Publishing type：Research paper (scientific journal)   Publisher：Science Publishing Group  

DOI： 10.11648/j.bs.20190501.12

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases.

DOI： 10.1016/j.celrep.2018.03.141

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid beta-protein (A beta) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical A beta oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na+/K+-ATPase alpha 3 subunit (NAK alpha 3). ASPD-binding to NAK alpha 3 impaired NAK alpha 3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-A beta-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAK alpha 3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAK alpha 3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAK alpha 3 interaction.

DOI： 10.1073/pnas.1421182112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

DOI： 10.1073/pnas.1415525112

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Language：English   Publishing type：Part of collection (book)   Publisher：Wiley-VCH  

Multicolor fluorescent reporters are useful tools to visualize patterns of alternative splicing (AS) in cultured cells and in living organisms at a single-cell resolution. The multicolor reporters have been utilized to search for cis-elements and trans-acting factors involved in the regulation of AS, and to screen for chemical compounds affecting the splicing patterns. In this chapter, the technique is described of how to construct fluorescent AS reporter minigenes for the nematode Caenorhabditis elegans, cultured cells, and mice. The minigene construction is based on site-directed recombination, and various minigenes can be easily constructed by assembling modular DNA fragments such as a promoter, tag protein cDNAs, a genomic fragment of interest, fluorescent protein cDNAs, and a 3' cassette in separate vectors. Points to be considered when designing fluorescent AS reporters are also described. The splicing reporter system can, in theory, be applied to any other organisms. © 2012 Wiley-VCH Verlag GmbH &amp
Co. KGaA.

DOI： 10.1002/9783527636778.ch28

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc-/-) had normal kidneys. Combination of Fgfr2IIIc-/- with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1(Mes-/-)Fgfr2IIIc-/-) had small but identifiable MM at embryonic day (E) 10.5, expressing mesenchymal markers including Eya1, Six2, Pax2, and Gdnf (unlike Fgfr1/2(Mes-/-) mice that have no obvious MM). E11.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had rudimentary MM expressing only Eya1. Control, Fgfr2IIIc-/-, and Fgfr1(Mes-/-) Fgfr2IIIc-/- kidney mesenchymal tissues also express Fgfr2IIIb. In ureteric lineages, E10.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- embryos had ureteric outgrowth (sometimes multiple buds); however, by E11.5 Gdnf absence lead to no ureteric elongation or branching (similar to Fgfr1/2(Mes-/-) mice). Beyond E12.5, Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had no renal tissue. In conclusion, Fgfr2IIIc and Fgfr1 in kidney mesenchyme (together) are critical for normal early renal development. Developmental Dynamics 240: 240-249, 2011. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22501

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of "splice code''. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this "balance'' model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the "switch-like'' mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved "splice code,'' in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.

DOI： 10.1371/journal.pone.0010946

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

The laminar and area patterning of the mammalian neocortex are two organizing principles that define its functional architecture. Members of the immunoglobulin (1g) superfamily of cell adhesion molecules influence neural development by regulating cell adhesion, migration, and process growth. Here we describe the dynamic expression of the unique Ig-containing cell adhesion molecule, MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1), during forebrain development in mice and compare it with other markers. We show that MDGA1 is a layer-specific marker and an area-specific marker, being expressed in layers 2/3 throughout the neocortex, but within the primary somatosensory area (Sl), MDGAI is also uniquely expressed in layers 4 and 6a. Comparisons with other markers, including cadherins, serotonin, cytochrome oxidase, RORP, and COUP-TF1, reveal unique features of patterned expression of MDGA1 within cortex and S1 barrels. Further, our findings indicate that at earlier stages of development, MDGA1 is expressed by Reelin- and Tbr1-positive Caial-Retzius neurons that originate from multiple sources outside of neocortex and emigrate into it. At even earlier stages, MDGA1 is expressed by the earliest diencephalic and mesencephalic neurons, which appear to migrate from a MDGA1-positive domain of progenitors in the diencephalon and form a "preplate." These findings show that MDGAI is a unique marker for studies of cortical lamination and area patterning and together with recent reports suggest that MDGA1 has critical functions in forebrain/midbrain development.

DOI： 10.1093/cercor/bhl064

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

MAM( meprin/A5 protein/receptor protein tyrosine phosphatasemu) domain glycosylphosphatidylinositol anchor 1 ( MDGA1), a unique cell surface glycoprotein, is similar to Ig-containing cell adhesion molecules that influence neuronal migration and process outgrowth. We show in postnatal mice that MDGA1 is expressed by layer 2/3 neurons throughout the neocortex. During development, MDGA1 is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for MDGA1 in controlling their migration and settling in the superficial cortical plate. To test this hypothesis, we performed loss-of- function studies using RNA interference (RNAi) targeting different sequences of mouse MDGA1. RNAi or empty vectors were coelectroporated with an enhanced green fluorescent protein reporter in utero into the lateral ventricle at embryonic day 15.5 to transfect progenitors of superficial layer neurons; the distributions of transfected neurons were analyzed late on postnatal day 0. We found a direct correlation between effectiveness of an RNAi in suppressing MDGA1 expression and disrupting migration of superficial layer neurons. An RNAi with no effect on MDGA1 expression has no effect on the migration. In contrast, an RNAi that suppresses MDGA1 expression also blocks proper migration of transfected superficial layer neurons, with essentially all transfected cells found deep in the cortical plate or beneath it. This migration defect is rescued by cotransfection of a rat MDGA1 expression construct along with the effective RNAi, confirming that the RNAi effect is specific to diminishing mouse MDGA1 expression. RNAi transfections of deep layer neurons that do not express MDGA1 do not significantly affect their migration. We conclude thatMDGA1acts cell autonomously to control the migration of MDGA1- expressing superficial layer cortical neurons.

DOI： 10.1523/JNEUROSCI.4935-05.2006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FEDERATION AMER SOC EXP BIOL  

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34(-/-) mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiator factor 2alpha (eIF2alpha) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34(-/-) MEF, although following treatment with tunicamycin, the eIF2alpha phosphorylation level did not change in either GADD34(+/+) or GADD34(-/-) cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34(-/-) MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

DOI： 10.1096/fj.02-1184fje

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148(+/-) mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148(+/-) tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation(1). Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp(148+/-) embryonic stem cells and in fetal germ cells from chimeric Zfp148(+/-) embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

DOI： 10.1038/ng1072

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In a previous study, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed neuronal death owing to the cytotoxic effect of microglial nitric oxide (NO), and these neurons are finally eliminated by activated microglia. In this process, microglia are activated to release NO, increase in number, and accumulate toward the damaged area. In this study, we investigated the expression of macrophage colony-stimulating factor (M-CSF, also called colony stimulating factor-1; CSF-1) and other cytokines, which are reported to relate to activation, proliferation, or migration of microglia. The mRNA of M-CSF arose biphasically from 30 min to 1 hr and from 6 to 72 hr after the injury, as demonstrated by semiquantitative RT-PCR. However, another cytokine of granulocyte-macrophage CSF (GM-CSF) or interleukin-3 (IL-3), which causes proliferation of microglia in vitro, was nor detected. From immunohistochemical studies, positive staining of M-CSF was observed mainly in neuron-specific enolase (NSE)-positive cells from 1 to 12 hr after the injury, and after that M-CSF became positive in Griffonia simplicifolia isolectin-B4 (GSA-I-B4)-positive cells from 24 to 72 hr in the boundary area around necrosis. These results suggest that neurons around the damaged area express M-CSF in the early phase after injury, which may initially activate microglia, and these activated microglia also express M-CSF later, causing further proliferation or migration of microglia themselves to eliminate damaged neurons or necrotic brain tissue. J. Neurosci. Res. 65:38-44, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/jnr.1125

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia, In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells, It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury. (C) 2000 John Wiley & Sons, Inc.

DOI： 10.1002/1097-4695(200010)45:1<39::AID-NEU4>3.0.CO;2-A

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS  

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse pro alpha 2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse pro alpha 2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

DOI： 10.1042/0264-6021:3470055

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Expression of the manganese superoxide dismutase (Mn-SOD) is induced by pro-inflammatory cytokines, We investigated the cis-acting elements within a tumor necrosis factor-responsive element (TNFRE) which was identified in the second intron of the murine Mn-SOD gene. Site-directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF-kappa B and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis-acting elements may be required for induction of Mn-SOD gene expression mediated by lipopolysaccharide and interferon-gamma, (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)00408-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5-mu L ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti-iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA-I-B-4, ED-1 and OX-42. Haematoxylin-eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay on the first day after the injury. These TUNEL-positive neurons gradually disappeared toward the third day, while microglial cells increased. L-Ng-nitro-arginine methylester (L-NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.

DOI： 10.1046/j.1460-9568.1998.00168.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

We used the differential display technique in order to detect a new gene involved in murine type II collagen-induced arthritis (CIA). In this study, we have identified a novel gene, IF1, whose expression level is increased during the natural course of CIA. Northern blot analyses suggest that IF1 is involved in the natural course of CIA but is not involved as a trigger of CIA. IF1 is considered to be the murine ATPase inhibitor gene for several reasons. First, IF1 shows an extremely high homology to the rat ATPase inhibitor; the highly conserved region between rat and bovine amino acid residues 22-45, which is the minimum sequence showing ATPase inhibitory activities, is also highly conserved in IF1. Second, IF1 possesses a histidine-rich region in the same area, which is thought to be important for regulation of mammalian inhibitors. Third, the tissue distribution of IF1 is very suggestive. The expression of IF1 was very strong in energetic organs such as the heart, brain and kidney, and the development of arthritis requires great amounts of ATP. As arthritis develops rapidly, the cellular ATP pool may be decreased. Before the ATP pool is exhausted, the ATPase inhibitor may serve as a brake for ATP hydrolysis. If the supply of free energy can be reduced, the inflammation of arthritis may in turn be restored. Our hypothesis is that the ATPase inhibitor is involved in regulating the inflammatory responses.

DOI： 10.1046/j.1365-2567.1997.00370.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

In this study, we investigated the expression of genes for inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) of Kupffer cells in the presence of lipopolysaccharide (LPS), and the tissue expression of iNOS in a rat liver after LPS injection at various time intervals. The effects of L-N-G-nitroarginine-methyl-esther HCl (L-NAME), a NO inhibitor, also were examined. The mRNA transcripts of TNF-alpha, IL-1 beta, and IL-6 were rapidly detected no more than at 1 h after LPS stimulation, whereas the iNOS transcript was detectable from 3 h after LPS stimulation and maximally increased at 12 h. This fact suggested that these early induced cytokines were related to expression of iNOS. Using an anti-iNOS antiserum raised against recombinant iNOS protein, immunohistochemical analysis was made to reveal kinetics of NO producing cells. The cells immunoreactive for iNOS appeared at 6 h post-LPS injection in the sinusoids of the liver. By structural and immunohistochemical studies, almost all iNOS positive cells were identified as Kupffer cells and endothelial cells. The number of cells immunoreactive for iNOS increased until 12 h post-LPS injection. At 24 h after LPS injection, iNOS positive cells were restricted to the foci of spotty necrosis. Hepatic injury measured by released enzymes was increased by pretreatment of L-NAME before LPS injection. (C) 1997 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-4644(19970601)65:3<349::AID-JCB5>3.0.CO;2-S

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Radiation pneumonitis is a major complication of radiation therapy. To elucidate the mechanisms of radiation-induced pneumonitis, we studied nitric oxide (NO) produced from lung tissues using a model of unilaterally irradiated rats. Our results demonstrated that alveolar macrophages (AM) produced NO after irradiation, and the expression of inducible NO synthase (NOS) in both AM and alveolar epithelial cells was increased. Furthermore, the progression of radiation pneumonitis was reduced with the in vivo treatment of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). The effect of L-NAME was further confirmed by the inhibition of mRNA expression for procollagen-alpha1 type III of the lung. With these results, NO produced from AM and alveolar epithelial cells after irradiation may be an important mediator in the progression of radiation pneumonitis.

DOI： 10.1152/ajplung.1997.272.4.L651

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between -180 and -136 in the pro-alpha 2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the -180 to -152 segment of the mouse pro-cua(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-alpha 1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BBCOL1 participates in the transcriptional control of the two type I collagen genes.

DOI： 10.1074/jbc.272.8.4915

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