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Biomedical subjects

K Sugaya

Publications and source records attributed to K Sugaya.

At least 73 records · Page 4Linked to original sources
PubMed1997

Gene organization of human NOTCH4 and (CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3.

The cDNA and genomic clones for the human counterpart of the mouse mammary tumor gene Int3 were isolated and sequenced. We designated this human major histocompatibility complex (MHC) class III gene as NOTCH4, since very recently, by sequencing cDNA clones, the complete form of the mouse proto-oncogene Int3 has been clarified and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and by comparing it with the mouse Notch4 cDNA sequence, the exon/intron organization was clarified. The comparison of the predicted amino acid sequence of human NOTCH4 with those of other Notch homologues of a wide range of species revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)n repeats showing a variable number tandem repeat (VNTR) polymorphism for different human leukocyte antigen (HLA) haplotypes. Ten genes mapped on 6p21.3, including NOTCH4, were found to have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution. Similarity of genes on chromosomes 1, 6, 9 and 19 was also discussed.

Amino Acid Sequence↗
PubMed1997

Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors.

In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1alpha, EF-1beta, EF-1gamma, EF-2 and EF-3. With the exception of the EF-1gamma gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1alpha, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3' untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1alpha genes are located at different loci. Northern analysis indicated that one of three EF-1alpha genes was inducible with UV-irradiation, while the level of expression for another of three EF-1alpha genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1beta cDNA clone covered almost all the coding sequence (CDS) of EF-1beta except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3' UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.

Amino Acid Sequence↗
PubMed1997

The central neural pathways involved in micturition in the neonatal rat as revealed by the injection of pseudorabies virus into the urinary bladder.

Pseudorabies virus was injected into the wall of the urinary bladder in 2- and 12-day-old rats in order to examine developmental changes in the central neural pathways controlling micturition. Forty-eight hours after virus injection, virus-labeled neurons were identified in the lumbosacral spinal cord but not in the brain. Sixty to seventy-two hours after virus injection in both 2- and 12-day-old rats, infected neurons were detected in Barrington's nucleus, nucleus paragigantocellularis, nucleus reticularis gigantocellularis, A5 area, nucleus raphe obscurus, locus subcoeruleus, periaquaductal gray, red nucleus, paraventricular nucleus and cerebral cortex. These results in neonatal rats are similar to those reported in adult rats. Thus, it is likely that the supraspinal neural circuitry which underlies micturition in the adult animal is already organized in the neonatal rat during the early postnatal period even though the supraspinal micturition reflex pathway does not become functional until the third postnatal week.

Aging↗
PubMed1997

B220 expression by T lymphoid progenitor cells in mouse fetal liver.

The present study has characterized T lymphoid progenitor cells that reside in mouse fetal liver. Day 14 fetal liver contains progenitor cells that can differentiate into mature T cells upon being transferred into the thymus by hanging drop cultures. Fractionation of fetal liver cells indicated that T progenitor cells were confined in TER119- CD45+ FcR(low) cells. To our surprise, B220+ rather than B220- fraction in TER119- CD45+ FcR(low) fetal liver cells exhibited efficient progenitor activity generating T cells. Progenitor activity by the B220+ fetal liver cells was restricted to T cells, B cells, and macrophages at frequency approximately 1/10, approximately 1/10, and approximately 1/20, respectively, of isolated B220+ cells. B220+ fetal liver cells did not contain detectable D-J rearrangement of TCR-beta gene and were c-kit+ IL-7R+ Thy-1- CD3- CD4(low) CD8- CD25- CD44+. B220+ fetal liver cells expressed mRNAs encoding TCR-beta, pT alpha, Ig alpha, and VpreB. Interestingly, TCR beta-chains were expressed by B220+ fetal liver cells in the VDJ-rearranged TCR-beta-transgenic mice, indicating that TCR-beta transcription and B220 expression are activated simultaneously by the transgenic B220+ fetal liver cells. These results indicate that B220 is expressed by fetal liver lymphoid progenitor cells that can become T cells, and suggest that lymphoid progenitor cells in fetal liver concurrently undergo T- and B-specific molecular events within a single cell.

Animals↗
PubMed1997

Decrease in intravesical saline volume during isovolumetric cystometry in the rat.

This study was undertaken to examine the change in intravesical saline volume during isovolumetric cystometry in the urethane anesthetized female rat. The ureters were transected bilaterally and their distal ends were ligated. To prevent leakage of intravesical fluid, the proximal urethra was ligated after inserting a urethral catheter into the bladder. In 13 rats in which the bladder was filled with different volumes of saline between 0.3 and 2.0 ml and maintained under isovolumetric conditions for 3 hours, intravesical volume decreased (4-79%) when initial intravesical saline volume was equal to or greater than 0.6 ml (n = 9). In 9 rats in which intravesical volume decreased, mean final volume was 0.68 +/- 0.09 ml. The percentage change in intravesical volume (y) significantly depended on initial volume (x) (y = -42x + 17, n = 13). In 7 rats in which the proximal urethra was ligated and the bladder was filled with different volumes of soybean oil between 0.25 and 2.0 ml, intravesical volume did not change. In 8 rats in which the bladder neck was ligated around a urethral catheter to block blood flow to the bladder and in which the bladder was filled with different volumes of saline (0.3-1.8 ml), intravesical volume decreased when initial volume was equal to or greater than 1.35 ml. These results demonstrate that intravesical saline is absorbed from the normal bladder wall when intravesical volume is greater than the volume (0.6 ml) necessary to evoke the micturition reflex in urethane anesthetized rats.

Animals↗
PubMed1997

Use of capillary electrophoresis with laser-induced fluorescence detection to assess messenger ribonucleic acid molecules amplified by the polymerase chain reaction: applications in the cloning of cells.

Progressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter; i.e., basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter-specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c-NOS), and the growth-associated protein GAP-43, three genes known to be expressed in central cholinergic neurons. A "nondestructive" method of screening cultured cells for their expression of c-NOS was established using depolarization with medium containing 50 mM potassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E-115 (c-NOS-positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature-sensitive SV-40 transforming activity and neomycin resistance. Cell colonies surviving in G418-containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33 degrees C, differentiated by switching to a low-serum medium and growth at 39 degrees C, and screened for depolarization-induced accumulation of nitrite in the medium. The subset of putative c-NOS-positive clones (about 4%) were then screened for their expression of mRNAs using RT/PCR in combination with CE/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cells cloned from embryonic rat brainstem.

Animals↗
PubMed1997

A major non-LTR retrotransposon of Bombyx mori, L1Bm.

Repetitive sequences with oligo A tails were observed in Dra1 fragments of Bombyx mori genomic DNA. The full sequence of the element, an abundant non-LTR retrotransposon of B. mori, was determined by assembling inner restriction fragments. This element, designated L1Bm, contained two ORFs encoding a gag-like protein and reverse transcriptase (RT), respectively. An endonuclease domain was identified at the N-terminus of the RT sequence. The homology search of the amino acid sequences revealed that L1Bm belongs evolutionally to the same family as various other non-LTR retrotransposons of Drosophila and Mosquito. On the other hand, L1Bm resembles the L1 element of human genome in its high copy number and its frequent truncation at the 5'-side. Some units of the histone gene repeat in B. mori possess complete L1Bm elements in a 3'-flanking region of H2b.

Amino Acid Sequence↗
PubMed1997

Topographic associations between DNA fragmentation and Alzheimer's disease neuropathology in the hippocampus.

To identify whether the process of apoptosis bears a topographic relationship to selected aspects of Alzheimer's disease (AD) pathology, we used an in situ nick translation method (TUNEL) to map DNA fragmentation in hippocampal sections immunostained for abnormally phosphorylated tau, which exists in the neurofibrillary tangles (NFTs) and in the dystrophic neurites associated with senile plaques. To ascertain associations of DNA fragmentation with glia, TUNEL was combined with immunohistochemistry for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial antigen OX-42. Consistent with previous reports, the incidence of putative DNA fragmentation detected by TUNEL was much higher in the AD brain, compared to non-demented subjects. While most TUNEL-positive cells did not exhibit any systematic topographic relationship to senile plaques, which were visualized by immunostain of abnormally phosphorylated tau for dystrophic neurites, DNA fragmentation was found frequently within cells containing NFTs. In hippocampal sections prepared to visualize glia, DNA fragmentation was not observed in GFAP-positive astrocytes, but some OX-42-positive microglia exhibited TUNEL signals. Other TUNEL-positive cells were found frequently in proximity to glia. The data suggest that cells compromised by the deposition of NFTs are prone to initiate the process of apoptosis. Furthermore, some glial populations appear to be apoptotic in the AD brain.

Aged↗
PubMed1997

Proto-oncogene of int-3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis.

BACKGROUND: Notch and its homologues are key regulatory receptors of the cell fate decision in various developmental processes. The int-3 oncogene was originally identified as a frequent target in Mouse Mammary Tumour Virus (MMTV)-induced mammary tumours and has been regarded as a Notch homologue, based on its similarity to the intracellular domain of Notch. Studies with int-3 transgenic mice have suggested that the int-3 transgene affects the differentiation capacity of stem cells and leads to neoplastic proliferation in epithelial cells. However, the exact nature and the in vivo expression pattern of the int-3 proto-oncogene are unknown. The function of gene products in embryogenesis is also not clear. RESULTS: We isolated cDNA clones corresponding to the proto-oncogene of int-3 and analysed its overall structure. The predicted amino acid sequence of the int-3 proto-oncogene contains the conserved motif found in Notch family receptors. Therefore, we name Notch-4 for the int-3 proto-oncogene. However, Notch-4 has fewer EGF repeats and shows less similarity to Notch, compared with other mammalian Notch homologues. In embryogenesis, the expression of Notch-4 was detected in endothelial cells of blood vessels forming tissues such as the dorsal aorta, intersegmental vessels, yolk sac vessels, cephalic vessels, heart, vessels in branchial arches, and capillary plexuses. In these tissues, Notch-4 expression coincided with flk-1, the major regulatory gene of vasculogenesis and angiogenesis. We also found that Notch-4 expression was up-regulated in vitro during the differentiation of endothelial cells from embryonic stem cells (ES cells). CONCLUSION: The endothelial cell specific expression pattern of Notch-4, as well as its structural similarity of Notch, suggest that Notch-4 is an endothelial cell specific homologue of Notch and it may play a crucial role in vasculogenesis and angiogenesis.

Amino Acid Sequence↗
PubMed1997

Triplet repeat polymorphism in the NOTCH4 gene with the human major histocompatibility complex in a healthy population and patients with a salivary gland tumor in Japan.

The NOTCH4 gene, the human counterpart of the mouse mammary tumor gene, int-3, has been recently localized near the boundary of the HLA class II and class III regions. This gene is one of candidates for development of salivary gland tumor. Microsatellite polymorphism of (CTG)n repeat in the signal peptide domain of NOTCH4 was analyzed in Japanese including the patients with salivary gland tumor. Four alleles consisting of 6, 9, 10 and 11 repetitions of CTG (Leu) were observed and found to be in linkage disequilibria with HLA class I and class II alleles. No significant association of this microsatellite polymorphism with the disease were observed in 26 samples of salivary gland tumor. In this neoplasia, neither large-scale deletion nor translocation was detected around the NOTCH4 gene using genomic Southern hybridization analysis by the NOTCH4 cDNA as a probe.

Alleles↗
PubMed1997

Triplet repeat polymorphism within the NOTCH4 gene located near the junction of the HLA class II and class III regions in narcolepsy.

A polymorphic (CTG)n microsatellite repeat was found in the signal peptide domain of the NOTCH4 gene located near the junction of the class II and class III regions of the human major histocompatibility complex. This gene belongs to a multigene family of NOTCH originally identified as a differential factor of neuronal cells. To ascertain whether the NOTCH4 gene is involved in the development of neurogenic disease, narcolepsy, which is known to be tightly associated with HLA-DR15, this microsatellite polymorphism of the (CTG)n repeat was analyzed in Japanese patients with narcolepsy One allele, 9 repetitions of CTG (Leu) was significantly increased in the patient group. However, the significant increase of this allele in the patient group could be explained by a strong linkage disequilibrium with the HLA class II alleles, DRB1*1501, DQA1*0102 and DQB1*0602, which were more strongly associated with the disease. These results suggest that the (CTG)n repeat polymorphism in NOTCH4 does not primarily determine the susceptibility to narcolepsy.

Alleles↗
PubMed1997

Precise switching of DNA replication timing in the GC content transition area in the human major histocompatibility complex.

The human genome is composed of long-range G+C% (GC%) mosaic structures thought to be related to chromosome bands. We previously reported a boundary of megabase-sized GC% mosaic domains at the junction area between major histocompatibility complex (MHC) classes II and III, proposing it as a possible chromosome band boundary. DNA replication timing during the S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify to the nucleotide sequence level the replication switch point during the S phase, we determined the precise DNA replication timing for MHC classes II and III, focusing on the junction area. To do this, we used PCR-based quantitation of nascent DNA obtained from synchronized human myeloid leukemia HL60 cells. The replication timing changed precisely in the boundary region with a 2-h difference between the two sides, supporting the prediction that this region may be a chromosome band boundary. We supposed that replication fork movement terminates (pauses) or significantly slows in the switch region, which contains dense Alu clusters; polypurine/polypyrimidine tracts; di-, tri-, or tetranucleotide repeats; and medium-reiteration-frequency sequences. Because the nascent DNA in the switch region was recovered at low efficiency, we investigated whether this region is associated with the nuclear scaffold and found three scaffold-associated regions in and around the switch region.

Base Sequence↗
PubMed1996

Chromosomal localization of the proteasome Z subunit gene reveals an ancient chromosomal duplication involving the major histocompatibility complex.

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon gamma, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC.

Amino Acid Sequence↗
PubMed1996

Molecular indices of neuronal and glial plasticity in the hippocampal formation in a rodent model of age-induced spatial learning impairment.

Spatial learning ability was quantitated in young and aged Long-Evans rats, and molecular markers were assessed in the striatum and hippocampal formation using immunocytochemical, immunoblotting, and in situ hybridization histochemical procedures. The mRNA for beta-amyloid precursor protein (beta APP), most likely the transcript encoding the 695-amino acid form of this protein, was elevated in pyramidal and granule cells in the hippocampus of aged rats exhibiting poorer spatial learning. In immunoblots of hippocampal protein extracts, however, the level of beta APP-like immunoreactivity was depressed in the more impaired subjects. Similarly, the level in hippocampus of the mRNA for manganese-dependent superoxide dismutase (Mn-SOD), a marker of oxidative stress, was positively correlated with the degree of behavioral impairment, but immunoblotting revealed that Mn-SOD protein was depressed in the aged hippocampus compared with young. The mRNAs for the neuronal form of nitric oxide synthase and for the astrocyte marker glial fibrillary acidic protein (GFAP) were elevated in the hippocampus in correlation with the extent of learning impairment. In the striatum, the levels of mRNA and protein for several candidate genes, including GFAP, were elevated in parallel with the learning index, but these were age effects. Several hippocampal proteins were unchanged (GFAP) or depressed (beta APP and Mn-SOD) in level, despite elevations in corresponding mRNAs. In the aged cohort, hippocampal GFAP mRNA, Mn-SOD mRNA, and beta APP emerged as predictors of behavioral impairment, suggesting the involvement of these hippocampal systems in age-related cognitive impairment.

Aging↗
PubMed1996

Simplified RT/PCR quantitation of gene transcripts in cultured neuroblastoma (SN49) and microglial (BV-2) cells using capillary electrophoresis and laser-induced fluorescence.

We developed a simplified protocol for sensitive quantitation of mRNA using polymerase chain reaction (PCR) amplification of cDNA made by reverse transcriptase (RT), as resolved with capillary electrophoresis (CE) and detected with laser-induced fluorescence (LIF). The conditions required for adequate accuracy of the simplified version of the RT/PCR quantitation, in which a single concentration of external standard and amplification to within or near the plateau phase are used, were established for assay of mRNAs expressed at high, moderate, and low abundance. The mRNAs for the cytosolic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH) and the growth-associated protein GAP-43 in cultured SN49 neuroblastoma cells were used as target genes for high and moderate levels of expression, respectively. Using cultured mouse microglial cells (BV-2), we demonstrated the utility of this RT/PCR/CE/LIF protocol to quantitate a low-abundance mRNA, encoding a form of nitric oxide synthase (i-NOS) induced by treatment with endotoxin. The appearance of i-NOS mRNA after endotoxin treatment of BV-2 cells was confirmed by Northern blot analysis and in situ hybridization histochemistry, and functional enzyme activity was followed by release of nitric oxide (as nitrite) into the medium. The many advantages of the 'single-point' RT/PCR/CE/LIF protocol for quantitating mRNAs of interest include: simplified protocol, elimination of the use of radiotracers, high sensitivity and precision, and semi-automation of the quantitation phase of analysis.

Animals↗
PubMed1996

Heterogeneous expression of recombination activating genes and surface CD5 in CD3low CD4+ CD8+ thymocytes.

Clonal selection in the thymus occurs mostly in the CD4+ CD8+ (double positive; DP) stage. Within DP thymocytes, cells in the CD3low subset are believed to be involved in clonal selection, and this subset is generally considered as a homogeneous population. T-cell antigen-receptor (TCR) signals on DP thymocytes are known to (i) down-regulate recombination activating gene (RAG) expression; and (ii) up-regulate the expression of T-cell function-associated molecules, including the cell surface glycoprotein CD5. The present study examined the expression of RAG and CD5 molecules among the subpopulations of normal adult DP thymocytes. DP thymocytes were fractionated according to their cell surface expression levels of TCR-CD3 complex into CD3dim, CD3lo, CD3med, and CD3hi cells, since TCR expression is known to increase during thymocyte maturation. Down-regulation of RAG mRNA was located between the CD3low and CD3med DP populations. However, within the DP CD3low subpopulation, we found that CD5 varies from low to high expression levels. Upon fractionation of DP CD3low thymocytes into CD5low and CD5high subsets, we were able to detect down-regulation of RAG transcripts within the CD3low subpopulation. Thus, by the criteria of CD5 surface expression and RAG mRNA expression levels, DP CD3low thymocytes can be considered a heterogeneous thymocyte subpopulation.

Animals↗
PubMed1996

Mechanisms of the suppression of the bladder activity by flavoxate.

BACKGROUND: This study was designed to clarify the primary site of action of flavoxate, clinically used for the treatment of urinary frequency. METHODS: In rats, the effect of flavoxate on contractile responses in isolated detrusor strips, bladder contraction induced by pelvic nerve stimulation, isovolumetric rhythmic bladder contractions, and pelvic nerve activity were examined. In decerebrated cats, flavoxate was microinjected into the nuclei in the pons, and its effect on reflex micturition was observed. RESULTS: Flavoxate suppressed carbachol- and calcium ion (Ca2+)-induced contractions of isolated detrusor strips in a noncompetitive and a competitive manner, respectively. Intravenous flavoxate suppressed both initial phasic, and later tonic, bladder contractions induced by electrical stimulation of the distal end of the pelvic nerve. It abolished isovolumetric rhythmic bladder contractions and the associated efferent pelvic nerve activity, without affecting baseline vesical pressure and afferent pelvic nerve activity. When administered intracerebroventricularly or intrathecally, it abolished isovolumetric rhythmic bladder contractions. Flavoxate microinjected into the nucleus reticularis pontis oralis (PoO; pontine micturition inhibitory region) of decerebrated cats inhibited the reflex micturition, but had no effect when microinjected into the locus coeruleus alpha (pontine micturition center) or locus subcoeruleus (pontine urine storage center). CONCLUSIONS: Flavoxate suppressed the micturition reflex primarily by facilitating the inhibitory action of the PoO on the descending pathways from the pontine micturition center to the sacral parasympathetic intermediolateral nuclei.

Animals↗