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Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 19 recordsLinked to original sources
PubMed2005

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

3' Untranslated Regions↗
PubMed2004

Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis.

HutP is an RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus species by binding to cis-acting regulatory sequences on hut mRNA. We recently solved the HutP crystal structure, which revealed a novel fold where three dimers are arranged in a 3-fold axis to form the hexamer. We also identified a minimal RNA binding element sufficient for HutP binding: three UAG trinucleotide motifs, each separated by 4 nt, located just upstream of the terminator. In the present study we have identified important RNA chemical groups essential for HutP interactions, by combining an in vitro selection strategy and analyses by site-specific base substitutions. These analyses suggest that each HutP molecule recognizes one UAG motif, where the first base (U) can be substituted with other bases, while the second and third bases (A and G) are required for the interactions. Further analyses of the chemical groups of the A and G bases in the UAG motif by modified base analogs suggested the importance of the exocyclic NH2 group in these bases. Also, in this motif, only the 2'-OH group of A is important for HutP recognition. Considering the important chemical groups identified here, as well as the electrostatic potential analysis of HutP, we propose that Glu137 is one of the important residues for the HutP-RNA interactions.

Adenine↗
PubMed2003

The Ishiguro extension block technique for the treatment of mallet finger fracture: indications and clinical results.

The results of extension block Kirschner wire fixation for the treatment of mallet fractures of the distal phalanx were retrospectively assessed in 65 consecutive patients. The indications for this technique were the presence of a large bone fragment, and palmar subluxation or the loss of joint congruity of the distal interphalangeal joint. Using the Wehbé and Scheider classification there were 27 type IB, 19 type IIB, 17 type IA, and 2 type IIA fractures. According to the Crawford rating system there were 46% excellent, 32% good, 20% fair and 2% poor results. We believe that this technique, when properly applied, produces satisfactory results.

Adolescent↗
PubMed2003

Ultrasonic absorption in aqueous solutions of amino acids at neutral pH.

Ultrasonic absorption coefficients in aqueous solutions of glycine, L-alanine, imidazole, L-phenylalanine, L-histidine and L-tryptophan at neutral pH were measured in the range from 0.8 to 220 MHz at 25 degrees C. A characteristic ultrasonic relaxation phenomenon was observed only in the solution of L-histidine with a relaxation frequency at around 2 MHz at neutral pH. It was proposed from the concentration independent relaxation frequency and the linear concentration dependence of the maximum absorption per wavelength that the relaxation mechanism was associated with a perturbation of the rotational isomeric equilibrium of the L-histidine molecule. The existence of two rotational isomeric forms of L-histidine in water was examined by semiempirical quantum chemical methods, in order to determine the free energy difference between the two states. The forward and backward rate constants were determined from the relaxation frequency and the energy change. Also, the standard volume change of the reaction was estimated from the concentration dependence of the maximum absorption per wavelength. It was speculated that L-histidine fulfills a specific function among amino acids because of the rotational motion in the molecule, in addition to its well-established acid-base properties.

Absorption↗
PubMed2003

Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates.

Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.

Blotting, Western↗
PubMed2002

Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis.

HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. HutP and its mutant, which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging-drop vapor diffusion method. The space group was P2(1)3 with unit cell dimensions a=b=c=95.6A for HutP and a=b=c=96.8A for the mutant. Complete data sets of 3.0-A resolution for wild-type HutP and of 2.70-A resolution for the mutant HutP were collected.

Bacillus subtilis↗
PubMed2002

Treatment of soft tissue injuries to the dorsum of the metacarpophalangeal joint (Boxer's knuckle).

We retrospectively reviewed the surgical treatment for 16 cases of traumatic soft tissue injury to the metacarpophalangeal joint (Boxer's knuckle). A history of trauma was present in all cases and there was an associated extensor tendon dislocation in seven cases. Eight cases were initially treated conservatively, but their symptoms persisted. Intraoperative findings included rupture of the extensor hood or joint capsule in all cases. Surgical closure of the rupture of the joint capsule resulted in a successful outcome in all cases. We consider that conservative treatment of this injury may not be effective when the joint capsule of the metacarpophalangeal joint is ruptured. We recommend arthrography of the metacarpophalangeal joint to assist in the decision as to whether to proceed with surgical or conservative treatment.

Adolescent↗
PubMed2002

The carpal detachment injury of the triangular fibrocartilage complex.

Triangular fibrocartilage complex (TFCC) injuries were suspected clinically in 22 wrists of 21 patients, but arthrography and MRI assessments of this structure were normal. As conservative therapy for 2 months did not improve their symptoms, wrist arthroscopy was then performed. Although no abnormalities of the TFCC and ligaments were observed, meniscus homologue-like tissue which arose from TFCC was riding on the articular surface of the triquetrum. After resection of this soft tissue with a shaver and a punch, the symptoms disappeared in all cases. The arthroscopic findings suggested that a portion of TFCC that was originally attached to the ulnar side of the triquetrum had become detached.

Adolescent↗
PubMed2001

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Animals↗
PubMed2001

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells.

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

Animals↗
PubMed2001

Reovirus as an oncolytic agent against experimental human malignant gliomas.

BACKGROUND: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. METHODS: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. RESULTS: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P =.0002 for both U251N and U87) and in two intracerebral (P =.0004 for U251N and P =.0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P =.0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. CONCLUSIONS: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers.

Animals↗
PubMed2001

Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein E-deficient mice.

BACKGROUND: The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-alpha (PDGFR-alpha) and receptor-beta (PDGFR-beta) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-alpha and PDGFR-beta, respectively. METHODS AND RESULTS: Apolipoprotein E-deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced by 67% and 80%, respectively, compared with the control mice injected with irrelevant rat IgG. In contrast, the mice that received APA5 showed only minimal reduction of lesion size, and a large number of VSMCs were observed in the intima. In the intima of advanced lesions, APB5 immunolabeled VSMCs, whereas APA5 could detect VSMCs mainly in the media. CONCLUSIONS: These results indicate that PDGFR-beta plays a significant role in formation of fibrous atherosclerotic lesions and that regulation of the signal transduction through PDGFR-beta could affect atherogenesis in mice.

Animals↗
PubMed2001

A role of netrin-1 in the formation of the subcortical structure striatum: repulsive action on the migration of late-born striatal neurons.

The mammalian striatum arises in the basal telencephalon and contains morphologically homogenous neurons that can be divided into two distinct compartments, patches and the matrix. During development, patch neurons are generated first to form a striatal primordium. After a large influx of later-born matrix neurons into this region, the unique mosaic arrangement of these two neuronal phenotypes is established. The massive migration of matrix neurons continues during the embryonic period, and they eventually comprise 80-85% of the mature striatum. To elucidate the cellular mechanism or mechanisms underlying this critical event in striatal histogenesis, we examined the migration characteristics of striatal subventricular zone (SVZ) cells at embryonic day 18 when neurogenesis peaks for matrix neurons. Using gel cultures, we show that netrin-1, one of the diffusible guidance cues expressed in the striatal ventricular zone (VZ), exerts a repulsive action on migrating SVZ cells. This effect is blocked in the presence of antibodies against Deleted in colorectal cancer (DCC), a putative receptor for netrin-1. The expression patterns of netrin-1 and DCC strongly suggest the involvement of this effect in the outward migration of SVZ cells into the striatal postmitotic region. Our cell tracing study using living brain slices demonstrates that striatal SVZ cells migrate toward and disperse throughout the striatum, in which they differentiate into phenotypes of striatal projection neurons. We suggest that netrin-1 expressed in the striatal VZ serves to guide the large influx of striatal matrix neurons into the striatal primordium and is thereby involved in the initial formation of fundamental striatal structures.

Animals↗
PubMed2001

Compartmentalization of Peyer's patch anlagen before lymphocyte entry.

We have shown that Peyer's patch (PP) first develops as a simple and even cell aggregation during embryogenesis. To investigate when and how such a simple cell aggregation forms the complex PP architecture, we analyzed the distribution of cells expressing IL-7R alpha (PP inducer cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature lymphocytes by whole-mount immunostaining of 17.5 days post coitus to 2 days postpartum mouse gut. Our results show that compartmentalization of PP anlagen commences at day 18.5 of gestation by clustering and subsequent follicle formation of IL-7R alpha(+), VCAM-1(+), and CD11c(+) cells. This process adds the primitive architecture of PP anlage with several follicles in which IL-7R alpha(+) cells localize in the center, while VCAM-1(+) and CD11c(+) cells localize at the fringe. This follicle formation is accompanied by the establishment of PP-specific vascular network expressing mucosal addressin cellular adhesion molecule-1. Mature B and T lymphocytes entering in the PP anlage are distributed promptly to their own target zones; B cells to the follicle and T cells to nonfollicular zones. Our analysis of scid/scid mouse indicate that the initial processes including formation of PP-specific vascular network occur in the absence of lymphocytes. These observations indicate that the basic architecture of PP is formed by a set of cell lineages assembled during the initial phase of induction of PP anlagen before entry of mature lymphocytes.

Animals↗
PubMed2001

Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer's patch organogenesis.

Mice deficient in lymphotoxin beta receptor (LTbetaR) or interleukin 7 receptor alpha (IL-7Ralpha) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Ralpha(+) lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1(+)ICAM-1(+) nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1(+)ICAM-1(+) cells are mesenchymal cells that are activated by lymphoid cells through the LTbetaR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.

Animals↗