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Biomedical subjects

N Shimizu

Publications and source records attributed to N Shimizu.

At least 55 records · Page 3Linked to original sources
PubMed2002

Nitrate removal rate in a continuous column denitrification reactor using hydrogen generated by electrolysis with carbon anodes and stainless cathodes.

An autotrophic continuous denitrification process, using hydrogen generated by electrolysis with activated carbon anodes, was experimentally demonstrated to be an effective nitrate removal process. Several fixed bed columns with polypropylene packing and honeycomb shaped activated carbon anodes and stainless rod cathodes were set in a thermostat chamber at 30 degrees C, and potassium nitrate enriched tap water as influent was supplied at various flow rates and electric currents. Although the anode is in the same column where microbial biomass grows, sufficient nitrate removal was observed. For example, almost complete removal of nitrate and nitrite was observed at a hydraulic retention time (HRT) as short as 1.8 h. A model assuming successive denitrification reactions and plug-flow process, nitrate reduction rate = k1 [NO3-] [H2], and nitrite reduction rate = k2 [NO2-] [H2](1.5) was proposed. Calculated results with k1 = 1.3 mmol(-1) h(-1) and k2 = 3.3 mmol(-1.5) x h(-1) agreed well with all the experimental results.

Biofilms↗
PubMed2002

The mutagenicity of amino-derivatives of diphenyl ether herbicides in new Salmonella typhimurium YG tester strains.

In this study, we examined the mutagenicity of four diphenyl ether herbicides and their amino derivatives in Salmonella typhimurium TA tester strains and YG tester strains. YG tester strains have been newly developed for sensitive detection of specific chemicals. S. typhimurium YG 1021, YG 1024, YG 1026 and YG 1029 strains are sensitive to mutagenic nitoroarenes and hydroxyamines. S. typhimurium YG 3003 is a strain that is sensitive to some oxidative mutagens. And S. typhimurium YG 7108 is useful for detection of mutagenic alkylating agents. As a result, each amino derivative of diphenyl ether herbicides is more mutagenic than its parent herbicide in S. typhimurium TA and YG tester strains with metabolic activation by S9 mixture. Moreover, S. typhimurium YG tester strains are more useful for highly sensitive detection of mutagens than S. typhimurium TA tester strains. We also examined the production of amino derivatives in a water environment from parent herbicides. It was clear that diphenyl ether herbicides rapidly transform to amino derivatives in a water environment.

Amines↗
PubMed2001

Molecular cloning of a member of the facilitative glucose transporter gene family GLUT11 (SLC2A11) and identification of transcription variants.

We isolated a member of the facilitative glucose transporter (GLUT) gene family (GLUT11; SLC2A11 as a HGMW-approved symbol) based on the analysis of a human genomic BAC clone KB1125A3 located on band q11.2 of human chromosome 22. The gene GLUT11/SLC2A11 consists of 12 exons spanning over 29 kb in size and is located between two genes, SMARCB1 and MIF. The deduced amino acid sequence indicated the topological features of transmembrane helices and sequence motifs which are common to the GLUT protein family. The cDNA cloning revealed the presence of three types of variation in its transcripts. The first variation is caused by the existence of three distinct first exons (SLC2A11-a, -b, and -c). PCR analysis of multi-tissue-derived cDNA panels indicated the differential expression of these transcript variants. The second variation is caused by skipping over one exon (exon 6). The third variation is caused by the premature transcription termination at a site between exon 8 and exon 9. Both exon skipping and premature termination caused frameshift, resulting in the production of truncated GLUT11/SLC2A11 transcripts. These results suggested that transcription of GLUT11/SCL2A11 gene is controlled in a complex manner.

Amino Acid Motifs↗
PubMed2001

Parathyroid hormone (PTH)-(1-14) and -(1-11) analogs conformationally constrained by alpha-aminoisobutyric acid mediate full agonist responses via the juxtamembrane region of the PTH-1 receptor.

The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21)NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor.

Aminoisobutyric Acids↗
PubMed2001

Contribution of anaphylatoxin C5a to late airway responses after repeated exposure of antigen to allergic rats.

We attempted to elucidate the contribution of complement to allergic asthma. Rat sensitized to OVA received repeated intratracheal exposures to OVA for up to 3 consecutive days, and pulmonary resistance was then estimated for up to 6 h after the last exposure. Whereas the immediate airway response (IAR) in terms of R(L) tended to decrease in proportion to the number of OVA exposures, late airway response (LAR) became prominent only after three. Although premedication with two kinds of complement inhibitors, soluble complement receptor type 1 (sCR1) or nafamostat mesylate, resulted in inhibition of the IAR after either a single or a double exposure, the LAR was inhibited after the triple. Premedication with a C5a receptor antagonist (C5aRA) before every exposure to OVA also inhibited the LAR after three. Repeated OVA exposure resulted in eosinophil and neutrophil infiltration into the bronchial submucosa which was suppressed by premedication with sCR1 or C5aRA. Up-regulation of C5aR mRNA was shown in lungs after triple OVA exposure, but almost no up-regulation of C3aR. Pretreatment with sCR1 or C5aRA suppressed the up-regulation of C5aR expression as well as cytokine messages in the lungs. The suppression of LAR by pretreatment with sCR1 was reversed by intratracheal instillation of rat C5a desArg the action of which was inhibited by C5aRA. In contrast, rat C3a desArg or cytokine-induced neutrophil chemoattractant-1 induced cellular infiltration into the bronchial submucosa by costimulation with OVA, but these had no influence on the LAR. These differences might be explained by the fact that costimulation with OVA and C5a synergistically potentiated IAR, whereas that with OVA and either C3a or cytokine-induced neutrophil chemoattractant-1 did not. C5a generated by Ag-Ab complexes helps in the production of cytokines and contributes to the LAR after repeated exposure to Ag.

Airway Resistance↗
PubMed2001

Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.

Base Sequence↗
PubMed2001

Plasmids with a mammalian replication origin and a matrix attachment region initiate the event similar to gene amplification.

Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins.

Colorectal Neoplasms↗
PubMed2001

The genomic structure and promoter region of the human parkin gene.

Parkin has been identified as a causative gene of the autosomal recessive juvenile parkinsonism (AR-JP). In this study, we determined the genomic structure of the Parkin gene and identified a core promoter region based on the DNA sequence of 1.4 Mb. The 5'-flanking region contained no apparent TATA or CAAT box elements but several putative cis-elements for various transcription factors. The GC- and CpG-rich regions were observed not only in the 5'-flanking sequence but also in the 5'-part of the first intron of Parkin. We identified an exact starting point of Parkin transcription. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into human neuroblastoma cells. Furthermore, we located a neighboring novel gene in a head-to-head direction with Parkin with only a 198-bp interval.

Base Sequence↗
PubMed2001

Replication timing of amplified genetic regions relates to intranuclear localization but not to genetic activity or G/R band.

Amplified genes in many human cancer cells usually localize at the extrachromosomal double minutes (DMs). In the present study, we show that multiple DMs in the human colorectal tumor COLO 320DM line replicated semisynchronously during the early S phase. On the other hand, during longer passage of the cells with DMs, cells with the amplified genes at the chromosomal homogeneously staining region (HSR) generally dominate the population. We currently report that HSR was composed of a tandem array of DM-derived sequences, which was shown using a unique DM-painting probe. Nevertheless, we found that HSR was replicated much later during the S phase, unless the amplified c-myc genes were expressed almost equally from DMs and HSR. Therefore, this provided a novel instance in which the cytogenetic localization affected replication timing without alteration of expression. Furthermore, we unexpectedly found that HSR had a distinctive band structure with respect to replication timing. The replication band structure was usually associated with the chromosomal G/R bands; however, HSR was homogeneous in the G/R band and in the distribution of highly repetitive sequences. We discuss the mechanism by which the replication band may arise, in relation to the folding of chromatin inside the nucleus.

Bromodeoxyuridine↗
PubMed2001

Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.

The human ABCG1 gene encodes a member of the ATP-binding cassette (ABC) superfamily of transporter proteins and is highly induced when macrophages are incubated with oxysterols. Using mRNA from oxysterol-treated human THP-1 cells together with 5'-rapid amplification of cDNA ends and polymerase chain reaction, we identified a novel ABCG1 transcript that encodes a putative protein of 786 residues containing a new amino terminus of 203 amino acids. Characterization of the genomic organization and structure of the human ABCG1 gene demonstrates that: (i) the gene consists of 23 exons spanning 98 kilobase pairs (kb) on chromosome 21q22.3, (ii) the 203 amino acids are encoded on three previously unidentified exons, 8-10, and (iii) a promoter, containing a TATA box and two liver X receptor (LXR) alpha response elements (LXREs), is located upstream of exon 8. Northern analysis using exon-specific probes confirms that oxysterol treatment results in >10-fold induction of ABCG1 transcripts that are derived from either exons 8-23 or exons 5, 7, and 11-23. Electromobility shift assays demonstrate that LXRalpha and retinoid X receptor alpha bind to the two LXREs in intron 7. Cells were transiently transfected with reporter luciferase constructs under the control of either (i) 9 kb of genomic DNA corresponding to intron 7 and part of exon 8 and containing either wild-type or mutant LXREs or (ii) two copies of the wild-type or mutant LXRE. In all cases, the wild-type construct was regulated in an LXR- and oxysterol-dependent manner, and this regulation was attenuated when the LXREs were mutated. In conclusion, the human ABCG1 gene contains multiple promoters, spans more than 98 kb and comprises 23 exons that give rise to alternative transcripts encoding proteins with different amino-terminal sequences. Elucidation of the various roles of different ABCG1 isoforms will be important for our understanding of mammalian cholesterol homeostasis.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2001

The medaka rs-3 locus required for scale development encodes ectodysplasin-A receptor.

The bodies of most teleost fish species are covered with specialized subepithelial structures known as scales. The scale is an epithelial appendage that differentiates from the dermal mesenchyme. Mammals, on the other hand, have no scales, but instead their bodies are covered with hair. Although their appearances are quite different, scales and hair can be considered structurally similar in that both of them are epithelial appendages distributed over the body surface in an orderly pattern. This analogy suggests that they may have the same evolutionary origin. But, to date, no molecular evidence has been presented that links scales and hair. A mutation at the rs-3 locus of medaka (Oryzias latipes) leads to almost complete loss of scales. We demonstrated that the rs-3 locus encodes ectodysplasin-A receptor (EDAR), which is required for the initiation of hair development in mammals. We identified a novel transposon inserted in the first intron of EDAR, which causes aberrant splicing. This work shows that EDAR is required for scale development in fish and suggests that it is an evolutionarily conserved molecule that is required for the development of epithelial appendages in vertebrates.

Amino Acid Sequence↗
PubMed2001

Melt production beneath Mt. Shasta from boron data in primitive melt inclusions.

Most arc magmas are thought to be generated by partial melting of the mantle wedge induced by infiltration of slab-derived fluids. However, partial melting of subducting oceanic crust has also been proposed to contribute to the melt generation process, especially when young and hot lithosphere is being subducted. The isotopic composition of boron measured in situ in olivine-hosted primitive melt inclusions in a basaltic andesite from Mt. Shasta, California, is characterized by large negative values that are also highly variable (delta(11)B = -21.3 to -0.9 per mil). The boron concentrations, from 0.7 to 1.6 parts per million, are lower than in most other arc lavas. The relation between concentration and isotopic composition of boron observed here supports a hypothesis that materials left after dehydration of the subducting slab may have contributed to the generation of basaltic andesite lavas at Mt. Shasta.

Journal Article↗
PubMed2001

Hsp70 regulates the interaction between the peroxisome targeting signal type 1 (PTS1)-receptor Pex5p and PTS1.

The peroxisome targeting signal type 1 (PTS1) receptor, Pex5p, of the tetratricopeptide repeat (TPR) motif family is located mostly in the cytosol and mediates the translocation of PTS1 proteins to peroxisomes. As a step towards understanding the mechanisms of protein import into peroxisomes, we investigated the molecular mechanisms involved in PTS1 recognition by Pex5p with regard to requirement of energy and cytosolic factors, using cell-free synthesized acyl-CoA oxidase (AOx) as a PTS1 cargo protein, together with Pex5p and heat-shock protein (Hsp)70 from rat liver. Pex5p was partly associated with peroxisomes of rat liver, was resistant to washing with a high concentration of salt and to alkaline extraction and was inaccessible to protease added externally. Pex5p bound to AOx in an ATP-dependent manner. AOx synthesized in a cell-free translating system from rabbit reticulocyte lysate was imported into peroxisomes without being supplemented with Pex5p and Hsp70, implying that peroxisome-associated Pex5p was released from the membranes and functional in this in vitro import assay. Antibodies against Pex5p and Hsp70 inhibited AOx import. In contrast, AOx synthesized in a wheat-germ lysate required the external addition of Pex5p for import, in which Hsp70 augmented the AOx import. The TPR domain of Pex5p was revealed to bind to the N-terminal part in an Hsp70-independent manner, whereas mutual interaction of the TPR region was noted in the presence of Hsp70. Hsp70 interacted with the TPR domain of Pex5p. Moreover, Hsp70 and ATP synergistically enhanced the binding of Pex5p to the C-terminal PTS1-containing part of AOx, implying that Pex5p recognizes its cargo PTS1 protein by chaperone-assisted as well as energy-dependent mechanisms in vivo.

Adenosine Triphosphate↗
PubMed2001

Constitutive activation of nuclear factor-kappaB prevents TRAIL-induced apoptosis in renal cancer cells.

TRAIL has gained much attention for its specific induction of apoptosis in cancer cells but not in normal cells. This phenomenon has been explained thus: that cancer cells dominantly express death receptors while normal cells express decoy receptors. However, recent reports have shown that some cancer cell lines are resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and the presence of death receptors. This suggested the existance of an inhibitory factor. We herein showed that NF-kappaB is a key molecule underlying the TRAIL-resistant mechanism in renal cell carcinoma (RCC) cell lines. We observed that NF-kappaB is constitutively activated in resistant cell lines. Forced expression of antisense cDNA of IkappaBalpha, a specific inhibitor of NF-kappaB, in TRAIL-sensitive cell lines with a low NF-kappaB activity result in constitutive activation of NF-kappaB and resistance to TRAIL-induced apoptosis. Adenoviral expression of a stable form of IkappaBalpha in the TRAIL-resistant cell lines induced apoptosis. These data suggest that RCC can be classified into two subsets: TRAIL-sensitive RCC with a low NF-kappaB activity and TRAIL-resistant RCC with constitutively activated NF-kappaB. In the former group TRAIL can be a treatment option, while in the latter group a molecular approach targeting NF-kappaB appears to be a promising therapy.

Antineoplastic Agents↗
PubMed2001

Increased interleukin-6 of skin and serum in amyotrophic lateral sclerosis.

Studies of skin in amyotrophic lateral sclerosis (ALS) have demonstrated morphological and biochemical alterations. Interleukin-6 (IL-6) has been suggested to have a trophic effect in nerve cells and to have a direct pathogenic role in neurodegenerative central nervous system (CNS) disorders. However, little is known concerning IL-6 in ALS patients. We examined IL-6 immunoreactivity of biopsy specimens of skin and measured serum IL-6 levels from 11 ALS patients and 11 diseased control subjects. IL-6 immunoreactivity was markedly positive in the epidermis and dermal blood vessels and glands and was moderately positive in the reticular dermis in all ALS patients. These optical densities for IL-6 immunoreactivity in ALS patients were significantly higher than in control subjects, and were significantly increased with duration of illness. Serum IL-6 levels were detected in 8 (73%) of 11 ALS patients compared with only 1 (9%) of 11 diseased control subjects. Serum IL-6 levels were significantly correlated with duration of illness in ALS patients and immunoreactivity of IL-6 of the epidermis. These data suggest that the increased levels of serum IL-6 may reflect an increased IL-6 immunoreactivity of skin in ALS patients.

Adult↗
PubMed2001

Molecular mechanisms of human single-minded 2 (SIM2) gene expression: identification of a promoter site in the SIM2 genomic sequence.

We previously postulated that the single-minded 2 (SIM2) gene identified on the human chromosome 21q22.2 is a good candidate gene for the pathogenesis of mental retardation in Down syndrome because its mouse homolog exhibits preferential expression in the mouse diencephalon during early embryogenesis. We analyzed the genomic sequence of the entire SIM2 gene which consists of 11 exons and spans over 50 kb. As a step toward understanding the molecular mechanisms of SIM2 gene expression, we have analyzed the human SIM2 gene expression in nine established human cell lines. Three transcripts of 3.6, 4.4, and 6.0 kb were detected in the glioblastoma cell line, T98G, neuroblastoma cell line, TGW, and transformed embryonic kidney cell line, 293. The RACE analysis using SIM2-expressing human cell line T98G provided evidence for the transcription start site at approximately 1.2 kb upstream of the translation initiation site. The transfection assay using various deletion constructs with reporter gene suggested the presence of a presumptive promoter region. Transient transfection assay in T98G cell line revealed a significant promoter activity located in the 60 bp sequence between nt -1385 and -1325 upstream region of the translation initiation site. This 60 bp sequence contains cis-elements for c-myb, E47 and E2F transcription factors. Moreover, the gel retardation assay using oligo-DNA of various cis-element sequences indicated the presence of protein factor(s) which bind to the cis-element for c-myb. These results suggested that binding of a protein transcription factor(s) such as c-myb or that alike regulates transcription of the SIM2 gene by binding to a small upstream region.

Base Sequence↗
PubMed2001

Light induces destabilization of photoactive yellow protein.

To understand the effect of visible light on the stability of photoactive yellow protein (PYP), urea denaturation experiments were performed with PYP in the dark and with PYP(M) under continuous illumination. The urea concentrations at the midpoint of denaturation were 5.26 +/- 0.29 and 3.77 +/- 0.19 M for PYP and PYP(M), respectively, in 100 mM acetate buffer, and 5.26 +/- 0.24 and 4.11 +/- 0.12 M for PYP and PYP(M), respectively, in 100 mM citrate buffer. The free energy change upon denaturation (DeltaG(D)(H2O)), obtained from the denaturation curve, was 11.0 +/- 0.4 and 7.6 +/- 0.2 kcal/mol for PYP and PYP(M), respectively, in acetate buffer, and 11.5 +/- 0.3 and 7.8 +/- 0.1 kcal/mol for PYP and PYP(M), respectively, in citrate buffer. Even though the DeltaG(D)(H2O) value for PYP(M) is almost identical in the two buffer systems, the urea concentration at the midpoint of denaturation is lower in acetate buffer than in citrate buffer. Although their CD spectra indicate that the protein conformations of the denatured states of PYP and PYP(M) are indistinguishable, the configurations of the chromophores in their denatured structures are not necessarily identical. Both denatured states are interconvertible through PYP and PYP(M). Therefore, the free energy difference between PYP and PYP(M) is 3.4-3.7 kcal/mol for the protein moiety, plus the additional contribution from the difference in configuration of the chromophore.

Acetates↗