA into BaF3 cells, which were found to be highly resistant to HTLV-I infection, did not support the HTLV-I entry, and HSC70 expressed in NIH3T3 cells, which were found to be almost resistant to syncytium formation upon cocultivation with HTLV-I-producing cells but sensitive to infection with cell-free HTLV-I, enhanced cell fusion induced by HTLV-I-producing cells, but did not enhance the entry of cell-free HTLV-I into these cells. The mAb against HSC70 inhibited syncytium formation in NIH3T3 cells expressing HSC70, but showed little effect on infection of these cells with cell-free HTLV-I. These findings indicate that HSC70 markedly enhances syncytium formation induced by HTLV-I but does not facilitate HTLV-I entry into target cells.

3T3 Cells↗
PubMed1999

Changes in and discrepancies between cell tropisms and coreceptor uses of human immunodeficiency virus type 1 induced by single point mutations at the V3 tip of the env protein.

We examined the effect of amino acid substitutions of the GPGR (glycine-proline-glycine-arginine) tip sequence at the V3 domain of the Env protein of human immunodeficiency virus type 1 (HIV-1) on its cell tropism and coreceptor use. We changed the GPGR sequence of a T-cell line (T)- and macrophage (M)-tropic (R5-R3-X4) HIV-1 strain, GUN-1wt, to GA(alanine)GR (the resulting mutant was designated GUN-1/A), GL(leucine)GR (GUN-1/L), GP(proline)GR (GUN-1/P), GR(arginine)GR (GUN-1/R), GS(serine)GR (GUN-1/S), or GT(threonine)GR (GUN-1/T). GUN-1/A, GUN-1/S, and GUN-1/T mutants infected brain-derived cells such as a CD4-transduced glioma cell line, U87/CD4, and a brain-derived primary cell strain, BT-20/N, as well as T-cell lines in a CD4-dependent manner, although the plating of these mutants onto macrophages was inhibited. GUN-1/L, GUN-1/P, and GUN-1/R mutants showed both T- and M-tropism, but did not plate onto the brain-derived cells. A CCR3, CCR5, CCR8, or CXCR4 gene was introduced into a CD4-positive glioma cell line, NP-2/CD4, which demonstrated complete resistance to various HIV-1 strains. Not only HIV-1 strains, which were infectious to macrophages, such as GUN-1wt, GUN-1v, GUN-1/L, and GUN-1/P, but also an HIV-1 strain, GUN-1v, which was hardly infectious to macrophages, grew well in NP-2/CD4 cells expressing CCR3 or CCR5. However, the M-tropic GUN-1/R mutant could not efficiently use CCR5 nor CCR3. No point mutants, except GUN-1/L, grew well in NP-2/CD4 cells expressing CCR8. These findings indicate that the cell tropism of HIV-1 to macrophages and brain-derived cells and their use of the coreceptors were markedly, though not always concomitantly, affected by the tip sequence of the V3 domain.

Brain↗
PubMed1999

Substituent effect of a fluorine atom on the mutagenicity of nitroquinolines.

Some 16 nitroquinolines (NQs) and their fluorinated derivatives were tested for mutagenicity in Salmonella typhimurium TA100 without S9 mix to investigate the effect of fluorine-substitution on the mutagenicity. These NQs consist of 5-NQs, 5-nitroquinoline N-oxides (5-NQOs), N-methyl-5-nitroquinolinium methanesulfonates (N-Me-5-NQs) and 8-NQs, including three ortho-F-NQs, one meta-F-NQ, four para-F-NQs and four 3-F-NQs. For this purpose, eight F-NQs were newly synthesized. The data indicated that the ratio of the mutagenic activities (revertants/plate/nmol) of fluorinated NQs to those of the corresponding parent non-fluorinated compounds ranged from 0.6- to 119-fold. The fluorine atom located para to the nitro group markedly enhanced the mutagenicity (24-fold and more), while three ortho-fluorinated derivatives showed no significant increase in mutagenicity (enhancement ratio were 0.6, 0.8 and 1.7). With respect to 8-NQs, its meta-fluorinated derivative also had an enhanced mutagenicity over the parent compound (53-fold). In addition, although N-Me-5-NQ was less mutagenic than 5-NQ and 5-NQO, the mutagenicity of N-Me-5-NQ was most significantly enhanced by fluorine-substitution. These results suggest that introduction of a fluorine atom to the molecule in question may be a useful tool to modify their mutagenic potency and to better understand the mechanism of mutation.

Chromatography, Gel↗