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Biomedical subjects

S Ito

Publications and source records attributed to S Ito.

At least 109 records · Page 6Linked to original sources
PubMed2001

Extracellular signals that regulate the tangential migration of olfactory bulb neuronal precursors: inducers, inhibitors, and repellents.

Neuronal migration is an essential developmental step in the construction of the vertebrate nervous system, but the extracellular signals involved in initiating and regulating neuronal movement remain unclear. Here we report the identification of a novel astrocyte-derived migration-inducing activity (MIA). Using an in vitro assay, we show that MIA induces the migration of olfactory bulb interneuron precursors, increasing the number of migrating cells and the distance they move. We established quantitative criteria to distinguish between the biological effects of inducers, inhibitors, repellents, and attractants on migrating cells and used them to compare the effects of MIA with those of Slit, a putative repulsive guidance cue. Our analysis demonstrates that, by themselves, MIA induces and Slit inhibits migration from subventricular zone explants. However, when presented together with MIA, Slit acts as a repellent. This study shows that glial cells play a critical role in initiating and modulating the movement of neuronal precursors through the release of a diffusible protein. Moreover, this study provides evidence that the guidance of migrating neuronal precursors is an integrative process, resulting from the cooperation of distinct extracellular factors, and that the function of Slit is context dependent.

Animals↗
PubMed2001

Role of urotensin II in patients on dialysis.

Urotensin II is a potent vasoconstrictor, which also has some vasodilatory properties. We investigated its expression in various tissues and in the plasma of patients with renal dysfunction. Plasma concentrations of urotensin II-like immunoreactivity were 2-fold higher in patients not on dialysis and 3-fold higher in those on haemodialysis thanin healthy individuals. Messenger RNA encoding theurotensin II precursor and the urotensin II receptor precursor were expressed in various human tissues. The peptidemight act as an important regulator in the cardiovascularand renal systems. Urotensin II antagonists could, therefore, be useful in the treatment of diseases affecting theseorgans.

Adult↗
PubMed2001

Ocular toxicity and antenatal exposure to chloroquine or hydroxychloroquine for rheumatic diseases.

Chronic use of chloroquine and hydroxychloroquine inthe treatment of rheumatic disease carries a small risk of sight-threatening pigmentary retinopathy. To obtain safety data for its use in pregnancy, we did ophthalmic examinations in 21 children born to women who took these drugsduring pregnancy. Average daily maternal doses of the two drugs were 317 mg hydroxychloroquine and 332 mg chloroquine. The mean duration of gestational exposure was 7.2 months. No ophthalmic abnormality was detected in these children. Therapeutic use of these drugs during pregnancy may not pose a significant risk of ocular toxicity to offspring.

Antirheumatic Agents↗
PubMed2001

Hepatic stellate cells contain the functional estrogen receptor beta but not the estrogen receptor alpha in male and female rats.

In an earlier study, we showed that estradiol (E2) inhibits proliferation and transformation in cultured rat hepatic stellate cells (HSCs) and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on an investigation of the cellular localization of ER subtypes ERalpha and ERbeta using immunohistochemistry in experimental fibrotic liver rats and of each ER subtype expression in cultured rat HSCs by evaluating the produced mRNA and protein. The results indicate that high levels of ERbeta expression and low or no levels of ERalpha expression were observed in normal and fibrotic livers and in quiescent and activated HSCs from both males and females. The specificity of E2-mediated antiapoptotic induction through the ERbeta was shown by dose-dependent inhibition by the pure ER antagonist ICI 182,780 in HSCs which were undergoing early apoptosis. These findings demonstrate for the first time that rat HSCs possess functional Erbeta, but not Eralpha, to respond directly to E2 exposure.

Animals↗
PubMed2001

Extraction of human Langerhans cells: a method for isolation of epidermis-resident dendritic cells.

Langerhans cells (LCs) are immature dendritic cells in the epidermis that play a central role in T-lymphocyte mediated skin immunity. Upon activation with antigenic stimuli, they differentiate drastically into mature dendritic cells while migrating from the epidermis to regional lymph nodes. Thus, in order to study biological details of immature LCs, it is crucial to isolate epidermis-resident, immature LCs without dermal dendritic cell contamination. Methods for extracting LCs from human skin as well as in vitro derivation of LC-like cells from hematopoietic progenitor cells have been described previously, but the cell preparations can potentially contain a significant number of dendritic cells that are not identical to epidermal LCs. Here, we describe a technique by which purely epidermis-resident LCs are extracted from human skin. Following digestion of human skin with dispase, the epidermis was separated mechanically without any attached dermal component. The trypsinized epidermal cells were then fractionated by centrifugation with a discontinuous density gradient composed of bovine albumin and sodium metrizoate. The LC-enriched preparation thus obtained contained 80% to >90% CD1a+, E-cadherin+ cells that expressed Birbeck granules and the Lag protein. Consistent with their being at an immature stage, the freshly isolated LCs lacked the expression of CD83, a marker for mature dendritic cells. The purified LCs were able to activate allogeneic T cells, indicating that the cells retained T-cell stimulation ability even after extraction. Thus, the present work offers an opportunity for precise in vitro studies of epidermal LCs.

Antigens, CD↗
PubMed2001

Preserved phosphorylation of RET receptor protein in spinal motor neurons of patients with amyotrophic lateral sclerosis: an immunohistochemical study by a phosphorylation-specific antibody at tyrosine 1062.

Ret oncoprotein is a functional receptor for the glial cell line-derived neurotrophic factor (GDNF) family and it is expressed in motor neurons, playing an important role in the motor neuron function. In this study, we examined the expression of the phosphorylation state of tyrosine residue 1062 (Tyr-1062) of Ret in the spinal cords of amyotrophic lateral sclerosis (ALS), using the phosphorylation state specific antibody at Tyr-1062 of Ret. The immunohistochemical study demonstrated that Tyr-1062 of Ret was phosphorylated to variable extents in the surviving motor neurons of all the ALS as well as controls studied. This is the first report that the phosphorylation of Tyr-1062 occurred in neurons with nononcogenic type of Ret. The Ret-signaling pathway by Tyr-1062 autophosphorylation is constitutively activated via the phosphatidylinositol 3-kinase and/or mitogen-activated protein kinase cascade for motoneuron survival even in the ALS motor neurons, supporting the view that GDNF is a candidate for therapeutic approach to ALS.

Aged↗
PubMed2001

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease.

Serum amyloid P component (SAP) is a major acute-phase reactant in mice. Recently, it was reported that SAP-deficient mice spontaneously developed anti-nuclear antibodies and severe glomerulonephritis. Because the SAP-deficient mice we generated display no obvious phenotypic abnormalities, we investigated whether our SAP-deficient mice would also spontaneously develop autoimmune responses. In accordance with the report, our mice produced high titers of anti-nuclear antibody but did not develop severe glomerulonephritis. On the other hand, it was recently reported that SAP bound to gram-negative bacteria via lipopolysaccharide (LPS) prevented LPS-mediated activation of a classical complement pathway. Thus, we asked if SAP-deficient mice would show altered responses to an intraperitoneal injection of LPS from Salmonella typhimirium. SAP-deficiency did afford resistance to lethality induced by high-dose LPS. Our experiments clearly showed that contrary to documented data, SAP-deficient mice do not develop serious autoimmune disease and we suggest that SAP has a critical role in LPS toxicity.

Animals↗
PubMed2001

Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme.

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

Adaptation, Physiological↗
PubMed2001

Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line.

Initial arrest, attachment, extravasation and subsequent extravascular growth of tumor cells in the secondary organs are believed to be crucial events for hematogenous metastasis, but the actual processes in living animals remain unclear. For the present study, we established green fluorescent protein (GFP)-expressing rat tongue carcinoma cell lines (RSC3) that permit real-time analysis of micrometastasis formation in combination with intravital video microscopy (IVVM). With this system, GFP-expressing metastatic (LM-EGFP) and non-metastatic (E2-EGFP) cell lines could be visualized at the cellular level in live mice for more than 1 month. Real-time IVVM analysis of liver metastases after intraportal injection of cells via a mesenteric vein revealed that both LM-EGFP and E2-EGFP tumor cells arrest similarly in sinusoidal vessels near terminal portal venules within 0.4 sec, during which time no evidence of a "rolling"-like movement along endothelial cell surfaces was observed. Quantitative analysis of GFP-positive foci showed that E2-EGFP cells were completely sheared from the liver sinusoid within 3 days, with no solitary dormant cells, whereas a substantial number of LM-EGFP cells remained in the liver, probably due to stable attachment to the sinusoidal wall. Confocal laser scanning microscopic study in combination with laminin immunohistochemistry revealed that only LM-EGFP cells started growth at 3 to 4 days after inoculation and that most of the growing foci were surrounded by subsinusoidal basement membrane. Our results suggest that micrometastasis formation by LM-EGFP cells consists of initial tumor cell arrest due to size constraints of the vessel, stable attachment to subsinusoidal basement membrane and subsequent intravascular growth before extravasation. The difference in metastatic potential between the 2 lines may reside in their capacity to attach stably to the vessel wall rather than their potential for initial cell arrest or subsequent growth. The system used in the present study may be a powerful tool for analyzing targets for various anti-metastatic agents in the sequential process of metastasis.

Animals↗
PubMed2001

The 5'-untranslated region of the mouse glial cell line-derived neurotrophic factor gene regulates expression at both the transcriptional and translational levels.

We previously cloned mouse glial cell line-derived neurotrophic factor (GDNF) cDNA and genomic DNA and found that the mouse gene contains a 1086-bp 5'-untranslated region (5'-UTR). We investigated the contributions of the 5'-UTR to promoter activity and found one positive regulatory region and two negative regulatory regions in the 5'-UTR. In the present study, using gel retardation assays and mutation analyses, two novel cis-elements that interact with nuclear extracts from mouse astrocytes were identified. The first cis-element (nucleotides (nt) +70 to +81) enhances promoter activity, whereas the second cis-element (nt +239 to +247) attenuates promoter activity in a position- and orientation-dependent manner. Suppression of gene expression by a third region (nt +509 to +580) occurs at the translational level. The ATG sequence (nt +547 to +549) has the potential to initiate translation and to attenuate the efficiency of translation for the GDNF precursor coding region. Furthermore, we identified an alternative promoter in the 5'-UTR that is driven by an Sp1 element, circumventing the translational suppression. Taken together, the 5'-UTR of mouse GDNF contains two novel cis-elements, a short upstream open reading frame and an alternative promoter that influences gene expression at both the transcriptional and translational levels.

5' Untranslated Regions↗
PubMed2001

Purification and properties of a high-molecular-weight, alkaline exopolygalacturonase from a strain of Bacillus.

1An exopolygalacturonase [exo-PG; poly (1,4-alpha-D-galacturonide) digalacturonohydrolase, EC 3.2.1.82] was found in a culture of Bacillus sp. strain KSM-P576. The purified exo-PG had a molecular weight of approximately 115,000 and an isoelectric point of pH 4.6. The N-terminal amino acid sequence was Thr-Glu-Val-Ser-Pro-Lys-Ser-Pro-Ala-Ser-Pro-Val. Maximum activity toward polygalacturonic acid (PGA) was observed at 55 degrees C and pH 8.0 in 100 mM Tris-HCl buffer. The exo-PG was quite stable in various pH buffers between pH 6 and 12 when incubated at 30 degrees C for 1 h. Mg(2+,) Mn(2+,) Pd(2+) and Ca(2+) ions stimulated the enzyme activity. The exo-PG released digalacturonic acid from PGA, tri-, tetra-, and penta-galacturonic acids. The apparent K(m) values for oligogalacturonic acids were almost identical, and k(cat) values increased with the chain length of the substrates.

Journal Article↗
PubMed2001

Transcriptional regulation of the murine acetyl-CoA synthetase 1 gene through multiple clustered binding sites for sterol regulatory element-binding proteins and a single neighboring site for Sp1.

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5'-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (approximately 2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility "supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.

Acetate-CoA Ligase↗
PubMed2001

Analysis of Tag1-like elements in Arabidopsis thaliana and their distribution in other plants.

Analysis of genomic DNA of Arabidopsis Columbia (Col.) ecotype using a transposon Tag1-specific primer showed the presence of Tag1 homologues which was confirmed by Southern hybridization with a Tag1 probe. Further analysis showed that the homologue, 0.75 kb in length, had inverted repeats at both ends, 8-bp duplicated sequences at the site at which it is located and about 80% homology with Tag1, and was randomly distributed in the Arabidopsis genome. Based on these results, we concluded that these elements are non-autonomous variants of Tag1 and we termed this element sTag1. Using the polymerase chain reaction fragment hybridization technique, we found the distribution of such homologues in other plant species.

Arabidopsis↗
PubMed2001

Increased oxidative stress in childhood atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of unknown etiology. To examine the involvement of impaired homeostasis of oxygen/nitrogen radicals in childhood AD, we compared the levels of urinary 8-hydroxy-2'-deoxyguanosine (marker of oxidative stress), nitrite/nitrate (marker of nitric oxide synthesis) and selenium (marker of selenium store) in 27 children with AD to those of 25 healthy control children. Urinary 8-hydroxy-2'-deoxyguanosine was significantly higher and nitrite/nitrate levels were significantly lower in patients with AD than in the control. Urinary selenium levels were similar in both groups. Our findings suggest that impaired homeostasis of oxygen/nitrogen radicals and increased oxidative stress are involved in the pathophysiology of childhood AD, and indicate that suppression of oxidative stress might be a potentially useful strategy for the treatment of AD.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Crystallization and preliminary X-ray diffraction studies of a novel alkaline serine protease (KP-43) from alkaliphilic Bacillus sp. strain KSM-KP43.

A novel alkaline serine protease (KP-43) which belongs to a new class of the subtilisin superfamily was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 43.50 (2), b = 110.4 (1), c = 168.9 (1) A. The crystals diffract X-rays beyond 1.9 A resolution using Cu Kalpha radiation from a rotating-anode generator and are suitable for high-resolution crystal structure analysis.

Bacillus↗
PubMed2001

Renal tubule-specific transcription and chromosomal localization of rat thiazide-sensitive Na-Cl cotransporter gene.

The molecular mechanism underlying the renal expression localization of the thiazide-sensitive Na-Cl cotransporter (TSC) gene was studied. The TSC gene was localized to chromosome 19p12-14. In cultured cells, tissue-specific transcription activity of the 5'-flanking region of the rat rTSC gene (5'FL/rTSC) was demonstrated, and the major promoter region was located between position -580 and -141. To further examine the tissue-specific transcription, transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene were generated. Immunohistochemical analysis clearly showed that LacZ gene expression was co-localized to distal convoluted tubules (DCT) with TSC, indicating that the 5'FL/rTSC regulates the renal tubule-specific TSC expression. Because a transcription factor, HFH-3 (hepatocyte nuclear factor-3/folk head homologue-3), had also been localized to DCT, a possible role of the putative cis-acting element (HFH-3/rTSC, -400/-387 position) for HFH-3 binding in the tissue-specific transcription was examined. Deletion and mutation analyses suggested that transcription of the HFH-3/rTSC was actually responsive to HFH-3, and electrophoretic mobility shift assay showed a direct binding of in vitro synthesized HFH-3 to the HFH-3/rTSC. In conclusion, the rTSC gene is localized to rat chromosome 19p12--24. The transcription regulatory region of the TSC gene confers DCT-specific gene expression. DCT-specific transcription factor HFH-3 may be involved in the renal tubule-specific transcription of TSC gene.

Animals↗
PubMed2001

Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase.

Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.

Amino Acid Sequence↗