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Biomedical subjects

N Arai

Publications and source records attributed to N Arai.

At least 145 records · Page 8Linked to original sources
PubMed1995

Expression of prostaglandin E receptor subtypes in bone: expression of EP2 in bone development.

Prostaglandin (PG) E2 displays physiological and pharmacological action in various tissues including bone. It increases intracellular Ca, and stimulates or inhibits cAMP production through the PGE receptor subtypes EP1, EP2, and EP3, respectively. These receptor subtypes have been recently cloned. In the present study, we investigate the expression of these receptor subtypes in bone tissue. RT-PCR revealed that EP1, EP2, and EP3 were expressed in rat calvariae and that osteoblastic cells (MC3T3-E1) expressed EP1 and EP2. In situ hybridization analysis using cryosection of neonatal calvariae revealed that EP2 was expressed by osteoblasts and cells not in contact with bone, probably including preosteoblasts. EP2 expression was observed at an early stage in calvarial development, at 14 days prenatal. EP2 expression was also observed at day 3 in rat bone marrow cell culture in which bone-like mineralized nodules are formed at day 8. It has been established that PGE2 response accompanying cAMP production is one of the characteristics of osteoblasts. The present results indicate that this phenotype appears at an early stage of osteoblastic differentiation and bone development.

3T3 Cells↗
PubMed1995

Regulation of expression of the IL-2 and IL-5 genes and the role of proteins related to nuclear factor of activated T cells.

Cyclic adenosine monophosphate (cAMP) inhibits phorbol 12-myristate 13-acetate (PMA)-induced IL-2 production while it inhibits IL-5 production at the transcriptional level in EL-4, a mouse lymphoma line. The -321 to +46 region of the mouse IL-2 promoter is required for activation by PMA and inhibition by cAMP. This promoter region contains several elements that interact with transcription factors, such as nuclear factor of activated T cells (NF-AT), NF-kappa B, AP-1, and octamer. With use of reporter plasmid carrying multiple copies of each element, we found that the construct that contained the NF-AT site was most effective for responding to PMA activation and cAMP inhibition. In electrophoretic mobility shift assay (EMSA), PMA-induced NF-AT binding complex was altered by cAMP. Furthermore, overexpression of the cytoplasmic component of NF-AT abrogated the inhibitory action of cAMP. These results indicate that the NF-AT site is a target of the inhibitory action of cAMP. We have previously reported that the -1200 to +33 region of the mouse IL-5 promoter can mediate transcriptional stimulation by PMA and cAMP in EL-4 cells. Here we identified the element IL-5P, which is required for maximal activation of the IL-5 promoter. We found that this element is homologous to the binding site for NF-AT and interacted with NF-AT-related factors induced by PMA and cAMP. Thus it appears that an NF-AT factor is involved in the regulation of IL-5 gene transcription.

Animals↗
PubMed1995

Grumose or foamy spheroid bodies involving astrocytes in the human brain.

Grumose or foamy spheroid bodies (GFSB), which correspond to the classical pathological description, 'grumose degeneration', are described. By light microscopy, GFSB are faintly eosinophilic and spheroidal structures with a foamy appearance in haematoxylin and eosin stains; they vary from 10 to 50 microns in diameter and contain amorphous debris-like material. Some GFSB, however, contain a varying amount of eosinophilic grumose aggregates, some of which are randomly stained with periodic acid Schiff (PAS), Schmorl, Berlin blue, Grimelius or silver methods. The Gallyas stain, on the other hand, usually stains the contents of GFSB black or brown. Immunohistochemically, most GFSB are ubiquitin-positive. Characteristically glial fibrillary acidic protein is associated with some GFSB giving a foamy appearance. Ultrastructurally, GFSB consist of dense bodies of various sizes and configuration. Glial fibrillary bundles and astrocytic punctate adhesions are occasionally observed associated with GFSB. Anatomically, GFSB are observed preferentially in the rostroventral parts of both the substantia nigra pars reticulata and the globus pallidus in a number of human neurodegenerative diseases and aged brains. GFSB, however, appear outside the above regions in various circumstances such as trauma, infarction and astrocytomas. In conclusion, GFSB are ubiquitinated structures closely related to astrocytes in their formation and with a preferred location in the deep regions of the basal ganglia.

Astrocytes↗
PubMed1995

NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus.

The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of cytokine genes. Here, we report the cDNA cloning, chromosomal localization, and initial characterization of a transcription factor related to NFATp and NFATc. The novel molecule, designated NFATx, exhibits in its middle a region very similar to the Rel homology domain in NFATc and NFATp. The amino-terminal region of NFATx also shows significant similarities to corresponding sequences in NFATc and NFATp and contains three copies of a conspicuous 17-residue motif of unknown function. We provide evidence showing that NFATx can reconstitute binding to the NFAT-binding site from the interleukin 2 promoter when combined with AP1 (c-Fos/c-Jun) polypeptides and that NFATx is capable of activating transcription of the interleukin 2 promoter in COS-7 cells when stimulated with phorbol ester and calcium ionophore. NFATx mRNA is preferentially and remarkably found in the thymus and at lower levels in peripheral blood leukocytes. The expression pattern of NFATx, together with its functional activity, strongly suggests that NFATx plays a role in the regulation of gene expression in T cells and immature thymocytes.

Amino Acid Sequence↗
PubMed1995

Phenopyrrozin, a new radical scavenger produced by Penicillium sp. FO-2047.

A new radical scavenger, named phenopyrrozin, was isolated from the culture broth of Penicillium sp. FO-2047. Phenopyrrozin was purified from whole broth solvent extraction, silica gel chromatography, and HPLC. The structure of phenopyrrozin was elucidated as 5,6,7,7a-tetrahydro-2-hydroxy-1-phenyl-3H-pyrrolizin-3-one. The IC50 of phenopyrrozin against lipid peroxidation induced by Cr2K2O7 was 73 micrograms/ml. Phenopyrrozin also reduced chromosomal aberrations induced by paraquat.

Animals↗
PubMed1995

Isochromophilones III-VI, inhibitors of acyl-CoA:cholesterol acyltransferase produced by Penicillium multicolor FO-3216.

New azaphilones named isochromophilones III-VI were isolated from the culture broth of Penicillium multicolor FO-3216 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). Their structures were elucidated by NMR and other spectroscopic analyses. The IC50 values of isochromophilones III, IV, V and VI for ACAT activity in an enzyme assay using rat liver microsomes were calculated to be 110, 50, 50 and 120 microM, respectively.

Animals↗
PubMed1995

New antibiotics phthoxazolins B, C and D produced by Streptomyces sp. KO-7888.

New antibiotics, phthoxazolins B, C and D were isolated from the fermentation broth of Streptomyces sp. KO-7888. They are geometrical isomers of 10-hydroxyphthoxazolin A. They showed selective antifungal activity against Phytophthora parasitica in vitro and modest herbicidal activity in a laboratory test, but the potencies were different among isomers.

Antifungal Agents↗
PubMed1994

The transcription factor Sp1 is required for induction of the murine GM-CSF promoter in T cells.

The cis-acting region, GM-kappa B/GC-box (positions -95 and -73), within the murine GM-CSF gene promoter is required for maximal induction by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in T cells. GM-kappa B defines a binding site for NF-kappa B, and GC-box defines a binding site for three (A1, A2, B) constitutive proteins. We report here that three copies of the GC-box can functionally compensate for the GM-kappa B/GC-box region, suggesting that the GC-motif can function independently of the GM-kappa B motif. The major GC-box binding activity A1 was purified and identified as the transcription factor Sp1. We show that depletion of Sp1 (A1) from nuclear extracts specifically decreases in vitro transcription activity on GM-CSF templates. Since the human GM-CSF promoter has a base difference within the GC-box, we speculate that this may explain why the human promoter is weak and that an upstream enhancer is required for the induction of the human GM-CSF gene.

Animals↗
PubMed1994

New observation on ubiquitinated neurons in the cerebral cortex of multiple system atrophy (MSA).

To investigate neuronal damage in the cerebral cortex in patients with multiple system atrophy (MSA), immunohistochemical stainings were carried out on the prefrontal cortex, the hippocampus, the precentral gyrus, the supplementary motor cortex and the occipital cortex in 6 cases of MSA and 6 controls, using antibodies against ubiquitin, tau protein and neurofilaments (BF10, RT97, 147). In MSA cases, a variable number of neuronal ubiquitinated inclusions were observed in the granule cell layer of the dentate gyrus (3/6) and the prefrontal cortex (3/6). An increased number of ubiquitinated dots-like structures were also observed in the parahippocampal gyrus of MSA cases (4/6) in comparison with controls. These results showed further evidence of neuronal damage in the cerebral cortex in MSA and strongly suggest a relationship between the cerebral cortical pathology and occasional manifestation of cognitive deficits in some MSA cases.

Atrophy↗
PubMed1994

Characterization of NF(P), the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to NF-kappa B and NF-AT.

The P sequence of the human interleukin-4 (IL-4) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the NF-kappa B and the NF-AT binding sites. We examined whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and NF-AT. NF-kappa B (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, NF(P) binding was inhibited by the unlabeled NF-AT binding site but not by the unlabeled AP1 binding site and purified NF-AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of NF-AT were similar to those of NF(P) and only a small amount of P65 was detected in NF(P) in crude nuclear extracts. These results indicate that the component(s) of NF-AT has the potential to reconstitute NF(P) whereas NF-kappa B alone cannot account for NF(P) in crude extracts. Unlike NF-AT, NF(P) does not contain AP1 as its DNA binding component.

Base Sequence↗
PubMed1994

Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells.

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)-expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+-signaling pathways downstream of T cell activation.

Animals↗
PubMed1994

Molecular basis for developmental changes of GM-CSF gene inducibility in embryonal carcinoma cells.

In a previous report, we reported that the induction of GM-CSF gene in differentiated P19 cells results from the maturation of the transcriptional machinery. Here, we identified a cis-DNA element which confers the activation of GM-CSF gene in response to PMA/A23187 stimulation in differentiated state. Analysis of the 5'-flanking region between -113 and -60 revealed two elements responsible for promotion and one for inhibition, and the overall effects led to the activation of GM-CSF gene mainly through the sequence between -95 and -73. Using the oligonucleotide between -94 and -73 as a probe in gel retardation assays, we identified a DNA-binding protein, NF-GM-P19, the binding activity of which was induced after differentiation in response to PMA/A23187 stimulation. These results indicate that the induction of GM-CSF gene after differentiation results from the maturation of the transcriptional machinery which recognizes the sequence between -95 and -73.

Animals↗
PubMed1994

Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin-5 genes in helper T cells.

Mouse thymoma line EL-4 cells produce cytokines such as interleukin (IL)-2, IL-3, IL-4, IL-10, and granulocyte-macrophage colony-stimulating factor in response to phorbol 12-myristate 13-acetate (PMA). EL-4 cells also produce low levels of IL-5 when stimulated by PMA alone; however, cAMP greatly augments PMA-dependent IL-5 production. A transient transfection assay revealed that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. In contrast, cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene, as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene is positively regulated by cAMP in a manner opposite to that for the IL-2 gene. One of the nuclear factors (NFs) that regulates the response of the IL-5 promoter to cAMP and PMA has properties similar to NF for activated t cell. The P sequence of the IL-4 gene, defined as a responsive element for PMA and calcium ionophore (A23187), shares sequence similarity with the NF kappa B and the NF-activated T cell binding sites. We attempted to determine whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and nuclear factor for activated T cell (NF-AT). In electromobility shift assays both NF-kappa B (P65 or P65/P50 heterodimer) and NF-AT bound to the P sequence. However, sequence specificity of NF-AT was more similar to that of NF(P), and only a small amount of P65 was detected in NF(P). These results indicate that a component or components of NF-AT have the potential to reconstitute NF(P), whereas NF-kappa B alone does not account for NF(P) in Jurkat crude extract. Taken together, these results suggest that NF-AT-like factors are involved in the regulation of IL-4 and IL-5 genes.

Animals↗
PubMed1994

Developmental changes of GM-CSF gene inducibility in embryonal carcinoma cells.

Murine embryonal carcinoma (EC) P19 cells, a tissue culture model of early embryonic development, failed to produce cytokines, such as interleukin-3 (IL-3), IL-4, granulocytemacrophage colony stimulating factor (GM-CSF) and interferon-beta (IFN-beta) at the mRNA level. Differentiation induced by retinoic acid (RA) released this repression to produce some cytokines. GM-CSF and IFN-beta genes were expressed in response to PMA/A23187, poly(I):poly(C), IL-1 alpha, forskolin, or LPS stimulation in differentiated P19 cells, whereas IL-3 and IL-4 genes were not expressed. To elucidate the mechanism of the GM-CSF gene induction after differentiation, we transfected a series of 5' deletion mutants of the mouse GM-CSF promoter fused to the bacterial CAT gene. The 740-bp fragment of the 5'-flanking region mediated the positive response. Deletion analysis revealed that the 5' boundary region of the DNA element required for activation lies between positions -95 and -84 and the region upstream of position -95 appears inhibitory. These results indicate that the maturation of the transcriptional machinery after differentiation results in the activation of the GM-CSF gene.

Animals↗
PubMed1994

In vitro effect of KCA-098, a derivative of coumestrol, on bone resorption of fetal rat femurs.

The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of 45Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of 45Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.

Animals↗
PubMed1994

Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

Animals↗