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Sample records for murine hoxc8 homeodomain

  1. The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

    International Nuclear Information System (INIS)

    Kong, Kyoung-Ah; Gadi, Jogeswar; Park, Hyoung Woo; Bok, Jinwoong; Kim, Myoung Hee

    2008-01-01

    Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins

  2. Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro

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    Mello Maria

    2003-04-01

    Full Text Available Abstract Background Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs. Results Cultured cells were characterized on the basis of morphology (light microscopy and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen. Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo 1, were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR. Conclusions The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro.

  3. Expression of cartilage developmental genes in Hoxc8- and Hoxd4-transgenic mice.

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    Claudia Kruger

    2010-02-01

    Full Text Available Hox genes encode transcription factors, which regulate skeletal patterning and chondrocyte differentiation during the development of cartilage, the precursor to mature bone. Overexpression of the homeobox transcription factors Hoxc8 and Hoxd4 causes severe cartilage defects due to delay in cartilage maturation. Matrix metalloproteinases (MMPs, bone morphogenetic proteins (BMPs and fibroblastic growth factors (FGFs are known to play important roles in skeletal development and endochondral bone formation and remodeling. In order to investigate whether these molecules are aberrantly expressed in Hoxc8- and/or Hoxd4-transgenic cartilage, we performed quantitative RT-PCR on chondrocytes from Hox-transgenic mice. Gene expression levels of Bmp4, Fgf8, Fgf10, Mmp9, Mmp13, Nos3, Timp3, Wnt3a and Wnt5a were altered in Hoxc8-transgenic chondrocytes, and Fgfr3, Ihh, Mmp8, and Wnt3a expression levels were altered in Hoxd4-transgenic chondrocytes, respectively. Notably, Wnt3a expression was elevated in Hoxc8- and reduced in Hoxd4-transgenic cartilage. These results suggest that both transcription factors affect cartilage maturation through different molecular mechanisms, and provide the basis for future studies into the role of these genes and possible interactions in pathogenesis of cartilage defects in Hoxc8- and Hoxd4-transgenic mice.

  4. Mouse Homologue of the Schizophrenia Susceptibility Gene ZNF804A as a Target of Hoxc8

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    Hyun Joo Chung

    2010-01-01

    Full Text Available Using a ChIP-cloning technique, we identified a Zinc finger protein 804a (Zfp804a as one of the putative Hoxc8 downstream target genes. We confirmed binding of Hoxc8 to an intronic region of Zfp804a by ChIP-PCR in F9 cells as well as in mouse embryos. Hoxc8 upregulated Zfp804a mRNA levels and augmented minimal promoter activity in vitro. In E11.5 mouse embryos, Zfp804a and Hoxc8 were coexpressed. Recent genome-wide studies identified Zfp804a (or ZNF804A in humans as a plausible marker for schizophrenia, leading us to hypothesize that this embryogenic regulatory control might also exert influence in development of complex traits such as psychosis.

  5. Molecular characterization of HOXC8 gene and methylation status analysis of its exon 1 associated with the length of cashmere fiber in Liaoning cashmere goat.

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    Bai, Wen L; Wang, Jiao J; Yin, Rong H; Dang, Yun L; Wang, Ze Y; Zhu, Yu B; Cong, Yu Y; Deng, Liang; Guo, Dan; Wang, Shi Q; Yang, Shu H; Xue, Hui L

    2017-02-01

    Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.

  6. Long Noncoding RNA HOXC-AS1 Suppresses Ox-LDL-Induced Cholesterol Accumulation Through Promoting HOXC6 Expression in THP-1 Macrophages.

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    Huang, Chuan; Hu, Yan-Wei; Zhao, Jing-Jing; Ma, Xin; Zhang, Yuan; Guo, Feng-Xia; Kang, Chun-Min; Lu, Jing-Bo; Xiu, Jian-Cheng; Sha, Yan-Hua; Gao, Ji-Juan; Wang, Yan-Chao; Li, Pan; Xu, Bang-Ming; Zheng, Lei; Wang, Qian

    2016-11-01

    Atherosclerosis is a common pathological basis of cardiovascular disease, which remains the leading cause of mortality. Long noncoding RNAs (lncRNAs) are newly studied non-protein-coding RNAs involved in gene regulation, but how lncRNAs exert regulatory effect on atherosclerosis remains unclear. In this study, we found that lncRNA HOXC cluster antisense RNA 1 (HOXC-AS1) and homeobox C6 (HOXC6) were downregulated in carotid atherosclerosis by performing microarray analysis. The results were verified in atherosclerotic plaques and normal arterial intima tissues by quantitative reverse transcription PCR and western blot analysis. Lentivirus-mediated overexpression of HOXC-AS1 induced HOXC6 expression at mRNA and protein levels in THP-1 macrophages. Besides, oxidized low-density lipoprotein (Ox-LDL) decreased expression of HOXC-AS1 and HOXC6 in a time-dependent manner. Induction of cholesterol accumulation by Ox-LDL could be partly suppressed by overexpression of HOXC-AS1.

  7. A novel mutation in homeobox DNA binding domain of HOXC13 gene underlies pure hair and nail ectodermal dysplasia (ECTD9) in a Pakistani family.

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    Khan, Anwar Kamal; Muhammad, Noor; Aziz, Abdul; Khan, Sher Alam; Shah, Khadim; Nasir, Abdul; Khan, Muzammil Ahmad; Khan, Saadullah

    2017-04-12

    Pure hair and nail ectodermal dysplasia (PHNED) is a congenital disorder of hair abnormalities and nail dysplasia. Both autosomal recessive and dominant inheritance fashion of PHNED occurs. In literature, to date, five different forms of PHNED have been reported at molecular level, having three genes known and two loci with no gene yet. In this study, a four generations consanguineous family of Pakistani origin with autosomal recessive PHNED was investigated. Affected members exhibited PHNED phenotypes with involvement of complete hair loss and nail dysplasia. To screen for mutation in the genes (HOXC13, KRT74, KRT85), its coding exons and exons-intron boundaries were sequenced. The 3D models of normal and mutated HOXC13 were predicted by using homology modeling. Through investigating the family to known loci, the family was mapped to ectodermal dysplasia 9 (ECTD9) loci with genetic address of 12q13.13. Mutation screening revealed a novel missense mutation (c.929A > C; p.Asn310Thr) in homeobox DNA binding domain of HOXC13 gene in affected members of the family. Due to mutation, loss of hydrogen bonding and difference in potential energy occurs, which may resulting in alteration of protein function. This is the first mutation reported in homeodomain, while 5 th mutation reported in HOXC13 gene causing PHNED.

  8. DNA Methylation and the HOXC6 Paradox in Prostate Cancer

    International Nuclear Information System (INIS)

    Vinarskaja, Anna; Yamanaka, Masanori; Ingenwerth, Marc; Schulz, Wolfgang A.

    2011-01-01

    Overexpression of the classical homeobox transcription factor HOXC6 is frequent in prostate cancers and correlates with adverse clinical parameters. Since surprisingly many HOXC6 target genes are downregulated in prostate cancer, it has been posited that oncogenic effects of HOXC6 in prostate cancer may be unmasked by concurrent epigenetic downregulation of target genes exerting tumor suppressive effects. To test this hypothesis, we have studied the expression of three HOXC6 target genes, CNTN1 (encoding a cell adhesion protein), DKK3 and WIF1 (encoding WNT growth factor antagonists) as well as DNA methylation of DKK3 and WIF1. HOXC6 upregulation and association with poor prognosis were confirmed in our tissue series. The three target genes were each significantly downregulated in cancer tissues and expression of each one correlated inversely with that of HOXC6. Cases with lower WIF1 expression showed significantly earlier recurrence (p = 0.021), whereas no statistical significance was reached for CNTN1 and DKK3. Hypermethylation of DKK3 or WIF1 gene promoters was observed in a subset of cancers with downregulated expression, but was often weak. Our data support the hypothesis that HOXC6 target genes exerting tumor-suppressive effects are epigenetically downregulated in prostate cancer, but DNA methylation appears to follow or bolster rather than to cause their transcriptional inactivation

  9. Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer.

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    Hussain, Imran; Bhan, Arunoday; Ansari, Khairul I; Deb, Paromita; Bobzean, Samara A M; Perrotti, Linda I; Mandal, Subhrangsu S

    2015-06-01

    HOXC6 is a homeobox-containing gene associated with mammary gland development and is overexpressed in variety of cancers including breast and prostate cancers. Here, we have examined the expression of HOXC6 in breast cancer tissue, investigated its transcriptional regulation via estradiol (E2) and bisphenol-A (BPA, an estrogenic endocrine disruptor) in vitro and in vivo. We observed that HOXC6 is differentially over-expressed in breast cancer tissue. E2 induces HOXC6 expression in cultured breast cancer cells and in mammary glands of Sprague Dawley rats. HOXC6 expression is also induced upon exposure to BPA both in vitro and in vivo. Estrogen-receptor-alpha (ERα) and ER-coregulators such as MLL-histone methylases are bound to the HOXC6 promoter upon exposure to E2 or BPA and that resulted in increased histone H3K4-trimethylation, histone acetylation, and recruitment of RNA polymerase II at the HOXC6 promoter. HOXC6 overexpression induces expression of tumor growth factors and facilitates growth 3D-colony formation, indicating its potential roles in tumor growth. Our studies demonstrate that HOXC6, which is a critical player in mammary gland development, is upregulated in multiple cases of breast cancer, and is transcriptionally regulated by E2 and BPA, in vitro and in vivo. Published by Elsevier B.V.

  10. The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation.

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    Potter, Christopher S; Pruett, Nathanael D; Kern, Michael J; Baybo, Mary Ann; Godwin, Alan R; Potter, Kathleen A; Peterson, Ron L; Sundberg, John P; Awgulewitsch, Alexander

    2011-04-01

    Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13(tm1Mrc)) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1(nu) (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13(tm1Mrc) and Foxn1(nu) mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13(tm1Mrc) mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.

  11. HOXC9-Induced Differentiation in Neuroblastoma Development

    Science.gov (United States)

    2014-10-01

    by HOXC9-binding peaks with the motif-finding program MEME revealed that the most enriched binding motif (T/ATTTAT, E value = 1.6e-35) corresponds to...sequences from predicted peak summits and ran MEME for identify- ing statistically overrepresented motifs. We performed MAST to search motifs in the

  12. Murine homeobox-containing gene, Msx-1: analysis of genomic organization, promoter structure, and potential autoregulatory cis-acting elements.

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    Kuzuoka, M; Takahashi, T; Guron, C; Raghow, R

    1994-05-01

    Detailed molecular organization of the coding and upstream regulatory regions of the murine homeodomain-containing gene, Msx-1, is reported. The protein-encoding portion of the gene is contained in two exons, 590 and 1214 bp in length, separated by a 2107-bp intron; the homeodomain is located in the second exon. The two-exon organization of the murine Msx-1 gene resembles a number of other homeodomain-containing genes. The 5'-(GTAAGT) and 3'-(CCCTAG) splicing junctions and the mRNA polyadenylation signal (UAUAA) of the murine Msx-1 gene are also characteristic of other vertebrate genes. By nuclease protection and primer extension assays, the start of transcription of the Msx-1 gene was located 256 bp upstream of the first AUG. Computer analysis of the promoter proximal 1280-bp sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements for Ap-1, Ap-2, Ap-3, Sp-1, a possible binding site for RAR:RXR, and a number of TCF-1 consensus motifs. Importantly, a perfect reverse complement of (C/G)TTAATTG, which was recently shown to be an optimal binding sequence for the homeodomain of Msx-1 protein (K.M. Catron, N. Iler, and C. Abate (1993) Mol. Cell. Biol. 13:2354-2365), was also located in the murine Msx-1 promoter. Binding of bacterially expressed Msx-1 homeodomain polypeptide to Msx-1-specific oligonucleotide was experimentally demonstrated, raising a distinct possibility of autoregulation of this developmentally regulated gene.

  13. A Novel Homozygous Missense Mutation in HOXC13 Leads to Autosomal Recessive Pure Hair and Nail Ectodermal Dysplasia.

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    Li, Xiaoxiao; Orseth, Meredith Lee; Smith, J Michael; Brehm, Mary Abigail; Agim, Nnenna Gebechi; Glass, Donald Alexander

    2017-03-01

    Pure hair and nail ectodermal dysplasia (PHNED) is a rare disorder that presents with hypotrichosis and nail dystrophy while sparing other ectodermal structures such as teeth and sweat glands. We describe a homozygous novel missense mutation in the HOXC13 gene that resulted in autosomal recessive PHNED in a Hispanic child. The mutation c.812A>G (p.Gln271Arg) is located within the DNA-binding domain of the HOXC13 gene, cosegregates within the family, and is predicted to be maximally damaging. This is the first reported case of a missense HOXC13 mutation resulting in PHNED and the first reported case of PHNED identified in a North American family. Our findings illustrate the critical role of HOXC13 in human hair and nail development. © 2017 Wiley Periodicals, Inc.

  14. Role of Hoxc genes in the development of the limb integumentary organ (nail, claw, or hoof).

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    Lasierra, Maria Angeles Ros; Fernández-Guerrero, Marc; Delisle, Lucille; Yakushiji-Kaminatsui, Nayuta; Darbellay, Fabrice; Pérez-Gómez, Rocío; Duboule, Denis

    2018-04-01

    The developing vertebrate limb has long proved as an excellent system for studying the mechanisms involved in pattern formation and morphogenesis and more recently in transcriptional regulation and morphological evolution. To elucidate the stage-specific expression profiles of the components of the developing limb, we have generated the temporal transcriptome of the limb progenitors and of the overlying ectoderm separately. Our study has uncovered a collinear activation of Hoxc genes in the limb ectoderm that we have validated by in situ hybridization. However, while members of the HoxA and HoxD clusters show complex and dynamic patterns of expression during limb development that correlate with the morphology of the different limb segments, no specific function for the HoxC or HoxB clusters has been identified ( 1 - 3 ). To investigate the function of Hoxc genes in the limb ectoderm, we have reexamined the HoxC cluster null mice. Remarkably, and despite exhibiting normal terminal phalanges, these mice didn't form claws (anonychia). Morphological and immunohistochemical analysis identified a failure in the differentiation of the main components of the nail/claw organ. To unravel the transcriptional regulation of Hoxc genes in the limb ectoderm, we used the ATACseq technique. Using this approach, we identified two putative regulatory regions which activity was tested in mouse transgenic enhancer assays. It is currently considered that Hox genes have played a key role in the evolution of morphological traits, probably associated with changes in their regulatory landscapes ( 4 ). Given that the form and size of the distal limb integumentary organ (nail, claw or hoof) correlates with that of the distal phalanx and that the development of hooves was a major innovation in the evolution of a cursorial lifestyle, we are also exploring the possible implication of Hoxc genes in the nail/claw/hoof transition. This abstract is from the Experimental Biology 2018 Meeting. There

  15. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

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    Hoffmann, H M; Catron, K M; van Wijnen, A J; McCabe, L R; Lian, J B; Stein, G S; Stein, J L

    1994-12-20

    The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

  16. Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Moon, Sunjin; Lee, Yong Woo; Kim, Woo Taek; Lee, Weontae

    2014-01-01

    Highlights: •We have determined solution structures of CEH-37 homedomain. •CEH-37 HD has a compact α-helical structure with HTH DNA binding motif. •Solution structure of CEH-37 HD shares its molecular topology with that of the homeodomain proteins. •Residues in the N-terminal region and HTH motif are important in binding to Caenorhabditis elegans telomeric DNA. •CEH-37 could play an important role in telomere function via DNA binding. -- Abstract: The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding

  17. Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Sunjin [Structural Biochemistry and Molecular Biophysics Lab, Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Yong Woo; Kim, Woo Taek [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Structural Biochemistry and Molecular Biophysics Lab, Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2014-01-10

    Highlights: •We have determined solution structures of CEH-37 homedomain. •CEH-37 HD has a compact α-helical structure with HTH DNA binding motif. •Solution structure of CEH-37 HD shares its molecular topology with that of the homeodomain proteins. •Residues in the N-terminal region and HTH motif are important in binding to Caenorhabditis elegans telomeric DNA. •CEH-37 could play an important role in telomere function via DNA binding. -- Abstract: The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.

  18. Crystal structure of the Msx-1 homeodomain/DNA complex.

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    Hovde, S; Abate-Shen, C; Geiger, J H

    2001-10-09

    The Msx-1 homeodomain protein plays a crucial role in craniofacial, limb, and nervous system development. Homeodomain DNA-binding domains are comprised of 60 amino acids that show a high degree of evolutionary conservation. We have determined the structure of the Msx-1 homeodomain complexed to DNA at 2.2 A resolution. The structure has an unusually well-ordered N-terminal arm with a unique trajectory across the minor groove of the DNA. DNA specificity conferred by bases flanking the core TAAT sequence is explained by well ordered water-mediated interactions at Q50. Most interactions seen at the TAAT sequence are typical of the interactions seen in other homeodomain structures. Comparison of the Msx-1-HD structure to all other high resolution HD-DNA complex structures indicate a remarkably well-conserved sphere of hydration between the DNA and protein in these complexes.

  19. YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues.

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    Liu, Ming; Zhao, Shuangyun; Lin, Qingjie; Wang, Xiu-Ping

    2015-04-01

    Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains

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    Owens Raymond J

    2010-05-01

    Full Text Available Abstract Background Zhx1 to 3 (zinc-fingers and homeoboxes form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination. Results A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer. Conclusions Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain, by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally

  1. MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8

    DEFF Research Database (Denmark)

    Lüttichau, H R; Gerstoft, J; Schwartz, T W

    2001-01-01

    The viral CC chemokines MC148, encoded by the poxvirus molluscum contagiosum, and viral macrophage inflammatory protein (vMIP)-I and vMIP-II, encoded by human herpesvirus 8, were probed on the murine CC receptor (CCR) 8 in parallel with human CCR8. In calcium mobilization assays, vMIP-I acted...... as a high-affinity agonist, whereas vMIP-II acted as a low-affinity antagonist on the murine CCR8 as well as the human CCR8. MC148 was found to bind and block responses through the human CCR8 with high affinity, but surprisingly MC148 was unable to bind and block responses through the murine CCR8. Because...

  2. Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8.

    Directory of Open Access Journals (Sweden)

    Min Du

    Full Text Available The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

  3. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    International Nuclear Information System (INIS)

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-01-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL

  4. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Kausik [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Ramagopal, Udupi A. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Almo, Steven C., E-mail: nathenso@aecom.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States)

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  5. Crystal structure and DNA binding of the homeodomain of the stem cell transcription factor Nanog.

    Science.gov (United States)

    Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C; Kolatkar, Prasanna R

    2008-02-22

    The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

  6. Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

    Energy Technology Data Exchange (ETDEWEB)

    Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C.; Kolatkar, Prasanna R. (GI-Singapore); (Scripps)

    2010-02-08

    The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

  7. Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano

    2010-01-01

    antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...... of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O......-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr(77)), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin...

  8. Structural and electrostatic regularities in interactions of homeodomains with operator DNA

    International Nuclear Information System (INIS)

    Chirgadze, Yu.N.; Ivanov, V.V.; Polozov, R.V.; Zheltukhin, E.I.; Sivozhelezov, V.S.

    2008-01-01

    Interfaces of five DNA-homeodomain complexes, selected by similarity of structures and patterns of contacting residues, were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 Angstroem away from molecular surfaces of both protein and DNA. For proteins, clear positive potential is displayed only at the side contacting DNA, while grooves of DNA display a strong negative potential. Thus, one functional role of electrostatics is guiding the protein into the DNA major groove. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in formation of protein-DNA atomic contacts in the interface. The protein's recognizing α-helix was shown to form both invariant and variable contacts with DNA by means of the certain specific side groups, with water molecules participating in some of the contacts. The invariant contacts included the highly specific Asn-Ade hydrogen bonds, nonpolar contacts of hydrophobic amino acids serving as barriers for fixing the protein on DNA, and interface water molecule cluster providing local mobility necessary for the dissociation of the protein-DNA complex. One of the water molecules is invariant and located at the center of the interface. Invariant contacts of the proteins are mostly formed with the TAAT motive of promoter DNA's forward strand. They distinguish the homeodomain family from other DNA-binding proteins. Variable contacts are formed with the reverse strand and are responsible for the binding specificity within the homeodomain family

  9. Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia

    Directory of Open Access Journals (Sweden)

    Tim D.D. Somerville

    2018-01-01

    Full Text Available The Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML, ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

  10. Protein kinase A phosphorylates serine 267 in the homeodomain of engrailed-2 leading to decreased DNA binding

    DEFF Research Database (Denmark)

    Hjerrild, Majbrit; Stensballe, Allan; Jensen, Ole N

    2004-01-01

    Engrailed-2 (En-2) belongs to an evolutionarily conserved family of DNA binding homeodomain-containing proteins that are expressed in mammalian brain during development. Here, we demonstrate that serine 267 in the homeodomain of En-2 is phosphorylated by protein kinase A (PKA) in forskolin......-treated COS-7 cells. Furthermore, we analyze the physiological function of En-2 phosphorylation by PKA. The nuclear localization of En-2 is not influenced by the phosphorylation of serine 267. However, substitution of serine 267 with alanine resulted in increased binding of En-2 to DNA, while replacing serine...

  11. Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs.

    Science.gov (United States)

    Towler, D A; Bennett, C D; Rodan, G A

    1994-05-01

    A detailed analysis of the transcriptional machinery responsible for osteoblast-specific gene expression should provide tools useful for understanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATTGTGT) present in the region between -200 and -170 that binds a multisubunit CP1/NFY/CBF-like CAAT factor complex contributes significantly to high level basal activity and presumably functions as the CAAT box for the rat OC promoter. We show that the region -121 to 32 is sufficient to confer osteoblastic cell type specificity in transient transfection assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-100 to -74) contains a CAAT motif, we could not detect CP1-like CAAT factor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-end of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognizes this motif appears to be present in osteoblastic ROS 17/2.8 cells, which produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-rich DNA cognates of the homeodomain-containing POU family of transcription factors. Taken together, these data suggest that a ubiquitous CP1-like CAAT factor and a cell type-restricted homeodomain containing (Msx or POU family) transcription factor interact with the proximal rat OC promoter to direct appropriate basal OC transcription in osteoblastic cells.

  12. Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

    Science.gov (United States)

    Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Mardesic Brakus, Snjezana; Saraga-Babic, Mirna

    2017-10-02

    Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19 INK4d . p19 INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19 INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19 INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19 INK4d throughout the investigated period indicates that p19 INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19 INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

  13. Hydrogen-deuterium exchange studies of the rat thyroid transcription factor 1 homeodomain

    International Nuclear Information System (INIS)

    Esposito, Gennaro; Fogolari, Federico; Damante, Giuseppe; Formisano, Silvestro; Tell, Gianluca; Leonardi, Antonio; Di Lauro, Roberto; Viglino, Paolo

    1997-01-01

    The 1 H NMR solution structure of the rat thyroid transcription factor 1 homeodomain (TTF-1 HD) showed that the molecule folds like classical homeodomains. The C-terminal extension of helix III (fragment 51-59) appeared to adopt a helical geometry, albeit not as rigid as the preceding portion, but the hydrogen-deuterium exchange of backboneamides and the NOE data provided evidence of a discontinuity between the two moieties of helix III at the highly conserved fragmentAsn 51 -His 52 -Arg 53 .Analysis of quantitative measurements of isotope exchange rates allows one to recognize the general occurrence, in that region of HD motifs, of opposite effects to helix III stability. Asparagine, histidine and arginine residues occur most frequently at the beginning and end of protein helices.In TTF-1 HD a local fluctuation is observed in the fragment 51-53 which either kinks or tightens the α-helix. A search through the protein structure database reveals that the three most common variants of HD fragments 51-53 are often involved in helices and, frequently, in helix initiation or termination. For homeodomains in general, the nature of the fragment 51-53 may be related to the conformational dynamics of their DNA-recognition helix (helix III). Besides the specific results on fragment 51-53, the complete isotope exchange analysis of TTF-1 HD data shows that the partially solvent-exposed recognition helix is stabilized by hydrophobic interactions, like most of the structured regions of the molecule. Hydrophobic stabilization of the contacting regions meets the requirements of a DNA-interaction mechanism which, as shown with other DNA-protein complexes, should entail negative heat capacity variations due to changes in solvent exposure of the nonpolar protein surface

  14. Developmental regulation of gonadotropin-releasing hormone gene expression by the MSX and DLX homeodomain protein families.

    Science.gov (United States)

    Givens, Marjory L; Rave-Harel, Naama; Goonewardena, Vinodha D; Kurotani, Reiko; Berdy, Sara E; Swan, Christo H; Rubenstein, John L R; Robert, Benoit; Mellon, Pamela L

    2005-05-13

    Gonadotropin-releasing hormone (GnRH) is the central regulator of the hypothalamic-pituitary-gonadal axis, controlling sexual maturation and fertility in diverse species from fish to humans. GnRH gene expression is limited to a discrete population of neurons that migrate through the nasal region into the hypothalamus during embryonic development. The GnRH regulatory region contains four conserved homeodomain binding sites (ATTA) that are essential for basal promoter activity and cell-specific expression of the GnRH gene. MSX and DLX are members of the Antennapedia class of non-Hox homeodomain transcription factors that regulate gene expression and influence development of the craniofacial structures and anterior forebrain. Here, we report that expression patterns of the Msx and Dlx families of homeodomain transcription factors largely coincide with the migratory route of GnRH neurons and co-express with GnRH in neurons during embryonic development. In addition, MSX and DLX family members bind directly to the ATTA consensus sequences and regulate transcriptional activity of the GnRH promoter. Finally, mice lacking MSX1 or DLX1 and 2 show altered numbers of GnRH-expressing cells in regions where these factors likely function. These findings strongly support a role for MSX and DLX in contributing to spatiotemporal regulation of GnRH transcription during development.

  15. Developmental Regulation of Gonadotropin-releasing Hormone Gene Expression by the MSX and DLX Homeodomain Protein Families*

    Science.gov (United States)

    Givens, Marjory L.; Rave-Harel, Naama; Goonewardena, Vinodha D.; Kurotani, Reiko; Berdy, Sara E.; Swan, Christo H.; Rubenstein, John L. R.; Robert, Benoit; Mellon, Pamela L.

    2010-01-01

    Gonadotropin-releasing hormone (GnRH) is the central regulator of the hypothalamic-pituitary-gonadal axis, controlling sexual maturation and fertility in diverse species from fish to humans. GnRH gene expression is limited to a discrete population of neurons that migrate through the nasal region into the hypothalamus during embryonic development. The GnRH regulatory region contains four conserved homeodomain binding sites (ATTA) that are essential for basal promoter activity and cell-specific expression of the GnRH gene. MSX and DLX are members of the Antennapedia class of non-Hox homeodomain transcription factors that regulate gene expression and influence development of the craniofacial structures and anterior forebrain. Here, we report that expression patterns of the Msx and Dlx families of homeodomain transcription factors largely coincide with the migratory route of GnRH neurons and co-express with GnRH in neurons during embryonic development. In addition, MSX and DLX family members bind directly to the ATTA consensus sequences and regulate transcriptional activity of the GnRH promoter. Finally, mice lacking MSX1 or DLX1 and 2 show altered numbers of GnRH-expressing cells in regions where these factors likely function. These findings strongly support a role for MSX and DLX in contributing to spatiotemporal regulation of GnRH transcription during development. PMID:15743757

  16. Genome wide binding (ChIP-Seq of murine Bapx1 and Sox9 proteins in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Sumantra Chatterjee

    2016-12-01

    Full Text Available This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.

  17. CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

    Science.gov (United States)

    Noval Rivas, Magali; Lee, Youngho; Wakita, Daiko; Chiba, Norika; Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Fishbein, Michael C; Lehman, Thomas J A; Crother, Timothy R; Arditi, Moshe

    2017-02-01

    Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans. © 2016, American College of Rheumatology.

  18. Two human homeobox genes, c1 and c8: structure analysis and expression in embryonic development.

    Science.gov (United States)

    Simeone, A; Mavilio, F; Acampora, D; Giampaolo, A; Faiella, A; Zappavigna, V; D'Esposito, M; Pannese, M; Russo, G; Boncinelli, E

    1987-07-01

    Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hydridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embryos and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.

  19. Two human homeobox genes, c1 and c8: structure analysis and expression in embryonic development

    International Nuclear Information System (INIS)

    Simeone, A.; Mavilio, F.; Acampora, D.

    1987-01-01

    Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomains identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hybridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embryos and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny

  20. The embryonic genes Dkk3, Hoxd8, Hoxd9 and Tbx1 identify muscle types in a diet-independent and fiber-type unrelated way

    Directory of Open Access Journals (Sweden)

    Boekschoten Mark V

    2010-03-01

    Full Text Available Abstract Background The mouse skeletal muscle is composed of four distinct fiber types that differ in contractile function, number of mitochondria and metabolism. Every muscle type has a specific composition and distribution of the four fiber types. To find novel genes involved in specifying muscle types, we used microarray analysis to compare the gastrocnemius with the quadriceps from mice fed a low fat diet (LFD or high fat diet (HFD for 8 weeks. Additional qPCR analysis were performed in the gastrocnemius, quadriceps and soleus muscle from mice fed an LFD or HFD for 20 weeks. Results In mice fed the 8-week LFD 162 genes were differentially expressed in the gastrocnemius vs. the quadriceps. Genes with the strongest differences in expression were markers for oxidative fiber types (e.g. Tnni1 and genes which are known to be involved in embryogenesis (Dkk3, Hoxd8,Hoxd9 and Tbx1. Also Dkk2, Hoxa5, Hoxa10, Hoxc9, Hoxc10, Hoxc6 and Tbx15 were detectably, but not differentially expressed in adult muscle tissue. Expression of differentially expressed genes was not influenced by an 8-week or 20-week HFD. Comparing gastrocnemius, quadriceps and soleus, expression of Hoxd8 and Hoxd9 was not related with expression of markers for the four different fiber types. We found that the expression of both Hoxd8 and Hoxd9 was much higher in the gastrocnemius than in the quadriceps or soleus, whereas the expression of Dkk3 was high in quadriceps, but low in both gastrocnemius and soleus. Finally, expression of Tbx1 was high in quadriceps, intermediate in soleus and low in gastrocnemius. Conclusions We found that genes from the Dkk family, Hox family and Tbx family are detectably expressed in adult mouse muscle. Interestingly, expression of Dkk3, Hoxd8, Hoxd9 and Tbx1 was highly different between gastrocnemius, quadriceps and soleus. In fact, every muscle type showed a unique combination of expression of these four genes which was not influenced by diet. Altogether

  1. Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains

    NARCIS (Netherlands)

    Bird, L.E.; Ren, J.; Nettleship, J.E.; Folkers, G.E.|info:eu-repo/dai/nl/162277202; Owens, RJ; Stammers, D.K.

    2010-01-01

    Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor

  2. Roles of the LHX3 and LHX4 LIM-Homeodomain Factors in Pituitary Development

    OpenAIRE

    Mullen, Rachel D.; Colvin, Stephanie C.; Hunter, Chad S.; Savage, Jesse J.; Walvoord, Emily C.; Bhangoo, Amrit P.S.; Ten, Svetlana; Weigel, Johannes; Pfäffle, Roland W.; Rhodes, Simon J.

    2007-01-01

    The LHX3 and LHX4 LIM-homeodomain transcription factors play essential roles in pituitary gland and nervous system development. Mutations in the genes encoding these regulatory proteins are associated with combined hormone deficiency diseases in humans and animal models. Patients with these diseases have complex syndromes involving short stature, and reproductive and metabolic disorders. Analyses of the features of these diseases and the biochemical properties of the LHX3 and LHX4 proteins wi...

  3. P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.

    OpenAIRE

    Szeto, D P; Ryan, A K; O'Connell, S M; Rosenfeld, M G

    1996-01-01

    A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in t...

  4. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Frank Zach

    Full Text Available In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from

  5. Existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot

    Science.gov (United States)

    Kheirollahi, Majid; Khosravi, Fereshteh; Ashouri, Saeideh; Ahmadi, Alireza

    2016-01-01

    Background: Tetralogy of Fallot (TOF), the most common cyanotic heart defect and one of the most common congenital heart diseases, occurs mostly sporadically and nonsyndromically. The underlying molecular genetic mechanism is not known. Therefore, the existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot is evaluated. Materials and Methods: In the present study, we analyzed the peripheral blood samples of27 patients in order to find any mutation in the 180 bp homeodomain-encoding region of NKX2.5 gene, which is known to be involved in heart development and diseases. DNA was extracted and all the samples were amplified by polymerase chain reaction (PCR) and sequenced. Results: Twenty-seven patients were included in the study. Twenty-five of them were infants and children (6 days to 11 years of age), one was a teenager (14-years of age), and another was a 33-year-old man [mean ± standard deviation (SD): 5.80 ± 3.90 years]. Thirteen patents were males (mean ± SD: 6.587077 ± 5.02 years) and 14 were females (mean ± SD: 5.0726 ± 2.81 years). One synonymous variant, i.e., c.543G>A was identified in one patient. Conclusion: Mutations in the homeodomain-encoding region of NKX2.5 gene may not have an outstanding role in etiology of tetralogy of Fallot patients in Iran. PMID:27904570

  6. The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene

    NARCIS (Netherlands)

    Soppe, W.J.J.; Jacobsen, S.E.; Alonso-Blanco, C.; Jackson, J.P.; Kakutani, T.; Koornneef, M.; Peeters, A.J.M.

    2000-01-01

    The transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants. FWA was identified by loss-of-function mutations in normally flowering revertants of the fwa mutant, and it encodes a homeodomain-containing transcription factor. The DNA sequence of wild-type and fwa mutant alleles

  7. Genome-wide analysis of the homeodomain-leucine zipper (HD-ZIP) gene family in peach (Prunus persica).

    Science.gov (United States)

    Zhang, C H; Ma, R J; Shen, Z J; Sun, X; Korir, N K; Yu, M L

    2014-04-08

    In this study, 33 homeodomain-leucine zipper (HD-ZIP) genes were identified in peach using the HD-ZIP amino acid sequences of Arabidopsis thaliana as a probe. Based on the phylogenetic analysis and the individual gene or protein characteristics, the HD-ZIP gene family in peach can be classified into 4 subfamilies, HD-ZIP I, II, III, and IV, containing 14, 7, 4, and 8 members, respectively. The most closely related peach HD-ZIP members within the same subfamilies shared very similar gene structure in terms of either intron/exon numbers or lengths. Almost all members of the same subfamily shared common motif compositions, thereby implying that the HD-ZIP proteins within the same subfamily may have functional similarity. The 33 peach HD-ZIP genes were distributed across scaffolds 1 to 7. Although the primary structure varied among HD-ZIP family proteins, their tertiary structures were similar. The results from this study will be useful in selecting candidate genes from specific subfamilies for functional analysis.

  8. The Populus ARBORKNOX1 homeodomain transcription factor regulates woody growth through binding to evolutionarily conserved target genes of diverse function

    Science.gov (United States)

    Lijun Liu; Matthew S. Zinkgraf; H. Earl Petzold; Eric P. Beers; Vladimir Filkov; Andrew Groover

    2014-01-01

    The class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.

  9. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes

    International Nuclear Information System (INIS)

    Shi, Ge; Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin; Ou, Bai-sheng; Kim, Sooil; Lee, Young Ho; Yoon, Tae-Jin; Kim, Seong-Jin; Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon; Kim, Chang Deok

    2010-01-01

    Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

  10. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Ge [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China); Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Ou, Bai-sheng [Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China); Kim, Sooil; Lee, Young Ho [Department of Anatomy, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Yoon, Tae-Jin [Department of Dermatology, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of); Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of); Kim, Seong-Jin [Department of Dermatology, Chonnam National University Medical School, Gwangju, 501-757 (Korea, Republic of); Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Kim, Chang Deok, E-mail: cdkimd@cnu.ac.kr [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of)

    2010-11-15

    Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

  11. Chromosomal Alterations and Gene Expression Changes Associated with the Progression of Leukoplakia to Advanced Gingivobuccal Cancer

    Directory of Open Access Journals (Sweden)

    Priyanka G. Bhosale

    2017-06-01

    Full Text Available We present an integrative genome-wide analysis that can be used to predict the risk of progression from leukoplakia to oral squamous cell carcinoma (OSCC arising in the gingivobuccal complex (GBC. We find that the genomic and transcriptomic profiles of leukoplakia resemble those observed in later stages of OSCC and that several changes are associated with this progression, including amplification of 8q24.3, deletion of 8p23.2, and dysregulation of DERL3, EIF5A2, ECT2, HOXC9, HOXC13, MAL, MFAP5 and NELL2. Comparing copy number profiles of primary tumors with and without lymph-node metastasis, we identify alterations associated with metastasis, including amplifications of 3p26.3, 8q24.21, 11q22.1, 11q22.3 and deletion of 8p23.2. Integrative analysis reveals several biomarkers that have never or rarely been reported in previous OSCC studies, including amplifications of 1p36.33 (attributable to MXRA8, 3q26.31 (EIF5A2, 9p24.1 (CD274, and 12q13.2 (HOXC9 and HOXC13. Additionally, we find that amplifications of 1p36.33 and 11q22.1 are strongly correlated with poor clinical outcome. Overall, our findings delineate genomic changes that can be used in treatment management for patients with potentially malignant leukoplakia and OSCC patients with higher risk of lymph-node metastasis.

  12. Cooperative interactions between paired domain and homeodomain.

    Science.gov (United States)

    Jun, S; Desplan, C

    1996-09-01

    The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

  13. Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein BREVIPEDICELLUS is essential for differentiation of secondary xylem in Arabidopsis root.

    Science.gov (United States)

    Woerlen, Natalie; Allam, Gamalat; Popescu, Adina; Corrigan, Laura; Pautot, Véronique; Hepworth, Shelley R

    2017-06-01

    Repression of boundary genes by KNOTTED1-like homeodomain transcription factor BREVIPEDICELLUS promotes the differentiation of phase II secondary xylem in Arabidopsis roots. Plant growth and development relies on the activity of meristems. Boundaries are domains of restricted growth that separate forming organs and the meristem. Class I KNOX homeodomain transcription factors are important regulators of meristem maintenance. Members of this class including BREVIDICELLUS also called KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (BP/KNAT1) fulfill this function in part by spatially regulating boundary genes. The vascular cambium is a lateral meristem that allows for radial expansion of organs during secondary growth. We show here that BP/KNAT1 repression of boundary genes plays a crucial role in root secondary growth. In particular, exclusion of BLADE-ON-PETIOLE1/2 (BOP1/2) and other members of this module from xylem is required for the differentiation of lignified fibers and vessels during the xylem expansion phase of root thickening. These data reveal a previously undiscovered role for boundary genes in the root and shed light on mechanisms controlling wood development in trees.

  14. Large-scale identification of microRNA targets in murine Dgcr8-deficient embryonic stem cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew P A Davis

    Full Text Available Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.

  15. Expression and function of S100A8/A9 (calprotectin) in human typhoid fever and the murine Salmonella model.

    Science.gov (United States)

    De Jong, Hanna K; Achouiti, Ahmed; Koh, Gavin C K W; Parry, Christopher M; Baker, Stephen; Faiz, Mohammed Abul; van Dissel, Jaap T; Vollaard, Albert M; van Leeuwen, Ester M M; Roelofs, Joris J T H; de Vos, Alex F; Roth, Johannes; van der Poll, Tom; Vogl, Thomas; Wiersinga, Willem Joost

    2015-04-01

    Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model. S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury. S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S

  16. Expression and function of S100A8/A9 (calprotectin in human typhoid fever and the murine Salmonella model.

    Directory of Open Access Journals (Sweden)

    Hanna K De Jong

    2015-04-01

    Full Text Available Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8 and S100A9 (MRP14 form bioactive antimicrobial heterodimers (calprotectin that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT and S100A9-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response

  17. Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori

    Science.gov (United States)

    Takiya, Shigeharu; Tsubota, Takuya; Kimoto, Mai

    2016-01-01

    The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP) axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing “colinearity”. The central Hox class protein Antennapedia (Antp) directly regulates the expression of several middle silk gland–specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM)-homeodomain transcriptional factor Arrowhead (Awh) regulates the expression of posterior silk gland–specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins. PMID:29615585

  18. Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    Shigeharu Takiya

    2016-05-01

    Full Text Available The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing “colinearity”. The central Hox class protein Antennapedia (Antp directly regulates the expression of several middle silk gland–specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM-homeodomain transcriptional factor Arrowhead (Awh regulates the expression of posterior silk gland–specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins.

  19. Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, Hiroki [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Nakamura, Seikou [Department of Pharmacognosy, Kyoto Pharmaceutical University, Kyoto (Japan); Chisaki, Yugo [Education and Research Center for Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto (Japan); Takada, Tetsuya [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Toda, Yuki [Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto (Japan); Murata, Hiroaki [Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopedic Surgery, Matsushita Memorial Hospital, Osaka (Japan); Itoh, Kazuyuki [Department of Biology, Osaka Medical Center of Cancer and Cardiovascular Diseases, Osaka (Japan); Yano, Yoshitaka [Education and Research Center for Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto (Japan); Takata, Kazuyuki [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Ashihara, Eishi, E-mail: ash@mb.kyoto-phu.ac.jp [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan)

    2016-02-26

    Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

  20. Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

    International Nuclear Information System (INIS)

    Fukuda, Hiroki; Nakamura, Seikou; Chisaki, Yugo; Takada, Tetsuya; Toda, Yuki; Murata, Hiroaki; Itoh, Kazuyuki; Yano, Yoshitaka; Takata, Kazuyuki; Ashihara, Eishi

    2016-01-01

    Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

  1. Comparison of multiple crystal structures with NMR data for engrailed homeodomain

    Energy Technology Data Exchange (ETDEWEB)

    Religa, Tomasz L. [MRC Centre for Protein Engineering (United Kingdom)], E-mail: tlr25@mrc-lmb.cam.ac.uk

    2008-03-15

    Two methods are currently available to solve high resolution protein structures-X-ray crystallography and nuclear magnetic resonance (NMR). Both methods usually produce highly similar structures, but small differences between both solutions are always observed. Here the raw NMR data as well as the solved NMR structure were compared to the multiple crystal structures solved for the WT 60 residue three helix bundle engrailed homeodomain (EnHD) and single point mutants. There was excellent agreement between TALOS-predicted and crystal structure-observed dihedral angles and a good agreement for the {sup 3}J(H{sup N}H{sup {alpha}}) couplings for the multiple crystal structures. Around 1% of NOEs were violated for any crystal structure, but no NOE was inconsistent with all of the crystal structures. Violations usually occurred for surface residues or for residues for which multiple discreet conformations were observed between the crystal structures. Comparison of the disorder shown in the multiple crystal structures shows little correlation with dynamics under native conditions for this protein.

  2. Murine analogues of etanercept and of F8-IL10 inhibit the progression of collagen-induced arthritis in the mouse.

    Science.gov (United States)

    Doll, Fabia; Schwager, Kathrin; Hemmerle, Teresa; Neri, Dario

    2013-09-27

    Etanercept is a fusion protein consisting of the soluble portion of the p75-tumor necrosis factor receptor (TNFR) and the Fc fragment of human IgG1, which is often used for the treatment of patients with rheumatoid arthritis. F8-IL10 is a human immunocytokine based on the F8 antibody and interleukin-10, which is currently being investigated in rheumatoid arthritis with promising clinical results. We have aimed at expressing murine versions of these two fusion proteins, in order to assess their pharmaceutical performance in the collagen-induced model of rheumatoid arthritis in the mouse. Two fusion proteins (termed muTNFR-Fc and F8-muIL10) were cloned, expressed in chinese hamster ovary (CHO) cells, purified and characterized. Biological activity of muTNFR-Fc was assessed by its ability to inhibit TNF-induced killing of mouse fibroblasts, while F8-muIL10 was characterized in terms of muIL10 activity, of binding affinity to the cognate antigen of F8, the alternatively-spliced EDA domain of fibronectin, by quantitative biodistribution analysis and in vivo imaging. The therapeutic activity of both fusion proteins was investigated in a collagen-induced mouse model of arthritis. Mouse plasma was analyzed for anti-drug antibody formation and cytokine levels were determined by bead-based multiplex technology. The association of F8-IL10 proteins with blood cells was studied in a centrifugation assay with radiolabeled protein. Both fusion proteins exhibited excellent purity and full biological activity in vitro. In addition, F8-muIL10 was able to localize on newly-formed blood vessels in vivo. When used in a murine model of arthritis, the two proteins inhibited arthritis progression. The activity of muTNFR-Fc was tested alone and in combination with F8-huIL10. The chimeric version of F8-IL10 was not better then the fully human fusion protein and showed similar generation of mouse anti-fusion protein antibodies. Incubation studies of F8-muIL10 and F8-huIL10 with blood

  3. Three-dimensional alginate spheroid culture system of murine osteosarcoma.

    Science.gov (United States)

    Akeda, Koji; Nishimura, Akinobu; Satonaka, Haruhiko; Shintani, Ken; Kusuzaki, Katsuyuki; Matsumine, Akihiko; Kasai, Yuichi; Masuda, Koichi; Uchida, Atsumasa

    2009-11-01

    Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

  4. Differential activation of murine herpesvirus 68- and Kaposi's sarcoma-associated herpesvirus-encoded ORF74 G protein-coupled receptors by human and murine chemokines

    NARCIS (Netherlands)

    Verzijl, D.; Fitzsimons, C.P.; Van Dijk, M.; Stewart, J.P.; Timmerman, H.; Smit, M.J.; Leurs, R.

    2004-01-01

    Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred

  5. Chest radiographic findings of tsutsugamushi disease and murine typhus in Chunchon

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Heung Chul; Han, Tae Giun; Jang, Won Ho; Hwang, Woo Chul; Park, Man Soo; Lee, Myoung Gu; Kim, Yoon Won [School of Medicine, Hallym University, Chuncheon (Korea, Republic of); Park, Choong Ki [College of Medicine, Hanyang University, Guri (Korea, Republic of)

    1995-06-15

    To evaluate the chest radiographic findings of rickettsial disease including murine typhus and tsutsugamushi disease in Chunchon. Chest radiographic films of 81 cases diagnosed as rickettsial disease(55 cases of tsutsugamushi disease, 26 cases of murine typhus) by immunofluorescence test were retrospectively analyzed. Main serotypes of Rickettsia tsutsugamushi were Gilliam and Karp. Incidence rate of tsutsugamushi disease was 2.1 times greater than that of murine typhus. Chest radiographs were abnormal in 63.6% of tsutsugamushi disease, and in 30.8% of murine typhus. Radiographic findings were Kerly's B line, reticulonodular densities, hilar enlargement, pleural effusion, and splenomegaly in both entities, but pulmonary consolidation was only found in tsutsugamushi disease. The patients with the abnormal radiographic findings were statistically well correlated with cardiomegaly ({rho} < 0.01) and azygos engorgement ({rho} < 0.05), as compared to the patients with normal radiographic findings. Radiographic findings of both murine typhus and tsutsugamushi disease were interstitial pattern. But the chest radiographs in patients with tsutsugamushi disease showed more severe pattern with higher rate of abnormality.

  6. Chest radiographic findings of tsutsugamushi disease and murine typhus in Chunchon

    International Nuclear Information System (INIS)

    Kim, Heung Chul; Han, Tae Giun; Jang, Won Ho; Hwang, Woo Chul; Park, Man Soo; Lee, Myoung Gu; Kim, Yoon Won; Park, Choong Ki

    1995-01-01

    To evaluate the chest radiographic findings of rickettsial disease including murine typhus and tsutsugamushi disease in Chunchon. Chest radiographic films of 81 cases diagnosed as rickettsial disease(55 cases of tsutsugamushi disease, 26 cases of murine typhus) by immunofluorescence test were retrospectively analyzed. Main serotypes of Rickettsia tsutsugamushi were Gilliam and Karp. Incidence rate of tsutsugamushi disease was 2.1 times greater than that of murine typhus. Chest radiographs were abnormal in 63.6% of tsutsugamushi disease, and in 30.8% of murine typhus. Radiographic findings were Kerly's B line, reticulonodular densities, hilar enlargement, pleural effusion, and splenomegaly in both entities, but pulmonary consolidation was only found in tsutsugamushi disease. The patients with the abnormal radiographic findings were statistically well correlated with cardiomegaly (ρ < 0.01) and azygos engorgement (ρ < 0.05), as compared to the patients with normal radiographic findings. Radiographic findings of both murine typhus and tsutsugamushi disease were interstitial pattern. But the chest radiographs in patients with tsutsugamushi disease showed more severe pattern with higher rate of abnormality

  7. Chest radiographic findings of tsutsugamushi disease and murine typhus in Chunchon

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Heung Chul; Han, Tae Giun; Jang, Won Ho; Hwang, Woo Chul; Park, Man Soo; Lee, Myoung Gu; Kim, Yoon Won [School of Medicine, Hallym University, Chuncheon (Korea, Republic of); Park, Choong Ki [College of Medicine, Hanyang University, Guri (Korea, Republic of)

    1995-06-15

    To evaluate the chest radiographic findings of rickettsial disease including murine typhus and tsutsugamushi disease in Chunchon. Chest radiographic films of 81 cases diagnosed as rickettsial disease(55 cases of tsutsugamushi disease, 26 cases of murine typhus) by immunofluorescence test were retrospectively analyzed. Main serotypes of Rickettsia tsutsugamushi were Gilliam and Karp. Incidence rate of tsutsugamushi disease was 2.1 times greater than that of murine typhus. Chest radiographs were abnormal in 63.6% of tsutsugamushi disease, and in 30.8% of murine typhus. Radiographic findings were Kerly's B line, reticulonodular densities, hilar enlargement, pleural effusion, and splenomegaly in both entities, but pulmonary consolidation was only found in tsutsugamushi disease. The patients with the abnormal radiographic findings were statistically well correlated with cardiomegaly ({rho} < 0.01) and azygos engorgement ({rho} < 0.05), as compared to the patients with normal radiographic findings. Radiographic findings of both murine typhus and tsutsugamushi disease were interstitial pattern. But the chest radiographs in patients with tsutsugamushi disease showed more severe pattern with higher rate of abnormality.

  8. Enhancement of tumor radioresponse by combined chemotherapy in murine hepatocarcinoma

    International Nuclear Information System (INIS)

    Seong, Jin Sil; Kim, Sung Hee; Suh, Chang Ok

    2000-01-01

    The purpose of this study was to identify drugs that can enhance radioresponse of murine hepatocarcinoma. C3H/HeJ mice bearing 8 mm tumors of murine hepatocarcinoma, HCa-l, were treated with 25 Gy radiation and one of the following drugs: 5-Fu, 150 mg/kg; adriamycin, 8 mg/kg; cisplatin, 6 mg/kg; paclitaxel, 40 mg/kg; and gemcitabine, 50 mg/kg. Tumor response to the treatment was determined by tumor growth delay assay and by enhancement factor. Apoptotic level was assessed in tissue sections. Expression of regulating molecules was analyzed by western blotting for p53, 8c1-2, Sax, Bel-XL, Bd-XS, and p21 WAF1/CIP1 . Among the drugs tested, only gemcitabine enhanced the antitumor effect of radiation, with enhancement factor of 1.6. Induction of apoptosis by a combination of gerncitabine and radiation was shown as only additive level. In analysis of radiation-induced expression of regulating molecules, the most significant change by combining gemcitabine was activation of p21 WAF1/CIP1 . Gemcitabine is the first drug showing an enhancement of radioresponse in murine hepatocarcinoma, when combined with radiation. The key element of enhancement is thought to be p21 WAF1/CIP1

  9. Enhancement of tumor radioresponse by combined chemotherapy in murine hepatocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Jin Sil; Kim, Sung Hee; Suh, Chang Ok [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of)

    2000-12-01

    The purpose of this study was to identify drugs that can enhance radioresponse of murine hepatocarcinoma. C3H/HeJ mice bearing 8 mm tumors of murine hepatocarcinoma, HCa-l, were treated with 25 Gy radiation and one of the following drugs: 5-Fu, 150 mg/kg; adriamycin, 8 mg/kg; cisplatin, 6 mg/kg; paclitaxel, 40 mg/kg; and gemcitabine, 50 mg/kg. Tumor response to the treatment was determined by tumor growth delay assay and by enhancement factor. Apoptotic level was assessed in tissue sections. Expression of regulating molecules was analyzed by western blotting for p53, 8c1-2, Sax, Bel-XL, Bd-XS, and p21{sup WAF1/CIP1}. Among the drugs tested, only gemcitabine enhanced the antitumor effect of radiation, with enhancement factor of 1.6. Induction of apoptosis by a combination of gerncitabine and radiation was shown as only additive level. In analysis of radiation-induced expression of regulating molecules, the most significant change by combining gemcitabine was activation of p21 {sup WAF1/CIP1}. Gemcitabine is the first drug showing an enhancement of radioresponse in murine hepatocarcinoma, when combined with radiation. The key element of enhancement is thought to be p21{sup WAF1/CIP1}.

  10. Relationship between the murine Ah locus and 2,3,7,8-tetrachlorodibenzo-p-dioxin hepatic metabolism, enzyme induction, and toxicity

    International Nuclear Information System (INIS)

    Shen, E.S.

    1988-01-01

    The influence of the Ah locus and hepatic microsomal enzyme induction on 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) metabolism and hepatotoxicity was investigated using C57BL/6J (C57) and DBA/2J (DBA) mice. C57 mice are more sensitive to toxic and enzyme inductive effects of 2,3,7,8-TCDD than DBA mice. Characterization of interstrain differences in hepatic enzyme induction, 2,3,7,8-TCDD metabolism, and hepatotoxicity may aid in identifying the mechanism(s) of 2,3,7,8-TCDD toxicity. The hepatic uptake and metabolism of [ 14 C]2,3,7,8-TCDD were studied using isolated hepatocytes from control and 2,3,7,8-TCDD-pretreated C57 and DBA mice. Pretreated mice were injected with 2,3,7,8-TCDD at doses that maximally induce aryl hydrocarbon hydroxylase activity or at doses that approach the LD 50 value. Despite the induction of hepatic 7-ethoxyresorufin O-deethylase activity and benzo[a]pyrene metabolism, all 2,3,7,8-TCDD pretreatment doses failed to increase the rate of [ 14 C]2,3,7,8-TCDD metabolism for both C57 and DBA mice. These results suggest that the uptake and rate of hepatic metabolism of 2,3,7,8-TCDD do not correlate with genetic differences at the murine Ah locus

  11. Ecotropic murine leukemia virus-induced fusion of murine cells

    International Nuclear Information System (INIS)

    Pinter, A.; Chen, T.; Lowy, A.; Cortez, N.G.; Silagi, S.

    1986-01-01

    Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [ 3 H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells

  12. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  13. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  14. Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

    International Nuclear Information System (INIS)

    Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

    1990-01-01

    Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

  15. Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity

    DEFF Research Database (Denmark)

    Merelo, Paz; Ram, Hathi; Caggiano, Monica Pia

    2016-01-01

    A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-...... show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets....

  16. Msx1 Homeodomain Protein Represses the αGSU and GnRH Receptor Genes During Gonadotrope Development

    Science.gov (United States)

    Xie, Huimin; Cherrington, Brian D.; Meadows, Jason D.; Witham, Emily A.

    2013-01-01

    Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at −114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program. PMID:23371388

  17. Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development.

    Science.gov (United States)

    Xie, Huimin; Cherrington, Brian D; Meadows, Jason D; Witham, Emily A; Mellon, Pamela L

    2013-03-01

    Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at -114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program.

  18. Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid–stimulated migration of murine osteosarcoma LM8 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kubohara, Yuzuru, E-mail: ykuboha@juntendo.ac.jp [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Health Science, Juntendo University Graduate School of Health and Sports Science, Inzai 270-1695 (Japan); Komachi, Mayumi [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Homma, Yoshimi [Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295 (Japan); Kikuchi, Haruhisa; Oshima, Yoshiteru [Laboratory of Natural Product Chemistry, Tohoku University Graduate School of Pharmaceutical Sciences, Aoba-yama, Aoba-ku, Sendai 980-8578 (Japan)

    2015-08-07

    Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), −2, and −3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5–20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC{sub 50} values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC{sub 50} values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. - Highlights: • LPA induces cell migration (invasion) in murine osteosarcoma LM8 cells. • DIFs are novel lead anti-tumor agents found in Dictyostelium discoideum. • We examined the effects of DIF derivatives on LPA-induced LM8 cell migration in vitro. • Some of the DIF derivatives inhibited LPA-induced LM8 cell migration.

  19. Adherence of murine lymphocytes to high endothelial venules in vitro and its radiation effect

    Energy Technology Data Exchange (ETDEWEB)

    Lixin, Liu; Zijun, Mao; Zhiwei, Yin [Suzhou Medical Coll., JS (China). Dept. of Pathophysiology

    1991-02-01

    Using the assay of specific adhesion of lymphocytes to high endothelial venules (HEV) on cryostat sections of mesenteric lymph nodes (MLN), the effects of different doses (0, 1, 2, 4, 8 Gy) of {sup 60}Co {gamma}-ray irradiation of murine MLN lymphocytes in vitro on adhesion to normal HEV was observed. The results showed that in the irradiated murine MLN lymphocytes the ability to adhere to HEV of normal MLN was reduced. Statistical significance was revealed at 2, 4, 8 Gy irradiations. This results suggests that irradiation can inhibit the specific recognition and adhesion of lymphocytes to HEV to a certain extent.

  20. Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach.

    Science.gov (United States)

    Amato, Anastasia; Lucas, Xavier; Bortoluzzi, Alessio; Wright, David; Ciulli, Alessio

    2018-04-20

    Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.

  1. Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

    Science.gov (United States)

    Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

    2016-03-01

    Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = culture. Human embryo studies are needed. © The Author(s) 2015.

  2. Prep1, A Homeodomain Transcription Factor Involved in Glucose and Lipid Metabolism

    Directory of Open Access Journals (Sweden)

    Francesco Oriente

    2018-06-01

    Full Text Available The three-amino acid loop extension (TALE homeodomain proteins are a family of transcription factor including the mammalian Pbx, MEIS and Prep proteins. TALE proteins can bind other transcription factors such as Pdx-1 and play an important role in the regulation of glucose metabolism. Experiments performed in mutant mice have shown that while the single Pbx1 or Pdx-1 knockout mice feature pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia, the trans-heterozygous Pbx1+/−Pdx1+/− mice develop age-dependent overt diabetes mellitus. In contrast, Prep1 plays a different role with respect to these proteins. Indeed, Prep1 hypomorphic mice, expressing low levels of protein, feature pancreatic islet hypoplasia accompanied by hypoinsulinemia similar to Pbx1 or Pdx1. Nevertheless, these animals show increased insulin sensitivity in skeletal muscle, liver and adipose tissue accompanied by protection from streptozotocin-induced diabetes. In addition, Prep1 hypomorphic mice feature reduced triglyceride synthesis and do not develop steatohepatitis after a methionine and coline deficient diet. In this review we have underlined how important metabolic functions are controlled by TALE proteins, in particular by Prep1, leading to hypothesis that its suppression might represent beneficial effect in the care of metabolic diseases.

  3. A Petunia Homeodomain-Leucine Zipper Protein, PhHD-Zip, Plays an Important Role in Flower Senescence

    Science.gov (United States)

    Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S.; Jiang, Cai-Zhong

    2014-01-01

    Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence. PMID:24551088

  4. A Petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence.

    Science.gov (United States)

    Chang, Xiaoxiao; Donnelly, Linda; Sun, Daoyang; Rao, Jingping; Reid, Michael S; Jiang, Cai-Zhong

    2014-01-01

    Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO), and ABA (NCED) biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA) and abiotic stresses (dehydration, NaCl and cold). Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence.

  5. A Petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence.

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Chang

    Full Text Available Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO, and ABA (NCED biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29 was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA and abiotic stresses (dehydration, NaCl and cold. Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence.

  6. Recurrent DGCR8, DROSHA, and SIX Homeodomain Mutations in Favorable Histology Wilms Tumors

    NARCIS (Netherlands)

    A.L. Walz (Amy L.); A.H.A.G. Ooms (Ariadne ); S. Gadd (Samantha); D.S. Gerhard (Daniela S.); M.A. Smith (Malcolm A.); J.M. GuidryAuvil (Jamie M.); D. Meerzaman (Daoud); Q.-R. Chen (Qing-Rong); C. Hsu (ChihHao); C. Yan (Chunhua); C. Nguyen (Cu); Y. Hu (Ying); R. Bowlby (Reanne); D. Brooks (Denise); Y. Ma (Yussanne); A.A. Mungall (Andrew J.); R.A. Moore (Richard A.); J. Schein (Jacqueline); M.A. Marra (Marco A.); V. Huff (Vicki); J.S. Dome (Jeffrey); Y.-Y. Chi (Yueh-Yun); C.G. Mullighan (Charles); J. Ma (Jing); D.A. Wheeler (David A.); O.A. Hampton (Oliver A.); N. Jafari (Nadereh); N. Ross (Nicole); J.M. Gastier-Foster (Julie); E.J. Perlman (Elizabeth J.)

    2015-01-01

    textabstractWe report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/. 2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates

  7. The homeodomain protein ladybird late regulates synthesis of milk proteins during pregnancy in the tsetse fly (Glossina morsitans.

    Directory of Open Access Journals (Sweden)

    Geoffrey M Attardo

    2014-04-01

    Full Text Available Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina, the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1 by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This

  8. The conserved His8 of the Moloney murine leukemia virus Env SU subunit directs the activity of the SU-TM disulphide bond isomerase

    International Nuclear Information System (INIS)

    Li Kejun; Zhang, Shujing; Kronqvist, Malin; Ekstroem, Maria; Wallin, Michael; Garoff, Henrik

    2007-01-01

    Murine leukemia virus (MLV) fusion is controlled by isomerization of the disulphide bond between the receptor-binding surface (SU) and fusion-active transmembrane subunits of the Env-complex. The bond is in SU linked to a CXXC motif. This carries a free thiol that upon receptor binding can be activated (ionized) to attack the disulphide and rearrange it into a disulphide isomer within the motif. To find out whether His8 in the conserved SPHQ sequence of Env directs thiol activation, we analyzed its ionization in MLV vectors with wtEnv and Env with His8 deleted or substituted for Tyr or Arg, which partially or completely arrests fusion. The ionization was monitored by following the pH effect on isomerization in vitro by Ca 2+ depletion or in vivo by receptor binding. We found that wtEnv isomerized optimally at slightly basic pH whereas the partially active mutant required higher and the inactive mutants still higher pH. This suggests that His8 directs the ionization of the CXXC thiol

  9. Imported rickettsioses : think of murine typhus

    NARCIS (Netherlands)

    van der Kleij, FGH; Gansevoort, RT; Kreeftenberg, HG

    Murine typhus is a disease still prevalent in many parts of the world. Because the incidence in the US and Europe has declined rapidly, physicians in these continents have become unfamiliar with the clinical picture. Murine typhus is associated with significant morbidity and fatalities do occur,

  10. Biomechanical Properties of Murine Meniscus Surface via AFM-based Nanoindentation

    Science.gov (United States)

    Li, Qing; Doyran, Basak; Gamer, Laura W.; Lu, X. Lucas; Qin, Ling; Ortiz, Christine; Grodzinsky, Alan J.; Rosen, Vicki; Han, Lin

    2015-01-01

    This study aimed to quantify the biomechanical properties of murine meniscus surface. Atomic force microscopy (AFM)-based nanoindentation was performed on the central region, proximal side of menisci from 6- to 24-week old male C57BL/6 mice using microspherical tips (Rtip ≈ 5 μm) in PBS. A unique, linear correlation between indentation depth, D, and response force, F, was found on menisci from all age groups. This non-Hertzian behavior is likely due to the dominance of tensile resistance by the collagen fibril bundles on meniscus surface that are mostly aligned along the circumferential direction observed on 12-week old menisci. The indentation resistance was calculated as both the effective stiffness, Sind = dF/dD, and the effective modulus, Eind, via the isotropic Hertz model. Values of Sind and Eind were found to depend on indentation rate, suggesting the existence of poro-viscoelasticity. These values do not significantly vary with anatomical sites, lateral versus medial compartments, or mouse age. In addition, Eind of meniscus surface (e.g., 6.1 ± 0.8 MPa for 12 weeks of age, mean ± SEM, n = 13) was found to be significantly higher than those of meniscus surfaces in other species, and of murine articular cartilage surface (1.4 ± 0.1 MPa, n = 6). In summary, these results provided the first direct mechanical knowledge of murine knee meniscus tissues. We expect this understanding to serve as a mechanics-based benchmark for further probing the developmental biology and osteoarthritis symptoms of meniscus in various murine models. PMID:25817332

  11. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV.

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    Julie A Law

    2011-07-01

    Full Text Available DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2, is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs through a pathway termed RNA-directed DNA methylation (RdDM. Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV. However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1, the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2, CLASSY1 (CLSY1, and RNA-directed DNA methylation 4 (RDM4, suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1, was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.

  12. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    International Nuclear Information System (INIS)

    Abelson, H.T.; Gorka, C.

    1983-01-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no [3H]hypoxanthine uptake into tumor or tissue culture cells, no conversion of [3H]hypoxanthine to [3H]IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol

  13. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    International Nuclear Information System (INIS)

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

    1991-01-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells

  14. Studies on murine plasmocytoma treatment with mistletoe lectin I

    International Nuclear Information System (INIS)

    Raabe, F.; Storch, H.

    1987-01-01

    Mistletoe lectin I was tested in vivo and in vitro for its cytotoxic activity against murine plasmacytoma cells P3/X63-Ag8. As a result of this treatment, 30 to 60% of the BALB/c mice developed complete tumor regressions. 83% of the mice treated with mistletoe lectin I were resistant to viable tumor cell challenge after 100 days. The cytotoxic activity in vitro tested by 3 H-thymidine incorporation into P3/X63-Ag8 cells was very high. The rate was markedly reduced at concentrations up to 0.07 ng/ml. (author)

  15. Comparative transcriptome analyses indicate molecular homology of zebrafish swimbladder and mammalian lung.

    Directory of Open Access Journals (Sweden)

    Weiling Zheng

    Full Text Available The fish swimbladder is a unique organ in vertebrate evolution and it functions for regulating buoyancy in most teleost species. It has long been postulated as a homolog of the tetrapod lung, but the molecular evidence is scarce. In order to understand the molecular function of swimbladder as well as its relationship with lungs in tetrapods, transcriptomic analyses of zebrafish swimbladder were carried out by RNA-seq. Gene ontology classification showed that genes in cytoskeleton and endoplasmic reticulum were enriched in the swimbladder. Further analyses depicted gene sets and pathways closely related to cytoskeleton constitution and regulation, cell adhesion, and extracellular matrix. Several prominent transcription factor genes in the swimbladder including hoxc4a, hoxc6a, hoxc8a and foxf1 were identified and their expressions in developing swimbladder during embryogenesis were confirmed. By comparison of enriched transcripts in the swimbladder with those in human and mouse lungs, we established the resemblance of transcriptome of the zebrafish swimbladder and mammalian lungs. Based on the transcriptomic data of zebrafish swimbladder, the predominant functions of swimbladder are in its epithelial and muscular tissues. Our comparative analyses also provide molecular evidence of the relatedness of the fish swimbladder and mammalian lung.

  16. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes Internalin A for enhanced infectivity in the murine oral infection model

    LENUS (Irish Health Repository)

    Monk, Ian R

    2010-12-13

    Abstract Background Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human

  17. Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

    Directory of Open Access Journals (Sweden)

    Hill Colin

    2010-12-01

    Full Text Available Abstract Background Internalin A (InlA is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26, multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlAm*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlAm* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced

  18. Biological markers as predictors of radiosensitivity in syngeneic murine tumors

    International Nuclear Information System (INIS)

    Chang, Sei Kyung; Shin, Hyun Soo; Seong, Jin Sil; Kim, Sung Hee

    2006-01-01

    We investigated whether a relationship exists between tumor control dose 50 (TCD 50 ) or tumor growth delay (TGD) and radiation induced apoptosis (RIA) in syngeneic murine tumors. Also we investigated the biological markers that can predict radiosensitivity in murine tumor system through analysis of relationship between TCD 50 , TGD, RIA and constitutive expression levels of the genetic products regulating RIA. Syngeneic murine tumors such as ovarian adenocarcinoma, mammary carcinoma, squamous cell carcinoma, fibrosarcoma, hepatocarcinoma were used in this study. C3H/HeJ mice were bred and maintained in our specific pathogen free mouse colony and were 8 ∼ 12 weeks old when used for the experiments. The tumors, growing in the right hind legs of mice, were analyzed for TCD 50 , TGD, and RIA at 8 mm in diameter. The tumors were also analyzed for the constitutive expression levels of p53, p21 WAF1/CIP1 , BAX, Bcl-2, Bcl-x L , Bcl-x S , and p34. Correlation analysis was performed whether the level of RIA were correlated with TCD 50 or TGD, and the constitutive expression levels of genetic products regulating RIA were correlated with TCD 50 , TGD, RIA. The level of RIA showed a significant positive correlation (R = 0.922, ρ = 0.026) with TGD, and showed a trend to correlation (R = -0.848), marginally significant correlation with TCD 50 (ρ = 0.070). It indicates that tumors that respond to radiation with high percentage of apoptosis were more radiosensitive. The constitutive expression levels of p21 WAF1/CIP1 and p34 showed a significant correlation either with TCD 50 (R = 0.893, ρ = 0.041 and R = 0.904, ρ = 0.035) or with TGD (R = -0.922, ρ 0.026 and R = -0.890, ρ = 0.043). The tumors with high constitutive expression levels of p21 WAF1/CIP1 or p34 were less radiosensitive than those with low expression. Radiosensitivity may be predicted with the level of RIA in murine tumors. The constitutive expression levels of p21 WAF1/CIP1 or p34 can be used as biological

  19. Biological markers as predictors of radiosensitivity in syngeneic murine tumors

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Sei Kyung; Shin, Hyun Soo [Bundang CHA General Hospital, Seongnam (Korea, Republic of); Seong, Jin Sil; Kim, Sung Hee [Yonsei Cancer Center, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2006-06-15

    We investigated whether a relationship exists between tumor control dose 50 (TCD{sub 50}) or tumor growth delay (TGD) and radiation induced apoptosis (RIA) in syngeneic murine tumors. Also we investigated the biological markers that can predict radiosensitivity in murine tumor system through analysis of relationship between TCD{sub 50}, TGD, RIA and constitutive expression levels of the genetic products regulating RIA. Syngeneic murine tumors such as ovarian adenocarcinoma, mammary carcinoma, squamous cell carcinoma, fibrosarcoma, hepatocarcinoma were used in this study. C3H/HeJ mice were bred and maintained in our specific pathogen free mouse colony and were 8 {approx} 12 weeks old when used for the experiments. The tumors, growing in the right hind legs of mice, were analyzed for TCD{sub 50}, TGD, and RIA at 8 mm in diameter. The tumors were also analyzed for the constitutive expression levels of p53, p21{sup WAF1/CIP1}, BAX, Bcl-2, Bcl-x{sub L}, Bcl-x{sub S}, and p34. Correlation analysis was performed whether the level of RIA were correlated with TCD{sub 50} or TGD, and the constitutive expression levels of genetic products regulating RIA were correlated with TCD{sub 50}, TGD, RIA. The level of RIA showed a significant positive correlation (R = 0.922, {rho} = 0.026) with TGD, and showed a trend to correlation (R = -0.848), marginally significant correlation with TCD{sub 50} ({rho} = 0.070). It indicates that tumors that respond to radiation with high percentage of apoptosis were more radiosensitive. The constitutive expression levels of p21{sup WAF1/CIP1} and p34 showed a significant correlation either with TCD{sub 50} (R = 0.893, {rho} = 0.041 and R = 0.904, {rho} = 0.035) or with TGD (R = -0.922, {rho} 0.026 and R = -0.890, {rho} = 0.043). The tumors with high constitutive expression levels of p21{sup WAF1/CIP1} or p34 were less radiosensitive than those with low expression. Radiosensitivity may be predicted with the level of RIA in murine tumors. The

  20. Characterisation of Neutropenia-Associated Neutrophil Elastase Mutations in a Murine Differentiation Model In Vitro and In Vivo.

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    Michael Wiesmeier

    Full Text Available Severe congenital neutropenia (SCN is characterised by a differentiation block in the bone marrow and low neutrophil numbers in the peripheral blood, which correlates with increased risk of bacterial infections. Several underlying gene defects have been identified in SCN patients. Mutations in the neutrophil elastase (ELANE gene are frequently found in SCN and cyclic neutropenia. Both mislocalization and misfolding of mutant neutrophil elastase protein resulting in ER stress and subsequent induction of the unfolded protein response (UPR have been proposed to be responsible for neutrophil survival and maturation defects. However, the detailed molecular mechanisms still remain unclear, in part due to the lack of appropriate in vitro and in vivo models. Here we used a system of neutrophil differentiation from immortalised progenitor lines by conditional expression of Hoxb8, permitting the generation of mature near-primary neutrophils in vitro and in vivo. NE-deficient Hoxb8 progenitors were reconstituted with murine and human forms of typical NE mutants representative of SCN and cyclic neutropenia, and differentiation of the cells was analysed in vitro and in vivo. ER stress induction by NE mutations could be recapitulated during neutrophil differentiation in all NE mutant-reconstituted Hoxb8 cells. Despite ER stress induction, no change in survival, maturation or function of differentiating cells expressing either murine or human NE mutants was observed. Further analysis of in vivo differentiation of Hoxb8 cells in a murine model of adoptive transfer did not reveal any defects in survival or differentiation in the mouse. Although the Hoxb8 system has been found to be useful for dissection of defects in neutrophil development, our findings indicate that the use of murine systems for analysis of NE-mutation-associated pathogenesis is complicated by differences between humans and mice in the physiology of granulopoiesis, which may go beyond possible

  1. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans.

    Science.gov (United States)

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-21

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development.

  2. [The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

    Science.gov (United States)

    Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

    2003-09-01

    To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

  3. Decay-accelerating factor 1 deficiency exacerbates Trypanosoma cruzi-induced murine chronic myositis.

    Science.gov (United States)

    Solana, María E; Ferrer, María F; Novoa, María Mercedes; Song, Wen-Chao; Gómez, Ricardo M

    2012-10-01

    Murine infection with Trypanosoma cruzi (Tc) has been used to study the role of T-cells in the pathogenesis of human inflammatory idiopathic myositis. Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance murine T-cell responses and autoimmunity. To determine whether Daf1 deficiency can exacerbate Tc-induced myositis, C57BL/6 DAF(+/+) and DAF(-/-) mice were inoculated with 5 × 10(4) trypomastigotes, and their morbidity, parasitemia, parasite burden, histopathology, and T-cell expansion were studied in the acute and chronic stages. DAF(-/-) mice had lower parasitemia and parasite burden but higher morbidity, muscle histopathology, and increased number of CD44(+) (activated/memory phenotype) splenic CD4(+) and CD8(+) T-cells. An enhanced CD8(+) T-cell immune-specific response may explain the lower parasitemia and parasite burden levels and the increase in histopathological lesions. We propose that Tc-inoculated DAF(-/-) mice are a useful model to study T-cell mediated immunity in skeletal muscle tissues. Copyright © 2012 Wiley Periodicals, Inc.

  4. Identification of murine T-cell epitopes in Ebola virus nucleoprotein

    International Nuclear Information System (INIS)

    Simmons, Graham; Lee, Anee; Rennekamp, Andrew J.; Fan Xin; Bates, Paul; Shen Hao

    2004-01-01

    CD8 T cells play an important role in controlling Ebola infection and in mediating vaccine-induced protective immunity, yet little is known about antigenic targets in Ebola that are recognized by CD8 T cells. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding Ebola nucleoprotein (NP). CD8 T-cell responses were mapped to a H-2 d -restricted epitope (NP279-288) and two H-2 b -restricted epitopes (NP44-52 and NP288-296). The identification of these epitopes will facilitate studies of immune correlates of protection and the evaluation of vaccine strategies in murine models of Ebola infection

  5. Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

    1986-01-01

    Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN - ) for murine Cu-Zn-SOD was determined to be 6.8 x 10 -6 M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied

  6. The homeodomain derived peptide Penetratin induces curvature of fluid membrane domains.

    Directory of Open Access Journals (Sweden)

    Antonin Lamazière

    Full Text Available BACKGROUND: Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS: Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like membrane domains. CONCLUSIONS/SIGNIFICANCE: The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we

  7. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    International Nuclear Information System (INIS)

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45 + hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45 low c-Kit + cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo

  8. Determination of h2JNN and h1JHN coupling constants across Watson-Crick base pairs in the Antennapedia homeodomain-DNA complex using TROSY

    International Nuclear Information System (INIS)

    Pervushin, Konstantin; Fernandez, Cesar; Riek, Roland; Ono, Akira; Kainosho, Masatsune; Wuethrich, Kurt

    2000-01-01

    This paper describes NMR measurements of 15 N- 15 N and 1 H- 15 N scalar couplings across hydrogen bonds in Watson-Crick base pairs, h2 J NN and h1 J HN , in a 17 kDa Antennapedia homeodomain-DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of h1 J HN couplings in larger molecules. The h2 J NN and h1 J HN couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds

  9. Anatomy and Histology of the Human and Murine Prostate.

    Science.gov (United States)

    Ittmann, Michael

    2018-05-01

    The human and murine prostate glands have similar functional roles in the generation of seminal fluid to assist in reproduction. There are significant differences in the anatomy and histology of murine and human prostate and knowledge of the normal anatomy and histology of the murine prostate is essential to interpreting changes in genetically engineered mouse models. In this review, the normal anatomy and histology of both human and mouse prostate will be described. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

  10. Genome-wide DNA binding pattern of the homeodomain transcription factor Sine oculis (So in the developing eye of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Barbara Jusiak

    2014-12-01

    Full Text Available The eye of the fruit fly Drosophila melanogaster provides a highly tractable genetic model system for the study of animal development, and many genes that regulate Drosophila eye formation have homologs implicated in human development and disease. Among these is the homeobox gene sine oculis (so, which encodes a homeodomain transcription factor (TF that is both necessary for eye development and sufficient to reprogram a subset of cells outside the normal eye field toward an eye fate. We have performed a genome-wide analysis of So binding to DNA prepared from developing Drosophila eye tissue in order to identify candidate direct targets of So-mediated transcriptional regulation, as described in our recent article [20]. The data are available from NCBI Gene Expression Omnibus (GEO with the accession number GSE52943. Here we describe the methods, data analysis, and quality control of our So ChIP-seq dataset.

  11. Tenascin-C Prevents Articular Cartilage Degeneration in Murine Osteoarthritis Models.

    Science.gov (United States)

    Matsui, Yuriyo; Hasegawa, Masahiro; Iino, Takahiro; Imanaka-Yoshida, Kyoko; Yoshida, Toshimichi; Sudo, Akihiro

    2018-01-01

    Objective The objective of this study was to determine whether intra-articular injections of tenascin-C (TNC) could prevent cartilage damage in murine models of osteoarthritis (OA). Design Fluorescently labeled TNC was injected into knee joints and its distribution was examined at 1 day, 4 days, 1 week, 2 weeks, and 4 weeks postinjection. To investigate the effects of TNC on cartilage degeneration after surgery to knee joints, articular spaces were filled with 100 μg/mL (group I), 10 μg/mL (group II) of TNC solution, or control (group III). TNC solution of 10 μg/mL was additionally injected twice after 3 weeks (group IV) or weekly after 1 week, 2 weeks, and 3 weeks (group V). Joint tissues were histologically assessed using the Mankin score and the modified Chambers system at 2 to 8 weeks after surgery. Results Exogenous TNC was maintained in the cartilage and synovium for 1 week after administration. Histological scores in groups I and II were better than scores in group III at 4 and 6 weeks, but progressive cartilage damage was seen in all groups 8 weeks postoperatively. Sequential TNC injections (groups IV and V) showed significantly better Mankin score than single injection (group II) at 8 weeks. Conclusion TNC administered exogenously remained in the cartilage of knee joints for 1 week, and could decelerate articular cartilage degeneration in murine models of OA. We also showed that sequential administration of TNC was more effective than a single injection. TNC could be an important molecule for prevention of articular cartilage damage.

  12. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  13. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    International Nuclear Information System (INIS)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J.

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells

  14. Genome-Wide Identification, Evolution and Expression Analysis of the Grape (Vitis vinifera L. Zinc Finger-Homeodomain Gene Family

    Directory of Open Access Journals (Sweden)

    Hao Wang

    2014-04-01

    Full Text Available Plant zinc finger-homeodomain (ZHD genes encode a family of transcription factors that have been demonstrated to play an important role in the regulation of plant growth and development. In this study, we identified a total of 13 ZHD genes (VvZHD in the grape genome that were further classified into at least seven groups. Genome synteny analysis revealed that a number of VvZHD genes were present in the corresponding syntenic blocks of Arabidopsis, indicating that they arose before the divergence of these two species. Gene expression analysis showed that the identified VvZHD genes displayed distinct spatiotemporal expression patterns, and were differentially regulated under various stress conditions and hormone treatments, suggesting that the grape VvZHDs might be also involved in plant response to a variety of biotic and abiotic insults. Our work provides insightful information and knowledge about the ZHD genes in grape, which provides a framework for further characterization of their roles in regulation of stress tolerance as well as other aspects of grape productivity.

  15. A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

    Directory of Open Access Journals (Sweden)

    Mi Zhao

    Full Text Available We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

  16. A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

    Science.gov (United States)

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

  17. A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

    Science.gov (United States)

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

  18. A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

    OpenAIRE

    Paquette, Y; Doyon, L; Laperrière, A; Hanna, Z; Ball, J; Sekaly, R P; Jolicoeur, P

    1992-01-01

    The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to...

  19. Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents

    NARCIS (Netherlands)

    Hatting, M.; Zhao, G.; Schumacher, F.; Sellge, G.; Masaoudi, Al M.; Gaßler, N.; Boekschoten, M.V.; Müller, M.R.; Liedtke, C.; Cubero, F.J.; Trautwein, C.

    2013-01-01

    In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death-receptor-mediated apoptosis activity and therefore its modulation might be critical for

  20. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  1. Interleukin 37 limits monosodium urate crystal-induced innate immune responses in human and murine models of gout.

    Science.gov (United States)

    Liu, Lei; Xue, Yu; Zhu, Yingfeng; Xuan, Dandan; Yang, Xue; Liang, Minrui; Wang, Juan; Zhu, Xiaoxia; Zhang, Jiong; Zou, Hejian

    2016-11-18

    Interleukin (IL)-37 has emerged as a fundamental inhibitor of innate immunity. Acute gout is a self-limiting inflammatory response to monosodium urate (MSU) crystals. In the current study, we assessed the preventive and therapeutic effect of recombinant human IL-37 (rhIL-37) in human and murine gout models. We investigated the expression of IL-37 in patients with active and inactive gouty arthritis and assessed the effect of rhIL-37 in human and murine gout models: a human monocyte cell line (THP-1) and human synovial cells (containing macrophage-like and fibroblast-like synoviocytes) exposed to MSU crystals, a peritoneal murine model of gout and a murine gouty arthritis model. After inhibition of Mer receptor tyrosine kinase (Mertk), levels of IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL-2) were detected by ELISA and expression of mammalian homologs of the drosophila Mad gene 3 (Smad), suppressor of cytokine signaling 3 (SOCS3), NACHT-LRR-PYD-containing protein 3 (NLRP3), and IL-8R of THP-1 were assessed by qPCR and western blot to explore the molecular mechanisms. Our studies strongly indicated that rhIL-37 played a potent immunosuppressive role in the pathogenesis of experimental gout models both in vitro and in vivo, by downregulating proinflammatory cytokines and chemokines, markedly reducing neutrophil and monocyte recruitment, and mitigating pathological joint inflammation. In our studies, rhIL-37 suppressed MSU-induced innate immune responses by enhancing expression of Smad3 and IL-1R8 to trigger multiple intracellular switches to block inflammation, including inhibition of NLRP3 and activation of SOCS3. Mertk signaling participated in rhIL-37 inhibitory pathways in gout models. By inhibition of Mertk, the anti-inflammatory effect of rhIL-37 was partly abrogated, and IL-1R8, Smad3 and S​OCS3 expression were suppressed, whereas NLRP3 expression was reactivated. Our studies reveal that IL-37 limits runaway inflammation initiated by MSU crystal

  2. Murine Models of Gastric Corpus PreneoplasiaSummary

    Directory of Open Access Journals (Sweden)

    Christine P. Petersen

    2017-01-01

    Full Text Available Intestinal-type gastric adenocarcinoma evolves in a field of pre-existing metaplasia. Over the past 20 years, a number of murine models have been developed to address aspects of the physiology and pathophysiology of metaplasia induction. Although none of these models has achieved true recapitulation of the induction of adenocarcinoma, they have led to important insights into the factors that influence the induction and progression of metaplasia. Here, we review the pathologic definitions relevant to alterations in gastric corpus lineages and classification of metaplasia by specific lineage markers. In addition, we review present murine models of the induction and progression of spasmolytic polypeptide (TFF2–expressing metaplasia, the predominant metaplastic lineage observed in murine models. These models provide a basis for the development of a broader understanding of the physiological and pathophysiological roles of metaplasia in the stomach. Keywords: SPEM, Intestinal Metaplasia, Gastric Cancer, TFF2, Chief Cell, Hyperplasia

  3. Murine Typhus: An Important Consideration for the Nonspecific Febrile Illness

    Directory of Open Access Journals (Sweden)

    Gurjot Basra

    2012-01-01

    Full Text Available Murine typhus is a widely distributed flea-borne infection caused by Rickettsia typhi. Symptoms of murine typhus are nonspecific and mimic a variety of other infectious diseases. We herein report a case of murine typhus in an area where the broad use of DDT in the mid-20th century has now made it a rare disease. The patient described presented with headache, fever, and a faint macular rash. Initial laboratory studies revealed a slight transaminase elevation. Further questioning revealed exposure to opossums, prompting the consideration of murine typhus as a diagnosis. Although typhus group antibodies were not present during the patient’s acute illness, empiric therapy with doxycycline was initiated, and the patient defervesced. One month after convalescence, the patient returned to clinic with serum that contained typhus group antibodies with an IgG titer of 1 : 1024. Murine typhus is an important consideration during the workup of a patient with a nonspecific febrile illness. Exposure to reservoir hosts and the flea vector place humans at risk for this disease. Clinician recognition of this entity is required for diagnosis and effective therapy.

  4. Characterization of a Novel Murine Model to Study Zika Virus.

    Science.gov (United States)

    Rossi, Shannan L; Tesh, Robert B; Azar, Sasha R; Muruato, Antonio E; Hanley, Kathryn A; Auguste, Albert J; Langsjoen, Rose M; Paessler, Slobodan; Vasilakis, Nikos; Weaver, Scott C

    2016-06-01

    The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain-Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the interferon (IFN) alpha receptor (A129 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A129 mice, viremia peaked at ∼10(7) plaque-forming units/mL by day 2 postinfection (PI) and reached high titers in the spleen by day 1. ZIKV was detected in the brain on day 3 PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (3 weeks) succumbed to illness by day 7 PI. Older mice (11 weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG129 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A129 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines. © The American Society of Tropical Medicine and Hygiene.

  5. Topical Apigenin Alleviates Cutaneous Inflammation in Murine Models

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    Mao-Qiang Man

    2012-01-01

    Full Text Available Herbal medicines have been used in preventing and treating skin disorders for centuries. It has been demonstrated that systemic administration of chrysanthemum extract exhibits anti-inflammatory properties. However, whether topical applications of apigenin, a constituent of chrysanthemum extract, influence cutaneous inflammation is still unclear. In the present study, we first tested whether topical applications of apigenin alleviate cutaneous inflammation in murine models of acute dermatitis. The murine models of acute allergic contact dermatitis and acute irritant contact dermatitis were established by topical application of oxazolone and phorbol 12-myristate 13-acetate (TPA, respectively. Inflammation was assessed in both dermatitis models by measuring ear thickness. Additionally, the effect of apigenin on stratum corneum function in a murine subacute allergic contact dermatitis model was assessed with an MPA5 physiology monitor. Our results demonstrate that topical applications of apigenin exhibit therapeutic effects in both acute irritant contact dermatitis and allergic contact dermatitis models. Moreover, in comparison with the vehicle treatment, topical apigenin treatment significantly reduced transepidermal water loss, lowered skin surface pH, and increased stratum corneum hydration in a subacute murine allergic contact dermatitis model. Together, these results suggest that topical application of apigenin could provide an alternative regimen for the treatment of dermatitis.

  6. Non- invasive in vivo analysis of a murine aortic graft using high resolution ultrasound microimaging

    International Nuclear Information System (INIS)

    Rowinska, Zuzanna; Zander, Simone; Zernecke, Alma; Jacobs, Michael; Langer, Stephan; Weber, Christian; Merx, Marc W.; Koeppel, Thomas A.

    2012-01-01

    Introduction: As yet, murine aortic grafts have merely been monitored histopathologically. The aim of our study was to examine how these grafts can be monitored in vivo and non-invasively by using high-resolution ultrasound microimaging to evaluate function and morphology. A further aim was to prove if this in vivo monitoring can be correlated to immunohistological data that indicates graft integrity. Methods: Murine infrarenal aortic isografts were orthotopically transplanted into 14 female mice (C57BL/6-Background) whereas a group of sham-operated animals (n = 10) served as controls. To assess the graft morphology and hemodynamics, we examined the mice over a post-operative period of 8 weeks with a sophisticated ultrasound system (Vevo 770, Visual Sonics). Results: The non-invasive graft monitoring was feasible in all transplanted mice. We could demonstrate a regular post-transplant graft function and morphology, such as anterior/posterior wall displacement and wall thickness. Mild alterations of anterior wall motion dynamics could only be observed at the site of distal graft anastomosis (8 weeks after grafting (transplant vs. sham mice: 0.02 mm ± 0.01 vs. 0.03 mm ± 0.01, p < 0.05). However, the integrity of the entire graft wall could be confirmed by histopathological evaluation of the grafts. Conclusions: With regard to graft patency, function and morphology, high resolution ultrasound microimaging has proven to be a valuable tool for longitudinal, non-invasive, in vivo graft monitoring in this murine aortic transplantation model. Consequently, this experimental animal model provides an excellent basis for molecular and pharmacological studies using genetically engineered mice.

  7. Analysis of cardiomyocyte movement in the developing murine heart

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Hisayuki [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Tabata, Hidenori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Nakajima, Kazunori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Sakaue-Sawano, Asako; Miyawaki, Atsushi [Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Fukuda, Keiichi [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan)

    2015-09-04

    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  8. DMPD: The actions of bacterial DNA on murine macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10534106 The actions of bacterial DNA on murine macrophages. Sester DP, Stacey KJ, ... Show The actions of bacterial DNA on murine macrophages. PubmedID 10534106 Title The actions of bacterial DNA on murine macrophage

  9. Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

    International Nuclear Information System (INIS)

    Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Sang Ki; Lee, Sang Do; Park, Jin Bong; Chang, Seok Jong; Jeon, Byeong Hwa

    2011-01-01

    Highlights: → We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. → The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. → HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. → Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 o C was greater than in 4 o C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-α-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.

  10. Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

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    D'Orazi Gabriella

    2008-07-01

    Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

  11. Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

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    Jessica Mühlhaus

    2014-11-01

    Full Text Available The thyroid hormone derivative 3-iodothyronamine (3-T1AM exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1 agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8. By RT-qPCR and locked-nucleic-acid (LNA in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs.

  12. CD4+ and CD8+ T cells can act separately in tumour rejection after immunization with murine pneumotropic virus chimeric Her2/neu virus-like particles.

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    Kalle Andreasson

    Full Text Available BACKGROUND: Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained. METHODOLOGY AND RESULTS: During the effector phase in BALB/c mice, only depletion of CD4+ and CD8+ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4+, CD8+ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4+ and CD8+ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4-/-CD8-/- and CD4-/-, but not in CD8-/- mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4+, only CD8+, or CD4+ and CD8+ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNgamma response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge. CONCLUSION: Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.

  13. CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors

    International Nuclear Information System (INIS)

    Mason, Kathryn A.; Neal, Robert; Hunter, Nancy; Ariga, Hisanori; Ang, Kian; Milas, Luka

    2006-01-01

    Background and purpose: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074-7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361-9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). Materials and methods: CpG ODN 1826 (100 μg) was given sc three times: when leg tumors were 6 mm, when they grew to 8 mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10 Gy) were given when tumors were 8 mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. Results: Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Conclusion: Overall results show that CpG ODN 1826 can markedly improve tumor response

  14. The kin17 Protein in Murine Melanoma Cells

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    Anelise C. Ramos

    2015-11-01

    Full Text Available kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

  15. Preclinical Murine Models for Lung Cancer: Clinical Trial Applications

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    Amelia Kellar

    2015-01-01

    Full Text Available Murine models for the study of lung cancer have historically been the backbone of preliminary preclinical data to support early human clinical trials. However, the availability of multiple experimental systems leads to debate concerning which model, if any, is best suited for a particular therapeutic strategy. It is imperative that these models accurately predict clinical benefit of therapy. This review provides an overview of the current murine models used to study lung cancer and the advantages and limitations of each model, as well as a retrospective evaluation of the uses of each model with respect to accuracy in predicting clinical benefit of therapy. A better understanding of murine models and their uses, as well as their limitations may aid future research concerning the development and implementation of new targeted therapies and chemotherapeutic agents for lung cancer.

  16. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  17. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  18. Effects of trichostatins on differentiation of murine erythroleukemia cells

    International Nuclear Information System (INIS)

    Yoshida, M.; Nomura, S.; Beppu, T.

    1987-01-01

    The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation but not by treatment with dimethyl sulfoxide. This enhanced activity was completely inhibited by adding cycloheximide to the culture medium 2 h after exposure to TS, suggesting that TS are dimethyl sulfoxide-type inducers of erythroid differentiation. No inhibitory effect of TS was observed on macromolecular synthesis in cultured cells

  19. A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members' expression in tomato.

    Science.gov (United States)

    Zhang, Zhenzhu; Chen, Xiuling; Guan, Xin; Liu, Yang; Chen, Hongyu; Wang, Tingting; Mouekouba, Liana Dalcantara Ongouya; Li, Jingfu; Wang, Aoxue

    2014-01-01

    Homeodomain-leucine zipper (HD-Zip) proteins are a kind of transcriptional factors that play a vital role in plant growth and development. However, no detailed information of HD-Zip family in tomato has been reported till now. In this study, 51 HD-Zip genes (SlHZ01-51) in this family were identified and categorized into 4 classes by exon-intron and protein structure in tomato (Solanum lycopersicum) genome. The synthetical phylogenetic tree of tomato, Arabidopsis and rice HD-Zip genes were established for an insight into their evolutionary relationships and putative functions. The results showed that the contribution of segmental duplication was larger than that of tandem duplication for expansion and evolution of genes in this family of tomato. The expression profile results under abiotic stress suggested that all SlHZ I genes were responsive to cold stress. This study will provide a clue for the further investigation of functional identification and the role of tomato HD-Zip I subfamily in plant cold stress responses and developmental events.

  20. Formation and maturation of the murine meniscus.

    Science.gov (United States)

    Gamer, Laura W; Xiang, Lin; Rosen, Vicki

    2017-08-01

    Meniscal injuries are commonplace, but current surgical repair procedures do not prevent degenerative joint changes that occur after meniscal injury and often lead to osteoarthritis. Successful tissue regeneration in adults often recapitulates events that occur during embryogenesis, suggesting that understanding the regulatory pathways controlling these early processes may provide clues for developing strategies for tissue repair. While the mouse is now widely used to study joint diseases, detailed knowledge of the basic biology of murine meniscus is not readily available. Here, we examine meniscal morphogenesis in mice from embryonic day 13.5 (E13.5) to 6 months of age using histology, in situ hybridization, and immunohistochemistry. We find that the meniscus is a morphologically distinct structure at E16 when it begins to regionalize. At birth, the meniscus has a distinguishable inner, avascular, round chondrocyte cell region, an outer, vascularized, fibroblast cell region, and a surface superficial zone. Maturation begins at 2 weeks of age when the meniscus expresses type I collagen, type II collagen, type X collagen, and MMP-13 in specific patterns. By 4 weeks of age, small areas of ossification are detected in the anterior meniscal horn, a common feature seen in rodents. Maturation appears complete at 8 weeks of age, when the meniscus resembles the adult structure complete with ossifying tissue that contains bone marrow like areas. Our results provide, the first systematic study of mouse meniscal development and will be a valuable tool for analyzing murine models of knee joint formation and disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1683-1689, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  1. Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

    Science.gov (United States)

    Nomura, M; Tsujimura, A; Begum, N A; Matsumoto, M; Wabiko, H; Toyoshima, K; Seya, T

    2000-01-01

    The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46. PMID:11023821

  2. Current Translational Research and Murine Models For Duchenne Muscular Dystrophy

    Science.gov (United States)

    Rodrigues, Merryl; Echigoya, Yusuke; Fukada, So-ichiro; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscle degeneration. Mutations in the DMD gene result in the absence of dystrophin, a protein required for muscle strength and stability. Currently, there is no cure for DMD. Since murine models are relatively easy to genetically manipulate, cost effective, and easily reproducible due to their short generation time, they have helped to elucidate the pathobiology of dystrophin deficiency and to assess therapies for treating DMD. Recently, several murine models have been developed by our group and others to be more representative of the human DMD mutation types and phenotypes. For instance, mdx mice on a DBA/2 genetic background, developed by Fukada et al., have lower regenerative capacity and exhibit very severe phenotype. Cmah-deficient mdx mice display an accelerated disease onset and severe cardiac phenotype due to differences in glycosylation between humans and mice. Other novel murine models include mdx52, which harbors a deletion mutation in exon 52, a hot spot region in humans, and dystrophin/utrophin double-deficient (dko), which displays a severe dystrophic phenotype due the absence of utrophin, a dystrophin homolog. This paper reviews the pathological manifestations and recent therapeutic developments in murine models of DMD such as standard mdx (C57BL/10), mdx on C57BL/6 background (C57BL/6-mdx), mdx52, dystrophin/utrophin double-deficient (dko), mdxβgeo, Dmd-null, humanized DMD (hDMD), mdx on DBA/2 background (DBA/2-mdx), Cmah-mdx, and mdx/mTRKO murine models. PMID:27854202

  3. Hox code in embryos of Chinese soft-shelled turtle Pelodiscus sinensis correlates with the evolutionary innovation in the turtle.

    Science.gov (United States)

    Ohya, Yoshie Kawashima; Kuraku, Shigehiro; Kuratani, Shigeru

    2005-03-15

    Turtles have the most unusual body plan of the amniotes, with a dorsal shell consisting of modified ribs. Because this morphological change in the ribs can be described as an axial-level specific alteration, the evolution of the turtle carapace should depend on changes in the Hox code. To identify turtle-specific changes in developmental patterns, we cloned several Hox genes from the Chinese soft-shelled turtle, Pelodiscus sinensis, examined their expression patterns during embryogenesis, and compared them with those of chicken and mouse embryos. We detected possibly turtle-specific derived traits in Hoxc-6 expression, which is restricted to the paraxial part of the embryo; in the expression of Hoxa-5 and Hoxb-5, the transcripts of which were detected only at the cervical level; and in Hoxc-8 and Hoxa-7 expression, which is shifted anteriorly relative to that of the other two amniote groups. From the known functions of the Hox orthologs in model animals, these P. sinensis-specific changes apparently correlate with specializations in the turtle-specific body plan. Copyright 2005 Wiley-Liss, Inc.

  4. Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney.

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    Anja Lüttich

    Full Text Available The opportunistic fungal pathogen Candida albicans has a remarkable ability to adapt to unfavorable environments by different mechanisms, including microevolution. For example, a previous study has shown that passaging through the murine spleen can cause new phenotypic characteristics. Since the murine kidney is the main target organ in murine Candida sepsis and infection of the spleen differs from the kidney in several aspects, we tested whether C. albicans SC5314 could evolve to further adapt to infection and persistence within the kidney. Therefore, we performed a long-term serial passage experiment through the murine kidney of using a low infectious dose. We found that the overall virulence of the commonly used wild type strain SC5314 did not change after eight passages and that the isolated pools showed only very moderate changes of phenotypic traits on the population level. Nevertheless, the last passage showed a higher phenotypic variability and a few individual strains exhibited phenotypic alterations suggesting that microevolution has occurred. However, the majority of the tested single strains were phenotypically indistinguishable from SC5314. Thus, our findings indicate that characteristics of SC5314 which are important to establish and maintain kidney infection over a prolonged time are already well developed.

  5. Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

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    Annette Fink

    2014-02-01

    Full Text Available Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I glycoproteins, are often identified by “reverse immunology”, a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E phase protein, the m164 epitope is presented already during the Immediate Early (IE phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

  6. Characterization of the binding of radioiodinated hybrid recombinant IFN-alpha A/D to murine and human lymphoid cell lines

    International Nuclear Information System (INIS)

    Faltynek, C.R.; Princler, G.L.; Schwabe, M.; Shata, M.T.; Lewis, G.K.; Kamin-Lewis, R.M.

    1990-01-01

    The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors

  7. Newly Characterized Murine Undifferentiated Sarcoma Models Sensitive to Virotherapy with Oncolytic HSV-1 M002

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    Eric K. Ring

    2017-12-01

    Full Text Available Despite advances in conventional chemotherapy, surgical techniques, and radiation, outcomes for patients with relapsed, refractory, or metastatic soft tissue sarcomas are dismal. Survivors often suffer from lasting morbidity from current treatments. New targeted therapies with less toxicity, such as those that harness the immune system, and immunocompetent murine sarcoma models to test these therapies are greatly needed. We characterized two new serendipitous murine models of undifferentiated sarcoma (SARC-28 and SARC-45 and tested their sensitivity to virotherapy with oncolytic herpes simplex virus 1 (HSV-1. Both models expressed high levels of the primary HSV entry molecule nectin-1 (CD111 and were susceptible to killing by interleukin-12 (IL-12 producing HSV-1 M002 in vitro and in vivo. M002 resulted in a significant intratumoral increase in effector CD4+ and CD8+ T cells and activated monocytes, and a decrease in myeloid-derived suppressor cells (MDSCs in immunocompetent mice. Compared to parent virus R3659 (no IL-12 production, M002 resulted in higher CD8:MDSC and CD8:T regulatory cell (Treg ratios, suggesting that M002 creates a more favorable immune tumor microenvironment. These data provide support for clinical trials targeting sarcomas with oncolytic HSV-1. These models provide an exciting opportunity to explore combination therapies for soft tissue sarcomas that rely on an intact immune system to reach full therapeutic potential.

  8. Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor

    International Nuclear Information System (INIS)

    Gough, N.M.; Gearing, D.P.; King, J.A.; Willson, T.A.; Hilton, D.J.; Nicola, N.A.; Metcalf, D.

    1988-01-01

    A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the marine cDNA as a hybridization probe. The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence. After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1. Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF. This factor competed with 125 I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors

  9. Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

    Science.gov (United States)

    Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

    1992-05-15

    A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

  10. The murine Cd48 gene: allelic polymorphism in the IgV-like region.

    Science.gov (United States)

    Cabrero, J G; Freeman, G J; Reiser, H

    1998-12-01

    The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

  11. Analysis of the capacity to produce IL-3 in murine AIDS

    DEFF Research Database (Denmark)

    Neuenschwander, A U; Marker, O; Thomsen, Allan Randrup

    1994-01-01

    Adult C57BL/6 mice infected with LP-BM5 murine leukaemia virus represent a model of murine AIDS (MAIDS). In this study we have analysed the capacity of CD4+ T cells from infected mice to produce IL-3 following stimulation with ConA for 24-72 h. In contrast to the position with IL-2, the production...

  12. Reishi Protein LZ-8 Induces FOXP3+ Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice

    Directory of Open Access Journals (Sweden)

    Hsien-Yeh Hsu

    2013-01-01

    Full Text Available LZ-8, an immunomodulatory protein isolated from Ganoderma lucidum (also known as Ling-Zhi or Reishi, has been shown to promote cell proliferation and IL-2 production in T cells. In this study, we show that LZ-8 induces the expansion of both murine and human CD4+ T cells into FOXP3+ regulatory T (Treg cells. LZ-8 treatment was found to stimulate a 4-fold and a 10-fold expansion in the Treg populations of murine and human primary CD4+ T cells, respectively. In addition, the expression of CTLA-4 and IL-10 was induced in LZ-8-treated CD4+ T cells. Using neutralizing antibodies and gene-deficient T-cell lines, we also found that LZ-8 promotes Treg expansion through a CD45-mediated signaling pathway and that the CD18-dependent induction of IL-2 was involved in Treg formation and IL-10 production. The suppressive activity of LZ-8 was confirmed using a murine model of DSS-induced colitis; the disease was alleviated by the adoptive transfer of LZ-8-treated CD4+ T cells. In conclusion, a new regulatory function for LZ-8 was identified, and the molecular mechanisms underlying this function were elucidated.

  13. The Homeodomain Transcription Factor Cdx1 Does Not Behave as an Oncogene in Normal Mouse Intestine

    Directory of Open Access Journals (Sweden)

    Mary Ann S. Crissey

    2008-01-01

    Full Text Available The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium.

  14. Murine models of H. pylori-induced gastritis and gastric adenocarcinoma.

    Science.gov (United States)

    Krueger, Sabine; Roessner, Albert; Kuester, Doerthe

    2011-10-15

    Laboratory mice have become one of the best animal species for mechanistic studies in gastrointestinal research. Their abundant genetic information, the way of causing carcinogenesis easily by transgenic and gene knockout techniques, limited effort in time and costs, and their practicability provide advantages over other animal models. Meanwhile, several murine practical models have been established for the investigation of the initiation, expansion, and progression of gastritis and gastric carcinoma, for assessing the effects of bacterial, genetic and environmental factors, and for evaluating therapeutic and preventive strategies in gastric diseases. This article gives a review of murine models of gastritis and gastric cancer, placing emphasis on the models associated with Helicobacter pylori infection and techniques used in our laboratory. We discuss matters of murine gastric anatomy, as well as techniques of infection, tissue preparation, and histology. Copyright © 2011 Elsevier GmbH. All rights reserved.

  15. Murine transgenic embryonic stem cell lines for the investigation of sinoatrial node-related molecular pathways

    Directory of Open Access Journals (Sweden)

    Stefanie Schmitteckert

    2017-12-01

    Full Text Available The elucidation of molecular mechanisms that restrict the potential of pluripotent stem cells and promote cardiac lineage differentiation is of crucial relevance, since embryonic stem cells (ESCs hold great potential for cell based heart therapies. The homeodomain transcription factor Shox2 is essential for the development and proper function of the native cardiac pacemaker, the sinoatrial node. This prompted us to develop a cardiac differentiation model using ESC lines isolated from blastocysts of Shox2-deficient mice. The established cell model provides a fundamental basis for the investigation of molecular pathways under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

  16. Acute Febrile Illness and Complications Due to Murine Typhus, Texas, USA1,2.

    Science.gov (United States)

    Afzal, Zeeshan; Kallumadanda, Sunand; Wang, Feng; Hemmige, Vagish; Musher, Daniel

    2017-08-01

    Murine typhus occurs relatively commonly in southern Texas, as well as in California. We reviewed records of 90 adults and children in whom murine typhus was diagnosed during a 3-year period in 2 hospitals in southern Texas, USA. Most patients lacked notable comorbidities; all were immunocompetent. Initial signs and symptoms included fever (99%), malaise (82%), headache (77%), fatigue (70%), myalgias (68%), and rash (39%). Complications, often severe, in 28% of patients included bronchiolitis, pneumonia, meningitis, septic shock, cholecystitis, pancreatitis, myositis, and rhabdomyolysis; the last 3 are previously unreported in murine typhus. Low serum albumin and elevated procalcitonin, consistent with bacterial sepsis, were observed in >70% of cases. Rash was more common in children; thrombocytopenia, hyponatremia, elevated hepatic transaminases, and complications were more frequent in adults. Murine typhus should be considered as a diagnostic possibility in cases of acute febrile illness in southern and even in more northern US states.

  17. Anti-inflammatory effects of rebamipide eyedrop administration on ocular lesions in a murine model of primary Sjögren's syndrome.

    Directory of Open Access Journals (Sweden)

    Rieko Arakaki

    Full Text Available Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb, a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren's syndrome (SS. However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown.Reb eyedrops were administered to the murine model of SS aged 4-8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment.Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces.

  18. Reemergence of Murine Typhus in the US

    Centers for Disease Control (CDC) Podcasts

    2015-04-21

    Dr. Lucas Blanton discusses the Reemergence of Murine Typhus in Galveston Texas in 2013.  Created: 4/21/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 4/27/2015.

  19. Three-dimensional in vivo imaging of the murine liver: a micro-computed tomography-based anatomical study.

    Directory of Open Access Journals (Sweden)

    Teresa Fiebig

    Full Text Available Various murine models are currently used to study acute and chronic pathological processes of the liver, and the efficacy of novel therapeutic regimens. The increasing availability of high-resolution small animal imaging modalities presents researchers with the opportunity to precisely identify and describe pathological processes of the liver. To meet the demands, the objective of this study was to provide a three-dimensional illustration of the macroscopic anatomical location of the murine liver lobes and hepatic vessels using small animal imaging modalities. We analysed micro-CT images of the murine liver by integrating additional information from the published literature to develop comprehensive illustrations of the macroscopic anatomical features of the murine liver and hepatic vasculature. As a result, we provide updated three-dimensional illustrations of the macroscopic anatomy of the murine liver and hepatic vessels using micro-CT. The information presented here provides researchers working in the field of experimental liver disease with a comprehensive, easily accessable overview of the macroscopic anatomy of the murine liver.

  20. Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts

    International Nuclear Information System (INIS)

    Lapidot, T.; Terenzi, A.; Singer, T.S.; Salomon, O.; Reisner, Y.

    1989-01-01

    A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum

  1. Acute lethal toxicity following passive immunization for treatment of murine cryptococcosis.

    OpenAIRE

    Savoy, A C; Lupan, D M; Manalo, P B; Roberts, J S; Schlageter, A M; Weinhold, L C; Kozel, T R

    1997-01-01

    Passive immunization with monoclonal antibodies (MAbs) specific for the major capsular polysaccharide of Cryptococcus neoformans alters the course of murine cryptococcosis. During studies of passive immunization for treatment of murine cryptococcosis, we noted the occurrence of an acute, lethal toxicity. Toxicity was characterized by scratching, lethargy, respiratory distress, collapse, and death within 20 to 60 min after injection of antibody. The toxic effect was observed only in mice with ...

  2. Experiment list: SRX186705 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available t play a role a chromatin remodeling and histone demethylation. PHF8 also bears a...a chromatin remodeling and histone demethylation. PHF8 also bears a PHD (plant homeodomain)- type zinc-finge

  3. Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605

    Directory of Open Access Journals (Sweden)

    Ralph A. Pietrofesa

    2016-03-01

    Full Text Available The interaction of asbestos fibers with macrophages generates harmful reactive oxygen species (ROS and subsequent oxidative cell damage that are key processes linked to malignancy. Secoisolariciresinol diglucoside (SDG is a non-toxic, flaxseed-derived pluripotent compound that has antioxidant properties and may thus function as a chemopreventive agent for asbestos-induced mesothelioma. We thus evaluated synthetic SDG (LGM2605 in asbestos-exposed, elicited murine peritoneal macrophages as an in vitro model of tissue phagocytic response to the presence of asbestos in the pleural space. Murine peritoneal macrophages (MFs were exposed to crocidolite asbestos fibers (20 µg/cm2 and evaluated at various times post exposure for cytotoxicity, ROS generation, malondialdehyde (MDA, and levels of 8-iso Prostaglandin F2α (8-isoP. We then evaluated the ability of LGM2605 to mitigate asbestos-induced oxidative stress by administering LGM2605 (50 µM 4-h prior to asbestos exposure. We observed a significant (p < 0.0001, time-dependent increase in asbestos-induced cytotoxicity, ROS generation, and the release of MDA and 8-iso Prostaglandin F2α, markers of lipid peroxidation, which increased linearly over time. LGM2605 treatment significantly (p < 0.0001 reduced asbestos-induced cytotoxicity and ROS generation, while decreasing levels of MDA and 8-isoP by 71%–88% and 41%–73%, respectively. Importantly, exposure to asbestos fibers induced cell protective defenses, such as cellular Nrf2 activation and the expression of phase II antioxidant enzymes, HO-1 and Nqo1 that were further enhanced by LGM2605 treatment. LGM2605 boosted antioxidant defenses, as well as reduced asbestos-induced ROS generation and markers of oxidative stress in murine peritoneal macrophages, supporting its possible use as a chemoprevention agent in the development of asbestos-induced malignant mesothelioma.

  4. Expression of biologically active murine interleukin-18 in Lactococcus lactis.

    Science.gov (United States)

    Feizollahzadeh, Sadegh; Khanahmad, Hossein; Rahimmanesh, Ilnaz; Ganjalikhani-Hakemi, Mazdak; Andalib, Alireza; Sanei, Mohammad Hossein; Rezaei, Abbas

    2016-11-01

    The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL -1 and 0.6-0.7 ng mL -1 , respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Murine typhus in two travelers returning from Bali, Indonesia: an underdiagnosed disease.

    Science.gov (United States)

    Takeshita, Nozomi; Imoto, Kazuya; Ando, Shuji; Yanagisawa, Kunio; Ohji, Goh; Kato, Yasuyuki; Sakata, Akiko; Hosokawa, Naoto; Kishimoto, Toshio

    2010-01-01

    Two Japanese travelers from Bali were diagnosed with murine typhus in Japan during the same period. Although one had only mild illness, the other experienced liver and kidney dysfunction. Murine typhus may be missed not only in endemic areas around the world, but also in travelers, especially those returning from marine resorts in these areas. © 2010 International Society of Travel Medicine.

  6. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

    Science.gov (United States)

    Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

    2012-11-01

    Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

  7. Characterization of a Murine Model of Bioequivalent Bladder Wound Healing and Repair Following Subtotal Cystectomy

    Directory of Open Access Journals (Sweden)

    Mona Zarifpour

    2017-05-01

    Full Text Available Previous work demonstrated restoration of a bioequivalent bladder within 8 weeks of removing the majority of the bladder (subtotal cystectomy or STC in rats. The goal of the present study was to extend our investigations of bladder repair to the murine model, to harness the power of mouse genetics to delineate the cellular and molecular mechanisms responsible for the observed robust bladder regrowth. Female C57 black mice underwent STC, and at 4, 8, and 12 weeks post-STC, bladder repair and function were assessed via cystometry, ex vivo pharmacologic organ bath studies, and T2-weighted magnetic resonance imaging (MRI. Histology was also performed to measure bladder wall thickness. We observed a time-dependent increase in bladder capacity (BC following STC, such that 8 and 12 weeks post-STC, BC and micturition volumes were indistinguishable from those of age-matched non-STC controls and significantly higher than observed at 4 weeks. MRI studies confirmed that bladder volume was indistinguishable within 3 months (11 weeks post-STC. Additionally, bladders emptied completely at all time points studied (i.e., no increases in residual volume, consistent with functional bladder repair. At 8 and 12 weeks post-STC, there were no significant differences in bladder wall thickness or in the different components (urothelium, lamina propria, or smooth muscle layers of the bladder wall compared with age-matched control animals. The maximal contractile response to pharmacological activation and electrical field stimulation increased over time in isolated tissue strips from repaired bladders but remained lower at all time points compared with controls. We have established and validated a murine model for the study of de novo organ repair that will allow for further mechanistic studies of this phenomenon after, for example, genetic manipulation.

  8. Immune Modulating Topical S100A8/A9 Inhibits Growth of Pseudomonas aeruginosa and Mitigates Biofilm Infection in Chronic Wounds

    DEFF Research Database (Denmark)

    Trøstrup, Hannah; Lerche, Christian Johann; Christophersen, Lars

    2017-01-01

    in a murine model and P. aeruginosa growth in vitro. Seventy-six mice, inflicted with a full-thickness burn wound were challenged subcutaneously (s.c.) by 10⁶ colony-forming units (CFUs) of P. aeruginosa biofilm. Mice were subsequently randomized into two treatment groups, one group receiving recombinant...... murine S100A8/A9 and a group of vehicle controls (phosphate-buffered saline, PBS) all treated with s.c. injections daily for up to five days. Wounds were analyzed for quantitative bacteriology and contents of key inflammatory markers. Count of blood polymorphonuclear leukocytes was included. S100A8/A9...

  9. Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

    Science.gov (United States)

    Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

    1998-11-10

    Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

  10. Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

    Directory of Open Access Journals (Sweden)

    Mayumi Oakland

    2013-01-01

    Full Text Available Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.

  11. The Murine Factor H-Related Protein FHR-B Promotes Complement Activation

    Directory of Open Access Journals (Sweden)

    Marcell Cserhalmi

    2017-09-01

    Full Text Available Factor H-related (FHR proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH. While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3, and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

  12. The Homeodomain Transcription Factor Cdx1 Does Not Behave as an Oncogene in Normal Mouse Intestine1

    Science.gov (United States)

    Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

    2008-01-01

    The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium. PMID:18231635

  13. Early radiation effects in highly apoptotic murine lymphoma xenografts monitored by 31P magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Sakurai, Hideyuki; Mitsuhashi, Norio; Murata, Osamu; Kitamoto, Yoshizumi; Saito, Yoshihiro; Hasegawa, Masatoshi; Akimoto, Tetsuo; Takahashi, Takeo; Nasu, Sachiko; Niibe, Hideo

    1998-01-01

    Purpose: Phosphorus-31 magnetic resonance spectra ( 31 P-MRS) were obtained from highly apoptotic murine lymphoma xenografts before and up to 24 hr following graded doses of radiation ranging from 2 to 30 Gy. Radiation-induced apoptosis was also estimated up to 24 hr by scoring apoptotic cells in tumor tissue. Methods and Materials: Highly apoptotic murine lymphoma cells, EL4, were subcutaneously transplanted into C57/BL mice. At 7 days after transplantation, radiation was given to the tumor with a single dose at 3, 10, and 30 Gy. The β-ATP/Pi, PME/Pi, and β-ATP/PME values were calculated from the peak area of each spectrum. Radiation-induced apoptosis was scored with counting apoptotic cells on hematoxylin and eosin stained specimens (%apoptosis). Results: The values of % apoptosis 4, 8, and 24 hr after radiation were 21.8, 19.6, and 4.6% at 3 Gy, 35.1, 25.6, and 14.8% at 10 Gy, 38.4, 38.0, and 30.6% at 30 Gy, respectively (cf. 4.4% in control). There was no correlation between early change in β-ATP/Pi and % apoptosis at 4 hr after radiation when most of the apoptosis occurred. An early decrease in PME/Pi was observed at 4 hr after radiation dose at 30 Gy. For each dose, the values of β-ATP/Pi 24 hr after radiation were inversely related to radiation dose. Conclusion: The increase in β-ATP/Pi observed by 31 P-MRS was linked to the degree of histological recovery from radiation-induced apoptosis

  14. DMPD: Toll-like receptor 9 in murine lupus: more friend than foe! [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18241699 Toll-like receptor 9 in murine lupus: more friend than foe! Yu P, Musette ...us: more friend than foe! PubmedID 18241699 Title Toll-like receptor 9 in murine lupus...P, Peng SL. Immunobiology. 2008;213(2):151-7. Epub 2007 Sep 21. (.png) (.svg) (.html) (.csml) Show Toll-like receptor 9 in murine lup

  15. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    Directory of Open Access Journals (Sweden)

    Lefeng Wang

    2017-01-01

    Full Text Available Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC, leading to barrier dysfunction and acute respiratory distress syndrome (ARDS. Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix] of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

  16. NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells

    International Nuclear Information System (INIS)

    Nagel, Stefan; Scherr, Michaela; MacLeod, Roderick AF; Venturini, Letizia; Przybylski, Grzegorz K; Grabarczyk, Piotr; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; Schmidt, Christian A; Drexler, Hans G

    2009-01-01

    Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs. Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses. Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3

  17. Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

    Directory of Open Access Journals (Sweden)

    Want MY

    2017-03-01

    Full Text Available Muzamil Y Want,1 Mohammad Islammudin,1 Garima Chouhan,1 Hani A Ozbak,2 Hassan A Hemeg,2 Asoke P Chattopadhyay,3 Farhat Afrin2 1Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard, Hamdard University, New Delhi, India; 2Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; 3Department of Chemistry, University of Kalyani, Kalyani, India Abstract: Visceral leishmaniasis (VL is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin (NLA was prepared by thin-film hydration method and optimized using Box–Behnken design with a mean particle diameter of 83±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a

  18. [Virulence of Sporothrix globosa in murine models].

    Science.gov (United States)

    Cruz Choappa, Rodrigo; Pérez Gaete, Salomón; Rodríguez Badilla, Valentina; Vieille Oyarzo, Peggy; Opazo Sanchez, Héctor

    The sporothricosis disease is an infection caused by species included in Sporothrix schenkii complex. Verify the virulence of a strain of S. globosa using two different concentrations of inoculum by intraperitoneally and subcutaneously, into a mouse model. Nonrandomized pilot study, in murine inoculated with a strain of S. globosa (CBS 14.076M) by intraperitoneally and subcutaneously with inoculum concentrations of 0.5 and 4 McFarland. For this purpose 18 rodents CF-1 (ISP, Santiago, Chile) were used. The studied strain did not induce illness or injury on animals, they all survived and neither the tissue culture nor the histopathological analysis showed fungal growth or suggestive infection by organ abnormalities. The S. globosa strain did not present any virulence enough to cause disease at 0.5 and 4.0 McFarland concentration inoculum when inoculated in both intraperitoneally and subcutaneously, in murine models. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  19. Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line

    Directory of Open Access Journals (Sweden)

    Lu Yuanan

    2009-11-01

    Full Text Available Abstract Background Human Noroviruses are the predominant cause of non-bacterial gastroenteritis worldwide. To facilitate prevention and control, a norovirus isolated from mice can provide a model to understand human noroviruses. To establish optimal viral infectivity conditions for murine noroviruses, several cell lines of hematopoietic lineage, including murine BV-2, RAW 264.7, and TIB, as well as human CHME-5, were tested comparatively for their sensitivity to murine norovirus-1. Results Except for CHME-5, all three murine-derived cell lines were susceptible to MNV infection. Viral infection of these cells was confirmed by RT-PCR. Using both viral plaque and replication assays, BV-2 and RAW 264.7 cells were determined to have comparable sensitivities to MNV-1 infection. Comparisons of cell growth characteristics, general laboratory handling and potential in-field applications suggest the use of BV-2 to be more advantageous. Conclusion Results obtained from these studies demonstrate that an immortalized microglial cell line can support MNV-1 replication and provides a more efficient method to detect and study murine noroviruses, facilitating future investigations using MNV-1 as a model to study, detect, and control Human Norovirus.

  20. Dose-effect relationship of apoptosis induced by fission-neutron in murine thymocytes

    International Nuclear Information System (INIS)

    Yuan Bin; Li Liang; Xue Wencheng; Sun Jianmin; Wang Baoqin

    2000-01-01

    Objective: To investigate the effectiveness of high LET fission-neutron to induce apoptosis in murine thymocytes and to compare it with that of low LET 60 Co γ-ray. Methods: Apoptosis induction was studied qualitatively by light and transmission electron microscopy and DNA gel electrophoresis,also quantitatively by flow cytometry(FCM) and diphenylamine (DPA)methods. Results: DNA ladders of murine thymocytes were detectable, the typical apoptosis of thymocytes could be observed morphologically by means of light and electron microscopy at 6 h after fission-neutron irradiation with doses ranging from 0.5 to 5.0 Gy, meanwhile the percentages of apoptosis increased with increasing doses. After exposure to γ-rays with doses ranging from 1.0 to 30 Gy, the experimental results were similar to those from neutron radiation. The incidence of apoptosis peaked at about 20 Gy, the percentages did not increase further when doses increased. Conclusion: Apoptosis of murine thymocytes can be induced when mice are exposed to either fission-neutron (0.5-5.0 Gy) or to γ-ray (1-30 Gy). Although the relationship between apoptosis and radiation doses is similar, the percentage of apoptosis induced by neutron irradiation is higher than that induced by γ-irradiation. The RBE values of fission-neutron for inducing apoptosis murine thymocytes are 2.09 (by FCM method) and 2.37 (by DPA method), respectively. These results also suggest that fission-neutron-induced murine immune tissue is more severe than that induced by γ-rays at several hours post-irradiation and this might be the basis for heavy damage to immune tissues induced by fission-neutron-irradiation in later period

  1. Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

    Science.gov (United States)

    Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

    2015-08-01

    We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

    Science.gov (United States)

    Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

    2008-01-01

    The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

  3. Proliferation differentiation and therapeutic effect of short-term cultured murine bone marrow cells

    International Nuclear Information System (INIS)

    Zhao Zekun; Cong Jianbo

    1986-01-01

    Murine bone marrow cells were cultured in conditioned medium of muscle. After 24 hours of culture, both adherent and suspended cells appeared in the culture. The adherent cells mainly consisted of macrophages and the suspended cells were predominantly granulocytes. After 6 days, the total number of nucleated cells and CFU-C in the culture increased about 400% and 600% respectively, but CFU-S reduced to 21% approximately. Lymphocytes persisted only for 4 days. The stem cells (CFU-S) from 6-day culture were injected into the lethally irradiated syngenic mice. The 30 day survival rate of the treated mice was 89% whereas that of the controls was only 7%. The bone marrow cells in 2/8 of recipients sacrificed at 30 or 60 days were of donor type and 6/8 of the recipients were chimeras

  4. Azithromycin prophylaxis and treatment of murine toxoplasmosis.

    Science.gov (United States)

    Tabbara, Khalid F; Hammouda, Ehab; Tawfik, Abdulkader; Al-Omar, Othman M; Abu El-Asrar, Ahmed M

    2005-03-01

    To evaluate the azithromycin effects alone and in combination with other agents in the prophylaxis and treatment of murine toxoplasmosis. A total of 280 BALB/c mice were included, and 2 x 103 Toxoplasma organisms of the RH strain Toxoplasma gondii strain ATCC50174 were given intraperitoneally to each mouse. In experiment one, 40 animals were given azithromycin 200 milligram/kilogram/daily for 3 days starting the day of inoculation, 40 mice were control. In experiment 2, the treatment was started 48 hours after inoculation and given daily for 3 days: one group received azithromycin 200 milligram/kilogram/day, the second group received pyrimethamine 25 milligram/kilogram/day, and the sulfadiazine 100 milligram/kilogram/day. The third group was control. In experiment 3, 7 groups of animals received one of the following (1) none, (2) azithromycin 200 milligram/kilogram/day, (3) pyrimethamine 25 milligram/kilogram/day and sulfadiazine 100 milligram/kilogram/day, (4) azithromycin and sulfadiazine, (5) azithromycin and pyrimethamine, (6) azithromycin with sulfadiazine and pyrimethamine, (7) sulfadiazine alone. Treatment was initiated 72 hours after inoculation for 3 days. The study was conducted at the Animal Care Facility of King Saud University, Riyadh, Kingdom of Saudi Arabia. Animals that received azithromycin simultaneously with inoculation survived, and all control animals died. All animals died in groups receiving single drug therapy. Animals treated with azithromycin and sulfadiazine showed a survival rate of 40%, sulfadiazine and pyrimethamine 40%, or azithromycin with sulfadiazine and pyrimethamine 95% (p<0.0001). Azithromycin alone was found to be effective in the prophylaxis of murine toxoplasmosis. Combination therapy was effective in the treatment of murine toxoplasmosis.

  5. The POU homeodomain transcription factor POUM2 and broad complex isoform 2 transcription factor induced by 20-hydroxyecdysone collaboratively regulate vitellogenin gene expression and egg formation in the silkworm Bombyx mori.

    Science.gov (United States)

    Lin, Y; Liu, H; Yang, C; Gu, J; Shen, G; Zhang, H; Chen, E; Han, C; Zhang, Y; Xu, Y; Wu, J; Xia, Q

    2017-10-01

    Vitellogenin (Vg) is a source of nutrition for embryo development. Our previous study showed that the silkworm (Bombyx mori) transcription factor broad complex isoform 2 (BmBrC-Z2) regulates gene expression of the Vg gene (BmVg) by induction with 20-hydroxyecdysone (20E). However, the mechanism by which 20E regulates BmVg expression was not clarified. In this study, cell transfection experiments showed that the BmVg promoter containing the POU homeodomain transcription factor POUM2 (POUM2) and BrC-Z2 cis-response elements (CREs) showed a more significant response to 20E than that harbouring only the BrC-Z2 or POUM2 CRE. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that BmPOUM2 could bind to the POUM2 CRE of the BmVg promoter. Over-expression of BmPOUM2 and BmBrC-Z2 in B. mori embryo-derived cell line (BmE) could enhance the activity of the BmVg promoter carrying both the POUM2 and BrC-Z2 CREs following 20E induction. Quantitative PCR and immunofluorescence histochemistry showed that the expression pattern and tissue localization of BmPOUM2 correspond to those of BmVg. Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that BmPOUM2 interacts only with BmBrC-Z2 to regulate BmVg expression. Down-regulation of BmPOUM2 in female silkworm by RNA interference significantly reduced BmVg expression, leading to abnormal egg formation. In summary, these results indicate that BmPOUM2 binds only to BmBrC-Z2 to collaboratively regulate BmVg expression by 20E induction to control vitellogenesis and egg formation in the silkworm. Moreover, these findings suggest that homeodomain protein POUM2 plays a novel role in regulating insect vitellogenesis. © 2017 The Royal Entomological Society.

  6. Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity.

    Science.gov (United States)

    Hymus, Graham J; Cai, Suqin; Kohl, Elizabeth A; Holtan, Hans E; Marion, Colleen M; Tiwari, Shiv; Maszle, Don R; Lundgren, Marjorie R; Hong, Melissa C; Channa, Namitha; Loida, Paul; Thompson, Rebecca; Taylor, J Philip; Rice, Elena; Repetti, Peter P; Ratcliffe, Oliver J; Reuber, T Lynne; Creelman, Robert A

    2013-11-01

    Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.

  7. Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition

    Science.gov (United States)

    Taylor, A.M.; Preston, A.J.; Paulk, N.K.; Sutherland, H.; Keenan, C.M.; Wilson, P.J.M.; Wlodarski, B.; Grompe, M.; Ranganath, L.R.; Gallagher, J.A.; Jarvis, J.C.

    2012-01-01

    Objective Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. Design The homogentisate 1,2-dioxygenase Hgd+/−Fah−/− mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl’s reagent. Results Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. Conclusions Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy. PMID:22542924

  8. Chimeric anti-tenascin antibody 81C6: Increased tumor localization compared with its murine parent

    International Nuclear Information System (INIS)

    Zalutsky, Michael R.; Archer, Gary E.; Garg, Pradeep K.; Batra, Surinder K.; Bigner, Darell D.

    1996-01-01

    When labeled using the Iodogen method, a chimeric antibody composed of the human IgG 2 constant region and the variable regions of murine anti-tenascin 81C6 exhibited superior uptake in human glioma xenografts compared with its murine parent. In the current study, three paired-label experiments were performed in athymic mice with subcutaneous D-54 MG human glioma xenografts to evaluate further the properties of radioiodinated chimeric 81C6. These studies demonstrated that (a) the enhanced tumor uptake of chimeric 81C6 is specific; (b) when labeling was performed using N-succinimidyl 3-iodobenzoate, chimeric 81C6 again showed preferential accumulation in tumor compared with murine 81C6; and (c) the tumor uptake advantage observed previously with murine 81C6 for N-succinimidyl 3-iodobenzoate compared with Iodogen labeling did not occur with chimeric 81C6

  9. A hydrophobic residue in the TALE homeodomain of PBX1 promotes epithelial-to-mesenchymal transition of gastric carcinoma.

    Science.gov (United States)

    He, Changyu; Wang, Zhenqiang; Zhang, Li; Yang, Liyun; Li, Jianfang; Chen, Xuehua; Zhang, Jun; Chang, Qing; Yu, Yingyan; Liu, Bingya; Zhu, Zhenggang

    2017-07-18

    Pre-B-cell leukemia homeobox 1 (PBX1) was originally identified as a proto-oncogene in human leukemia. Although this protein has been shown to contribute to cellular development and tumorigenesis, the role of PBX1 in gastric carcinoma (GC) remains unclear. In this study, we observed increased expression of PBX1 in GC tissues compared with adjacent normal tissues. This increase in PBX1 expression levels negatively correlated with HOXB9 mRNA expression and was also associated with malignancy and metastasis. PBX1 promoted proliferation and metastasis of GC cells both in vitro and in vivo.These phenomena were also accompanied by epithelial-to-mesenchymal transition (EMT). Additionally, we observed that PBX1 promotes the expression of tumor growth and angiogenic factors. A structural model of the PBX1-HOX complex revealed that hydrophobic binding between PBX1 and the hexapeptide motif might be required for EMT induction. This analysis also demonstrated that the Phe252 residue in the first helix of the TALE homeodomain is involved in the latter hydrophobic binding reaction. In vitro data from PBX1 mutants suggest that PBX1 cannot promote tumorigenesis of GC cells via EMT induction when Phe252 residues lose hydrophobicity. It is likely that the presence of this residue is essential in facilitating hydrophobic binding with the hexapeptide motif. These findings suggest that PBX1 may be a potential target for GC treatment and this study provides a platform to elucidate the molecular mechanisms that underpin the role of PBX1 in GC tumorigenesis.

  10. Novel Application of Micro-Computerized Tomography for Morphologic Characterization of the Murine Penis.

    Science.gov (United States)

    O'Neill, Marisol; Huang, Gene O; Lamb, Dolores J

    2017-12-01

    The murine penis model has enriched our understanding of anomalous penile development. The morphologic characterization of the murine penis using conventional serial sectioning methods is labor intensive and prone to errors. To develop a novel application of micro-computerized tomography (micro-CT) with iodine staining for rapid, non-destructive morphologic study of murine penis structure. Penises were dissected from 10 adult wild-type mice and imaged using micro-CT with iodine staining. Images were acquired at 5-μm spatial resolution on a Bruker SkyScan 1272 micro-CT system. After images were acquired, the specimens were washed of any remaining iodine and embedded in paraffin for conventional histologic examination. Histologic and micro-CT measurements for all specimens were made by 2 independent observers. Measurements of penile structures were made on virtual micro-CT sections and histologic slides. The Lin concordance correlation coefficient demonstrated almost perfect strength of agreement for interobserver variability for histologic section (0.9995, 95% CI = 0.9990-0.9997) and micro-CT section (0.9982, 95% CI = 0.9963-0.9991) measurements. Bland-Altman analysis for agreement between the 2 modalities of measurement demonstrated mean differences of -0.029, 0.022, and -0.068 mm for male urogenital mating protuberance, baculum, and penile glans length, respectively. There did not appear to be a bias for overestimation or underestimation of measured lengths and limits of agreement were narrow. The enhanced ability offered by micro-CT to phenotype the murine penis has the potential to improve translational studies examining the molecular pathways contributing to anomalous penile development. The present study describes the first reported use of micro-CT with iodine staining for imaging the murine penis. Producing repeated histologic sections of identical orientation was limited by inherent imperfections in mounting and tissue sectioning, but this was

  11. NCBI nr-aa BLAST: CBRC-OPRI-01-1187 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-1187 ref|XP_344240.3| PREDICTED: similar to Paired mesoderm homeobox p...rotein 2B (Paired-like homeobox 2B) (PHOX2B homeodomain protein) (Neuroblastoma Phox) (NBPhox) [Rattus norvegicus] XP_344240.3 8.8 38% ...

  12. Reversal of Liver Fibrosis in Chronic Murine Schistosomiasis ...

    African Journals Online (AJOL)

    NO Al-Harbi, SA Bahashwan, MS Aboonq, MA Ramadan, AA Bahashwan. Abstract. Purpose: To evaluate the safety, pharmacological effect and mechanism of action of an antifibrotic compound, safironil (SAF)/praziquantel (PZQ) combination on reversal of liver fibrogenesis in chronic murine Schistosomiasis mansoni.

  13. A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

    Science.gov (United States)

    Bogaert, Lies; Woodham, Andrew W; Da Silva, Diane M; Martens, Ann; Meyer, Evelyne; Kast, W Martin

    2015-09-01

    Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.

  14. Establishment of a murine graft-versus-myeloma model using allogeneic stem cell transplantation.

    Directory of Open Access Journals (Sweden)

    Marilène Binsfeld

    Full Text Available Multiple myeloma (MM is a malignant plasma cell disorder with poor long-term survival and high recurrence rates. Despite evidence of graft-versus-myeloma (GvM effects, the use of allogeneic hematopoietic stem cell transplantation (allo-SCT remains controversial in MM. In the current study, we investigated the anti-myeloma effects of allo-SCT from B10.D2 mice into MHC-matched myeloma-bearing Balb/cJ mice, with concomitant development of chronic graft-versus-host disease (GvHD.Balb/cJ mice were injected intravenously with luciferase-transfected MOPC315.BM cells, and received an allogeneic (B10.D2 donor or autologous (Balb/cJ donor transplant 30 days later. We observed a GvM effect in 94% of the allogeneic transplanted mice, as the luciferase signal completely disappeared after transplantation, whereas all the autologous transplanted mice showed myeloma progression. Lower serum paraprotein levels and lower myeloma infiltration in bone marrow and spleen in the allogeneic setting confirmed the observed GvM effect. In addition, the treated mice also displayed chronic GvHD symptoms. In vivo and in vitro data suggested the involvement of effector memory CD4 and CD8 T cells associated with the GvM response. The essential role of CD8 T cells was demonstrated in vivo where CD8 T-cell depletion of the graft resulted in reduced GvM effects. Finally, TCR Vβ spectratyping analysis identified Vβ families within CD4 and CD8 T cells, which were associated with both GvM effects and GvHD, whereas other Vβ families within CD4 T cells were associated exclusively with either GvM or GvHD responses.We successfully established an immunocompetent murine model of graft-versus-myeloma. This is the first murine GvM model using immunocompetent mice that develop MM which closely resembles human MM disease and that are treated after disease establishment with an allo-SCT. Importantly, using TCR Vβ spectratyping, we also demonstrated the presence of GvM unique responses

  15. Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Mochizuki, D.Y.; Eisenman, J.R.; Conlon, P.J.; Park, L.S.; Urdal, D.L.

    1986-01-01

    The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1β, IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum together with recombinant GM-CSF that had been radiolabeled with 125 I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis

  16. Protective effects of astaxanthin from Paracoccus carotinifaciens on murine gastric ulcer models.

    Science.gov (United States)

    Murata, Kenta; Oyagi, Atsushi; Takahira, Dai; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Ishibashi, Takashi; Hara, Hideaki

    2012-08-01

    The purpose of this study was to investigate the effect of astaxanthin extracted from Paracoccus carotinifaciens on gastric mucosal damage in murine gastric ulcer models. Mice were pretreated with astaxanthin for 1 h before ulcer induction. Gastric ulcers were induced in mice by oral administration of hydrochloride (HCl)/ethanol or acidified aspirin. The effect of astaxanthin on lipid peroxidation in murine stomach homogenates was also evaluated by measuring the level of thiobarbituric acid reactive substance (TBARS). The free radical scavenging activities of astaxanthin were also measured by electron spin resonance (ESR) measurements. Astaxanthin significantly decreased the extent of HCl/ethanol- and acidified aspirin-induced gastric ulcers. Astaxanthin also decreased the level of TBARS. The ESR measurement showed that astaxanthin had radical scavenging activities against the 1,1-diphenyl-2-picrylhydrazyl radical and the superoxide anion radical. These results suggest that astaxanthin has antioxidant properties and exerts a protective effect against ulcer formation in murine models. Copyright © 2011 John Wiley & Sons, Ltd.

  17. Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

    DEFF Research Database (Denmark)

    Lundorf, Mikkel D.; Pedersen, Finn Skou; O'Hara, Bryan

    1998-01-01

    Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch...

  18. An EG-VEGF-dependent decrease in homeobox gene NKX3.1 contributes to cytotrophoblast dysfunction: a possible mechanism in human fetal growth restriction.

    Science.gov (United States)

    Murthi, P; Brouillet, S; Pratt, A; Borg, Aj; Kalionis, B; Goffin, F; Tsatsaris, V; Munaut, C; Feige, Jj; Benharouga, M; Fournier, T; Alfaidy, N

    2015-07-21

    Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that EG-VEGF (endocrine gland derived-vascular endothelial growth factor), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesise that EG-VEGF-dependent change in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a Homeobox gene cDNA array on placental explants of 8-12 weeks' gestation after stimulation with EG-VEGF in vitro for 24 hours. The Homeobox gene array identified a >5-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a >2 fold-decrease in mRNA expression compared to untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional role are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared to control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR8-SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 down-stream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

  19. Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

    Science.gov (United States)

    Paris, Daniel H; Stephan, Femke; Bulder, Ingrid; Wouters, Diana; van der Poll, Tom; Newton, Paul N; Day, Nicholas P J; Zeerleder, Sacha

    2015-01-01

    Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and

  20. Ascorbic Acid-Induced Cardiac Differentiation of Murine Pluripotent Stem Cells: Transcriptional Profiling and Effect of a Small Molecule Synergist of Wnt/β-Catenin Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Dina Ivanyuk

    2015-05-01

    Full Text Available Background: Reproducible and efficient differentiation of pluripotent stem cells (PSCs to cardiomyocytes (CMs is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. Methods: Differentiation was performed by embryoid body (EB-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. Results: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A only ascorbic acid (AA exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/β-catenin signaling pathway, was added to cultures after AA-treatment. Conclusion: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.

  1. Melanogenesis stimulation in murine B16 melanoma cells by Kava (Piper methysticum) rhizome extract and kavalactones.

    Science.gov (United States)

    Matsuda, Hideaki; Hirata, Noriko; Kawaguchi, Yoshiko; Naruto, Shunsuke; Takata, Takanobu; Oyama, Masayoshi; Iinuma, Munekazu; Kubo, Michinori

    2006-04-01

    Melanogenesis stimulation activity of aqueous ethanolic extracts obtained from several different parts of five Piper species, namely Piper longum, P. kadsura, P. methysticum, P. betle, and P. cubeba, were examined by using cultured murine B16 melanoma cells. Among them, the extract of P. methysticum rhizome (Kava) showed potent stimulatory effect on melanogenesis as well as P. nigrum leaf extract. Activity-guided fractionation of Kava extract led to the isolation of two active kavalactones, yangonin (2) and 7,8-epoxyyangonin (5), along with three inactive kavalactones, 5,6-dehydrokawain (1), (+)-kawain (3) and (+)-methysticin (4), and a glucosylsterol, daucosterin (6). 7,8-Epoxyyangonin (5) showed a significant stimulatory effect on melanogenesis in B16 melanoma cells. Yangonin (2) exhibited a weak melanogenesis stimulation activity.

  2. Reversal of CD8 T-Cell–Mediated Mucocutaneous Graft-Versus-Host-Like Disease by the JAK Inhibitor Tofacitinib

    Science.gov (United States)

    Okiyama, Naoko; Furumoto, Yasuko; Villarroel, Vadim A; Linton, Jay T; Tsai, Wanxia L; Gutermuth, Jan; Ghoreschi, Kamran; Gadina, Massimo; O'Shea, John J; Katz, Stephen I

    2014-01-01

    The utility of allogeneic hematopoietic stem cell transplantation is limited by graft-versus-host disease (GVHD), a significant cause of morbidity and mortality. Patients with GVHD exhibit cutaneous manifestations with histological features of interface dermatitis followed by scleroderma-like changes. JAK inhibitors represent a class of immunomodulatory drugs that inhibit signaling by multiple cytokines. Herein we report the effects of tofacitinib in a murine model of GVHD. Oral administration of tofacitinib prevented GVHD-like disease manifested by weight loss and mucocutaneous lesions. More importantly, tofacitinib was also effective in reversing established disease. Tofacitinib diminished the expansion and activation of murine CD8 T cells in this model, and had similar effects on IL-2-stimulated human CD8 T cells. Tofacitinib also inhibited the expression of IFN-γ-inducible chemoattractants by keratinocytes, and IFN-γ-inducible cell death of keratinocytes. Tofacitinib may be an effective drug for treatment against CD8 T-cell–mediated mucocutaneous diseases in patients with GVHD. PMID:24213371

  3. Adipocytes enhance murine pancreatic cancer growth via a hepatocyte growth factor (HGF)-mediated mechanism.

    Science.gov (United States)

    Ziegler, Kathryn M; Considine, Robert V; True, Eben; Swartz-Basile, Deborah A; Pitt, Henry A; Zyromski, Nicholas J

    2016-04-01

    Obesity accelerates the development and progression of pancreatic cancer, though the mechanisms underlying this association are unclear. Adipocytes are biologically active, producing factors such as hepatocyte growth factor (HGF) that may influence tumor progression. We therefore sought to test the hypothesis that adipocyte-secreted factors including HGF accelerate pancreatic cancer cell proliferation. Murine pancreatic cancer cells (Pan02 and TGP-47) were grown in a) conditioned medium (CM) from murine F442A preadipocytes, b) HGF-knockdown preadipocyte CM, c) recombinant murine HGF at increasing doses, and d) CM plus HGF-receptor (c-met) inhibitor. Cell proliferation was measured using the MTT assay. ANOVA and t-test were applied; p TGP-47 cell proliferation relative to control (59 ± 12% and 34 ± 12%, p TGP-47 cells remained unchanged. Recombinant HGF dose-dependently increased Pan02, but not TGP-47, proliferation (p TGP-47 cells. These experiments demonstrate that adipocyte-derived factors accelerate murine pancreatic cancer proliferation. In the case of Pan02 cells, HGF is responsible, in part, for this proliferation. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

  4. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

    1986-01-01

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  5. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Ryan W Carroll

    Full Text Available BACKGROUND: Cerebral malaria (CM is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies. METHODOLOGY/PRINCIPAL FINDINGS: A quantitative, rapid murine coma and behavior scale (RMCBS comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA. Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment. CONCLUSIONS/SIGNIFICANCE: Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field. The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.

  6. Identification of factors for physicians to facilitate early differential diagnosis of scrub typhus, murine typhus, and Q fever from dengue fever in Taiwan.

    Science.gov (United States)

    Chang, Ko; Lee, Nan-Yao; Ko, Wen-Chien; Tsai, Jih-Jin; Lin, Wei-Ru; Chen, Tun-Chieh; Lu, Po-Liang; Chen, Yen-Hsu

    2017-02-01

    Dengue fever, rickettsial diseases, and Q fever are acute febrile illnesses with similar manifestations in tropical areas. Early differential diagnosis of scrub typhus, murine typhus, and Q fever from dengue fever may be made by understanding the distinguishing clinical characteristics and the significance of demographic and weather factors. We conducted a retrospective study to identify clinical, demographic, and meteorological characteristics of 454 dengue fever, 178 scrub typhus, 143 Q fever, and 81 murine typhus cases in three Taiwan hospitals. Case numbers of murine typhus and Q fever correlated significantly with temperature and rainfall; the scrub typhus case number was only significantly related with temperature. Neither temperature nor rainfall correlated with the case number of dengue fever. The rarity of dengue fever cases from January to June in Taiwan may be a helpful clue for diagnosis in the area. A male predominance was observed, as the male-to-female rate was 2.1 for murine typhus and 7.4 for Q fever. Multivariate analysis revealed the following six important factors for differentiating the rickettsial diseases and Q fever group from the dengue fever group: fever ≥8 days, alanine aminotransferase > aspartate aminotransferase, platelets >63,000/mL, C-reactive protein >31.9 mg/L, absence of bone pain, and absence of a bleeding syndrome. Understanding the rarity of dengue in the first half of a year in Taiwan and the six differentiating factors may help facilitate the early differential diagnosis of rickettsial diseases and Q fever from dengue fever, permitting early antibiotic treatment. Copyright © 2015. Published by Elsevier B.V.

  7. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    Science.gov (United States)

    1995-04-13

    vaccinia virus-T7 RNA polymerase s y stem for e xpression of target genes . Mol . Cell . BioI . 7 : 2538-2544 . Gagneten , S ., Gout , 0 ., Dubois-Dalcq...glycoprotein. These results showed f or the first time that two murine CEA- related genes can be co-expressed in some cell lines from inbred mice...49 Southern Hybridization ................ . ............ 50 Subcloning of PCR Products and Gene Cloning ........ 51 Growth

  8. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.

    LENUS (Irish Health Repository)

    Monk, Ian R

    2010-01-01

    Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.

  9. Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

    Energy Technology Data Exchange (ETDEWEB)

    Kyle, J.W.; Vogler, C.; Hoffmann, J.W.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (USA)); Birkenmeier, E.H.; Gwynn, B. (Jackson Laboratory, Bar Harbor, ME (USA))

    1990-05-01

    The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{sup mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.

  10. Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy

    International Nuclear Information System (INIS)

    Colombo, F.R.; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D'Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P.

    2001-01-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various 99m Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding 99m Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [ 99m Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin

  11. Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg.

    Science.gov (United States)

    van de Vrugt, Henri J; Koomen, Mireille; Berns, Mariska A D; de Vries, Yne; Rooimans, Martin A; van der Weel, Laura; Blom, Eric; de Groot, Jan; Schepers, Rik J; Stone, Stacie; Hoatlin, Maureen E; Cheng, Ngan Ching; Joenje, Hans; Arwert, Fré

    2002-03-01

    Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway. We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA-G lymphoblasts fully corrects their cross-linker hypersensitivity. At mRNA and protein levels we detected the co-expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co-purify by immunoprecipitation. Upon transfection into Fanca-deficient mouse embryonic fibroblasts EGFP-Fancg chimeric protein was detectable in the nucleus. We identified a murine cDNA, Fancg, which cross-complements the cellular defect of human FA-G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.

  12. Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies

    NARCIS (Netherlands)

    Yang, M.; Reese, J.; Cotecchia, S.; Michel, M. C.

    1998-01-01

    Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with

  13. Signaling pathways regulating murine pancreatic development

    DEFF Research Database (Denmark)

    Serup, Palle

    2012-01-01

    The recent decades have seen a huge expansion in our knowledge about pancreatic development. Numerous lineage-restricted transcription factor genes have been identified and much has been learned about their function. Similarly, numerous signaling pathways important for pancreas development have...... been identified and the specific roles have been investigated by genetic and cell biological methods. The present review presents an overview of the principal signaling pathways involved in regulating murine pancreatic growth, morphogenesis, and cell differentiation....

  14. Signal one and two blockade are both critical for non-myeloablative murine HSCT across a major histocompatibility complex barrier.

    Directory of Open Access Journals (Sweden)

    Kia J Langford-Smith

    Full Text Available Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM:blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan. We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.

  15. Signal one and two blockade are both critical for non-myeloablative murine HSCT across a major histocompatibility complex barrier.

    Science.gov (United States)

    Langford-Smith, Kia J; Sandiford, Zara; Langford-Smith, Alex; Wilkinson, Fiona L; Jones, Simon A; Wraith, J Ed; Wynn, Robert F; Bigger, Brian W

    2013-01-01

    Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.

  16. Responses of murine and human macrophages to leptospiral infection: a study using comparative array analysis.

    Directory of Open Access Journals (Sweden)

    Feng Xue

    Full Text Available Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs and human peripheral blood monocytes (HBMs to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold. In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10 and tumor necrosis factor alpha (TNF-alpha, were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection.

  17. Responses of Murine and Human Macrophages to Leptospiral Infection: A Study Using Comparative Array Analysis

    Science.gov (United States)

    Yang, Yingchao; Zhao, Jinping; Yang, Yutao; Cao, Yongguo; Hong, Cailing; Liu, Yuan; Sun, Lan; Huang, Minjun; Gu, Junchao

    2013-01-01

    Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HBMs) to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold). In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD)-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha), were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection. PMID:24130911

  18. Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract

    Directory of Open Access Journals (Sweden)

    William R. Schwan

    2017-01-01

    Full Text Available Uropathogenic Escherichia coli (UPEC adhere to cells in the human urinary tract via type 1 pili that undergo phase variation where a 314-bp fimS DNA element flips between Phase-ON and Phase-OFF orientations through two site-specific recombinases, FimB and FimE. Three fim-lux operon transcriptional fusions were created and moved into the clinical UPEC isolate NU149 to determine their temporal regulation in UPEC growing in the urinary tract. Within murine urinary tracts, the UPEC strains demonstrated elevated transcription of fimA and fimB early in the infection, but lower transcription by the fifth day in murine kidneys. In contrast, fimE transcription was much lower than either fimA or fimB early, increased markedly at 24 h after inoculation, and then dropped five days after inoculation. Positioning of fimS was primarily in the Phase-ON position over the time span in UPEC infected bladders, whereas in UPEC infected murine kidneys the Phase-OFF orientation was favored by the fifth day after inoculation. Hemagglutination titers with guinea pig erythrocytes remained constant in UPEC growing in infected murine bladders but fell substantially in UPEC infected kidneys over time. Our results show temporal in vivo regulation of fim gene expression in different environmental niches when UPEC infects the murine urinary tract.

  19. Molecular cloning and functional characterization of two forms of Pax8 in the rainbow trout, Oncorhynchus mykiss

    Science.gov (United States)

    Katagiri, Nobuto; Uemae, Youji; Sakamoto, Joe; Hidaka, Yoshie; Susa, Takao; Kato, Yukio; Kimura, Shioko; Suzuki, Masakazu

    2014-01-01

    We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription. PMID:24380675

  20. Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

    Science.gov (United States)

    Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

    2015-01-01

    Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. © 2014 AlphaMed Press.

  1. The immunological effect of 8-methoxypsoralen and UVA treatment on murine T-cell leukemia

    International Nuclear Information System (INIS)

    Tingying Cheng; Fungwin Shen; Ronghwa Lin

    1996-01-01

    8-Methoxyproralen (8-MOP) plus long-wavelength UV radiation (UVA, 320-400 nm) have been used to treat various diseases such as cutaneous T-cell lymphoma, systemic scleroderma, rheumatoid arthritis and rejection of heart transplants. However, the immunological mechanism of this treatment remains unknown. In this report, we investigated the effect of 8-MOP/UVA on the modulation of the immunogenicity of a T-cell leukemia cell line (RL ''male'' 1 cells). The results demonstrated that the stimulator function of the in vitro 8-MOP/UVA-treated RL ''male'' 1 cells was enhanced in both RL ''male'' 1-specific allogeneic and syngeneic immune responses. Furthermore, the enhancement of the immunogenicity of the 8-MOP/UVA-treated RL ''male'' 1 cells was found to be strongly associated with the increase of intercellular adhesion molecule-1 expression on these 8-MOP/UVA-treated tumor cells. Therefore, our findings suggested that the alteration of the expression of the immune-related cell surface molecules might be an important effect of 8-MOP/UVA treatment on the elevation of the immunogenicity of the 8-MOP/UVA-treated tumor cells. (Author)

  2. The immunological effect of 8-methoxypsoralen and UVA treatment on murine T-cell leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Tingying Cheng; Fungwin Shen; Ronghwa Lin [National Taiwan Univ., Taipei (China)

    1996-09-01

    8-Methoxyproralen (8-MOP) plus long-wavelength UV radiation (UVA, 320-400 nm) have been used to treat various diseases such as cutaneous T-cell lymphoma, systemic scleroderma, rheumatoid arthritis and rejection of heart transplants. However, the immunological mechanism of this treatment remains unknown. In this report, we investigated the effect of 8-MOP/UVA on the modulation of the immunogenicity of a T-cell leukemia cell line (RL ``male`` 1 cells). The results demonstrated that the stimulator function of the in vitro 8-MOP/UVA-treated RL ``male`` 1 cells was enhanced in both RL ``male`` 1-specific allogeneic and syngeneic immune responses. Furthermore, the enhancement of the immunogenicity of the 8-MOP/UVA-treated RL ``male`` 1 cells was found to be strongly associated with the increase of intercellular adhesion molecule-1 expression on these 8-MOP/UVA-treated tumor cells. Therefore, our findings suggested that the alteration of the expression of the immune-related cell surface molecules might be an important effect of 8-MOP/UVA treatment on the elevation of the immunogenicity of the 8-MOP/UVA-treated tumor cells. (Author).

  3. Biodistribution studies of {sup 99m}Tc-labeled myoblasts in a murine model of muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Colombo, F.R. E-mail: colombof@policlinico.mi.it; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D' Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P

    2001-11-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various {sup 99m}Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding {sup 99m}Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [{sup 99m}Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

  4. Sustained CD8+ T-cell responses induced after acute parvovirus B19 infection in humans

    DEFF Research Database (Denmark)

    Norbeck, Oscar; Isa, Adiba; Pöhlmann, Christoph

    2005-01-01

    Murine models have suggested that CD8+ T-cell responses peak early in acute viral infections and are not sustained, but no evidence for humans has been available. To address this, we longitudinally analyzed the CD8+ T-cell response to human parvovirus B19 in acutely infected individuals. We...... observed striking CD8+ T-cell responses, which were sustained or even increased over many months after the resolution of acute disease, indicating that CD8+ T cells may play a prominent role in the control of parvovirus B19 and other acute viral infections of humans, including potentially those generated...

  5. Effects of the murine skull in optoacoustic brain microscopy.

    Science.gov (United States)

    Kneipp, Moritz; Turner, Jake; Estrada, Héctor; Rebling, Johannes; Shoham, Shy; Razansky, Daniel

    2016-01-01

    Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    Science.gov (United States)

    Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

    2017-01-01

    The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

  7. Transporters MCT8 and OATP1C1 maintain murine brain thyroid hormone homeostasis

    NARCIS (Netherlands)

    Mayerl, Steffen; Müller, Julia; Bauer, Reinhard; Richert, Sarah; Kassmann, Celia M.; Darras, Veerle M.; Buder, Katrin; Boelen, Anita; Visser, Theo J.; Heuer, Heike

    2014-01-01

    Allan-Herndon-Dudley syndrome (AHDS), a severe form of psychomotor retardation with abnormal thyroid hormone (TH) parameters, is linked to mutations in the TH-specific monocarboxylate transporter MCT8. In mice, deletion of Mct8 (Mct8 KO) faithfully replicates AHDS-associated endocrine abnormalities;

  8. P300/CBP acts as a coactivator to cartilage homeoprotein-1 (Cart1), paired-like homeoprotein, through acetylation of the conserved lysine residue adjacent to the homeodomain.

    Science.gov (United States)

    Iioka, Takashi; Furukawa, Keizo; Yamaguchi, Akira; Shindo, Hiroyuki; Yamashita, Shunichi; Tsukazaki, Tomoo

    2003-08-01

    The paired-like homeoprotein, Cart1, is involved in skeletal development. We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins. Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation. Cart1 encodes a paired-like homeoprotein expressed selectively in chondrocyte lineage during embryonic development. Although its target gene remains unknown, gene disruption studies have revealed that Cart1 plays an important role for craniofacial bone formation as well as limb development by cooperating with another homeoprotein, Alx4. In this report, we study the functional involvement of p300/CBP, coactivators with intrinsic histone acetyltransferase (HAT) activity, in the transcriptional control of Cart1. To study the transcription activity of Cart1, a reporter construct containing a putative Cart1 binding site was transiently transfected with the expression vectors of each protein. The interaction between p300/CBP and Cart1 was investigated by glutathione S-transferase (GST) pull-down, yeast two-hybrid, and immunoprecipitation assays. In vitro acetylation assay was performed with the recombinant p300-HAT domain and Cart1 in the presence of acetyl-CoA. p300 and CBP stimulate Cart1-dependent transcription activity, and this transactivation is inhibited by E1A and Tax, oncoproteins that suppress the activity of p300/CBP. Cart1 binds to p300 in vivo and in vitro, and this requires the homeodomain of Cart 1 and N-terminal 139 amino acids of p300. Confocal microscopy analysis shows that Cart1 recruits overexpressed and endogenous p300 to a Cart1-specific subnuclear compartment. Cart1 is acetylated in vivo and sodium butyrate and trichostatin A, histone deacetylase inhibitors, markedly enhance the transcription activity of Cart1. Deletion and mutagenesis analysis identifies the 131st

  9. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  10. A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP+-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

    2017-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP + -induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Differential expression of nanog1 and nanogp8 in colon cancer cells

    International Nuclear Information System (INIS)

    Ishiguro, Tatsuya; Sato, Ai; Ohata, Hirokazu; Sakai, Hiroaki; Nakagama, Hitoshi; Okamoto, Koji

    2012-01-01

    Highlights: ► Nanog is expressed in a majority of colon cancer cell lines examined. ► Both nanog1 and nanogp8 are expressed in colon cancer cells with varying ratios. ► Nanog mediates cell proliferation of colon cancer cells. ► Nanog predominantly localizes in cytoplasm of colon cancer cells. -- Abstract: Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

  12. Differential expression of nanog1 and nanogp8 in colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishiguro, Tatsuya; Sato, Ai; Ohata, Hirokazu; Sakai, Hiroaki [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Nakagama, Hitoshi, E-mail: hnakagam@ncc.go.jp [Division of Cancer Development System, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Okamoto, Koji, E-mail: kojokamo@ncc.go.jo [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Nanog is expressed in a majority of colon cancer cell lines examined. Black-Right-Pointing-Pointer Both nanog1 and nanogp8 are expressed in colon cancer cells with varying ratios. Black-Right-Pointing-Pointer Nanog mediates cell proliferation of colon cancer cells. Black-Right-Pointing-Pointer Nanog predominantly localizes in cytoplasm of colon cancer cells. -- Abstract: Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

  13. Hyper-radiation sensitivity of murine scid mutation and mapping of the human homologue HYRC1 gene

    International Nuclear Information System (INIS)

    Komatsu, Kenshi; Ohta, Tohru; Niikawa, Norio; Okumura, Yutaka; Kubota, Nobuo.

    1994-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a defect in lymphoid variable-(diversity)-joining(V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Present experiments also demonstrated the high sensitivity of scid cells to killing, because of a deficient repair of double strand breaks(DSB). Scid cells can repair only 60% of radiation-induced DSB for 3 hours, while normal cells repair 85% of the DSB. Significantly reduced Do and n values were obtained from survival curves of scid cells and were similar to ataxia-telangiectasia(AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Using these hybrid cells, fragments of human chromosome 8 were introduced into scid cells HPRT mutant via X-irradiation and somatic cell fusion. The resulting hybrid clones contained human DNA fragment(s) which complemented the hyper-radiosensitivity of the scid cells. Alu-PCR products from these hybrids were used for chromosome painting using the technique of chromosome in situ suppression hybridization, allowing assignment of the human HYRC1 (hyper-radiosensitivity of murine scid mutation, complementing 1) gene, a candidate for a V(D)J recombinant gene, to human chromosome 8q11. (author)

  14. CD8+ T cells provide immune protection against murine disseminated endotheliotropic Orientia tsutsugamushi infection.

    Directory of Open Access Journals (Sweden)

    Guang Xu

    2017-07-01

    Full Text Available Scrub typhus, caused by a Gram-negative obligately intracellular coccobacillus, Orientia tsutsugamushi, is a long neglected but important tropical disease. Orientia tsutsugamushi causes illness in one million people each year, and 1 billion people are at risk. Without appropriate diagnosis and treatment, the disease can cause severe multiorgan failure with a case fatality rate of 7-15%. The current gaps in knowledge of immunity include the unknown mechanisms of host immunity to O. tsutsugamushi. Using an intravenous (i.v. disseminated infection mouse model, we observed that more CD8+ T cells than CD4+ T cells were present in the spleen of infected mice at 12 dpi. We also determined that Treg cells and the proportion of T cells producing IL-10 were significantly increased from 6 dpi, which correlated with the onset of illness, body weight loss, and increased bacterial loads. We further studied CD8-/-, MHC I-/- and wild type control (WT C57BL/6J mice to determine the importance of CD8+ T cells and MHC I molecules. After infection with an ordinarily sub-lethal dose of O. tsutsugamushi, all CD8-/- and MHC I-/- mice were moribund between 12 and 15 dpi, whereas all WT mice survived. Bacterial loads in the lung, kidney, liver and spleen of CD8-/- and MHC I-/- mice were significantly greater than those in WT mice. Interferon-γ (IFN-γ and granzyme B mRNA levels in the liver of CD8-/- and MHC I-/- mice were significantly greater than in WT mice. In addition, more severe histopathologic lesions were observed in CD8-/- mice. Finally, adoptive transfer confirmed a major role of immune CD8+ T cells as well as a less effective contribution by immune CD8 T cell-depleted splenocytes in protection against O. tsutsugamushi infection. These studies demonstrated the critical importance of CD8+ T cells in the host immune response during O. tsutsugamushi infection.

  15. A novel combination treatment of armed oncolytic adenovirus expressing IL-12 and GM-CSF with radiotherapy in murine hepatocarcinoma

    International Nuclear Information System (INIS)

    Kim, Wonwoo; Seong, Jinsil; Oh, Hae-Jin; Koom, Woong-Sub; Choi, Kyung-Joo; Yun, Chae-Ok

    2011-01-01

    In this study, a novel combination treatment of armed oncolytic adenovirus expressing interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF) with radiation was investigated for antitumor and antimetastatic effect in a murine hepatic cancer (HCa-I) model. Tumor bearing syngeneic mice were treated with radiation, armed oncolytic virus Ad-ΔE1Bmt7 (dB7) expressing both IL-12 and GM-CSF (armed dB7), or a combination of both. The adenovirus was administered by intratumoral injection 1 x 10 8 plaque forming units (PFU) per tumor in 50 μl of phosphate buffered saline (PBS) four times every other day. Tumor response to treatment was determined by a tumor growth delay assay. Metastatic potential was evaluated by a lung metastasis model. To understand the underlying mechanism, the level of apoptosis was examined as well as the change in microvessel density and expression of immunological markers: CD4+, CD8+ and Cd11c. The combination of armed dB7 and radiation resulted in significant growth delay of murine hepatic cancer, HCa-1, with an enhancement factor of 4.3. The combination treatment also resulted in significant suppression of lung metastasis. Increase of apoptosis level as well as decrease of microvessel density was shown in the combination treatment, suggesting an underlying mechanism for the enhancement of antitumor effect. Expression of immunological markers: CD4+, CD8+ and Cd11c also increased in the combination treatment. This study showed that a novel combination treatment of radiotherapy with armed oncolytic adenovirus expressing IL-12 and GM-CSF was effective in suppressing primary tumor growth. (author)

  16. A novel mouse model of creatine transporter deficiency [v1; ref status: indexed, http://f1000r.es/4f8

    Directory of Open Access Journals (Sweden)

    Laura Baroncelli

    2014-09-01

    Full Text Available Mutations in the creatine (Cr transporter (CrT gene lead to cerebral creatine deficiency syndrome-1 (CCDS1, an X-linked metabolic disorder characterized by cerebral Cr deficiency causing intellectual disability, seizures, movement  and behavioral disturbances, language and speech impairment ( OMIM #300352. CCDS1 is still an untreatable pathology that can be very invalidating for patients and caregivers. Only two murine models of CCDS1, one of which is an ubiquitous knockout mouse, are currently available to study the possible mechanisms underlying the pathologic phenotype of CCDS1 and to develop therapeutic strategies. Given the importance of validating phenotypes and efficacy of promising treatments in more than one mouse model we have generated a new murine model of CCDS1 obtained by ubiquitous deletion of 5-7 exons in the Slc6a8 gene. We showed a remarkable Cr depletion in the murine brain tissues and cognitive defects, thus resembling the key features of human CCDS1. These results confirm that CCDS1 can be well modeled in mice. This CrT−/y murine model will provide a new tool for increasing the relevance of preclinical studies to the human disease.

  17. Pax3 stimulates p53 ubiquitination and degradation independent of transcription.

    Directory of Open Access Journals (Sweden)

    Xiao Dan Wang

    Full Text Available Pax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function.We employed murine embryonic stem cell (ESC-derived neuronal precursors as a cell culture model of embryonic neuroepithelium or neural crest. Pax3 reduced p53 protein stability, but had no effect on p53 mRNA levels or the rate of p53 synthesis. Full length Pax3 as well as fragments that contained either the DNA-binding paired box or the homeodomain, expressed as GST or FLAG fusion proteins, physically associated with p53 and Mdm2 both in vitro and in vivo. In contrast, Splotch Pax3, which causes neural tube and neural crest defects in homozygous embryos, bound weakly, or not at all, to p53 or Mdm2. The paired domain and homeodomain each stimulated Mdm2-mediated ubiquitination of p53 and p53 degradation in the absence of the Pax3 transcription regulatory domains, whereas Splotch Pax3 did not stimulate p53 ubiquitination or degradation.Pax3 inactivates p53 function by stimulating its ubiquitination and degradation. This process utilizes the Pax3 paired domain and homeodomain but is independent of DNA-binding and transcription regulation. Because inactivating p53 is the only required Pax3 function during neural tube closure and cardiac neural crest development, and inactivating p53 does not require Pax3-dependent transcription regulation, this indicates that Pax3 is not required to function as a transcription factor during neural tube closure and cardiac neural crest development. These findings further suggest novel explanations for PAX3 functions in human diseases, such as in neural crest-derived cancers and Waardenburg syndrome types 1 and 3.

  18. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  19. Comparison of vectorial ion transport in primary murine airway and human sinonasal air-liquid interface cultures, models for studies of cystic fibrosis, and other airway diseases.

    Science.gov (United States)

    Zhang, Shaoyan; Fortenberry, James A; Cohen, Noam A; Sorscher, Eric J; Woodworth, Bradford A

    2009-01-01

    The purpose of this study was to compare vectorial ion transport within murine trachea, murine nasal septa, and human sinonasal cultured epithelium. Our hypothesis is that murine septal epithelium, rather than trachea, will more closely mimic the electrophysiology properties of human sinonasal epithelium. Epithelium from murine trachea, murine septa, and human sinonasal tissue were cultured at an air-liquid interface to confluence and full differentiation. A limited number of homozygous dF508 epithelia were also cultured. Monolayers were mounted in modified Ussing chambers to investigate pharmacologic manipulation of ion transport. The change in forskolin-stimulated current (delta-I(SC), expressed as micro-A/cm(2)) in murine septal (n = 19; 16.84 +/- 2.09) and human sinonasal (n = 18; 12.15 +/- 1.93) cultures was significantly increased over murine tracheal cultures (n = 15; 6.75 +/- 1.35; p = 0.035 and 0.0005, respectively). Forskolin-stimulated I(SC) was inhibited by the specific cystic fibrosis transmembrane regulator (CFTR) inhibitor INH-172 (5 microM). No forskolin-stimulated I(SC) was shown in cultures of dF508 homozygous murine septal epithelium (n = 3). Murine septal I(SC) was largely inhibited by amiloride (12.03 +/- 0.66), whereas human sinonasal cultures had a very limited response (0.70 +/- 0.47; p < 0.0001). The contribution of CFTR to stimulated chloride current as measured by INH-172 was highly significantly different between all groups (murine septa, 19.51 +/- 1.28; human sinonasal, 11.12 +/- 1.58; murine trachea, 4.85 +/- 0.49; p < 0.0001). Human sinonasal and murine septal epithelial cultures represent a useful model for studying CFTR activity and may provide significant advantages over lower airway tissues for investigating upper and lower respiratory pathophysiology.

  20. Anti-CD45 radioimmunotherapy with 90Y but not 177Lu is effective treatment in a syngeneic murine leukemia model.

    Directory of Open Access Journals (Sweden)

    Johnnie J Orozco

    Full Text Available Radioimmunotherapy (RIT for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab labeled with 131I or 90Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing 90Y and 177Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J model. Biodistribution studies showed that both 90Y- and 177Lu-anti-murine CD45 Ab conjugates (DOTA-30F11 targeted hematologic tissues, as at 24 hours 48.8 ± 21.2 and 156 ± 14.6% injected dose per gram of tissue (% ID/g of 90Y-DOTA-30F11 and 54.2 ± 9.5 and 199 ± 11.7% ID/g of 177Lu-DOTA-30F11 accumulated in bone marrow (BM and spleen, respectively. However, 90Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi 90Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi 90Y-DOTA-30F11 had a median survival 66 days. 90Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, 177Lu- anti-CD45 RIT yielded no long-term survivors. Thus, 90Y was more effective than 177Lu for anti-CD45 RIT of AML in this murine leukemia model.

  1. PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

    Science.gov (United States)

    Zhang, Tiejun; Park, Kyeong Ah; Li, Yuwen; Byun, Hee Sun; Jeon, Juhee; Lee, Yoonjung; Hong, Jang Hee; Kim, Jin Man; Huang, Song-Mei; Choi, Seung-Won; Kim, Sun-Hwan; Sohn, Kyung-Cheol; Ro, Hyunju; Lee, Ji Hoon; Lu, Tao; Stark, George R.; Shen, Han-Ming; Liu, Zheng-gang; Park, Jongsun; Hur, Gang Min

    2014-01-01

    Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers. PMID:23797602

  2. Murine polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor.

    Directory of Open Access Journals (Sweden)

    Mathilda Eriksson

    Full Text Available Virus-like particles (VLPs consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV VLPs carrying the entire human Prostate Specific Antigen (PSA (PSA-MPyVLPs for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs. Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4(+ and CD8(+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4(+ and CD8(+ cells with a low induction of anti-VLP antibodies.

  3. Murine Polyomavirus Virus-Like Particles Carrying Full-Length Human PSA Protect BALB/c Mice from Outgrowth of a PSA Expressing Tumor

    Science.gov (United States)

    Eriksson, Mathilda; Andreasson, Kalle; Weidmann, Joachim; Lundberg, Kajsa; Tegerstedt, Karin

    2011-01-01

    Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4+ and CD8+ cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4+ and CD8+ cells with a low induction of anti-VLP antibodies. PMID:21858228

  4. Turnover of T cells in murine gammaherpesvirus 68-infected mice

    DEFF Research Database (Denmark)

    Hamilton-Easton, A M; Christensen, Jan Pravsgaard; Doherty, P C

    1999-01-01

    Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found...

  5. Local IL-23 expression in murine vaginal candidiasis and its relationship with infection and immune status.

    Science.gov (United States)

    Wu, Yan; Tan, Zhijian; Liu, Zhixiang; Xia, Dechao; Li, Jiawen

    2006-01-01

    To investigate the expression of vaginal IL-23 and its role in experimental murine vaginal candidiasis and its relationship with infection and immune status, immuno-competent (group A) and immuno-suppressed (group B) murine models of vaginal candidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls (group C). The level of IL-23 p19 mRNA in murine vaginal tissue was determined by RT-PCR. Significantly increased levels of IL-23p19mRNA were observed on the 4th, the 7th and 14th day after inoculation in immuno-competent group when compared with that in control group (Pvaginal candidiasis and has a protective function during infection. Low vaginal IL-23 level may correlate with the increased susceptibility to Candida albicans in immuno-suppressed group.

  6. [Evaluation of Fusarium spp. pathogenicity in plant and murine models].

    Science.gov (United States)

    Forero-Reyes, Consuelo M; Alvarado-Fernández, Angela M; Ceballos-Rojas, Ana M; González-Carmona, Lady C; Linares-Linares, Melva Y; Castañeda-Salazar, Rubiela; Pulido-Villamarín, Adriana; Góngora-Medina, Manuel E; Cortés-Vecino, Jesús A; Rodríguez-Bocanegra, María X

    The genus Fusarium is widely recognized for its phytopathogenic capacity. However, it has been reported as an opportunistic pathogen in immunocompetent and immunocompromised patients. Thus, it can be considered a microorganism of interest in pathogenicity studies on different hosts. Therefore, this work evaluated the pathogenicity of Fusarium spp. isolates from different origins in plants and animals (murine hosts). Twelve isolates of Fusarium spp. from plants, animal superficial mycoses, and human superficial and systemic mycoses were inoculated in tomato, passion fruit and carnation plants, and in immunocompetent and immunosuppressed BALB/c mice. Pathogenicity tests in plants did not show all the symptoms associated with vascular wilt in the three plant models; however, colonization and necrosis of the vascular bundles, regardless of the species and origin of the isolates, showed the infective potential of Fusarium spp. in different plant species. Moreover, the pathogenicity tests in the murine model revealed behavioral changes. It was noteworthy that only five isolates (different origin and species) caused mortality. Additionally, it was observed that all isolates infected and colonized different organs, regardless of the species and origin of the isolates or host immune status. In contrast, the superficial inoculation test showed no evidence of epidermal injury or colonization. The observed results in plant and murine models suggest the pathogenic potential of Fusarium spp. isolates in different types of hosts. However, further studies on pathogenicity are needed to confirm the multihost capacity of this genus. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. Sex differences in the MB49 syngeneic, murine model of bladder cancer.

    Science.gov (United States)

    White-Gilbertson, Shai; Davis, Megan; Voelkel-Johnson, Christina; Kasman, Laura M

    The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro . MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro . The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice.

  8. Nanoelectroablation therapy for murine basal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nuccitelli, Richard, E-mail: rich@bioelectromed.com [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States); Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States); Chang, Kris S.; Epstein, Ervin H. [The Children' s Hospital Oakland Research Institute, Oakland, CA 94609 (United States); Tang, Jean Y. [The Children' s Hospital Oakland Research Institute, Oakland, CA 94609 (United States); Stanford University, Stanford, CA 94305 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  9. Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis.

    Directory of Open Access Journals (Sweden)

    Rose McGready

    2014-11-01

    Full Text Available There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus.Data were analyzed from: i pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii case series of published studies in PubMed using the search terms "scrub typhus" (ST, "murine typhus" (MT, "Orientia tsutsugamushi", "Rickettsia tsutsugamushi", "Rickettsia typhi", "rickettsiae", "typhus", or "rickettsiosis"; and "pregnancy", until February 2014 and iii an unpublished case series. Fever clearance time (FCT and pregnancy outcome (miscarriage and delivery were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn.There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours were 25 [16-42] for azithromycin (n=5, 34 [20-53] for antimalarials (n=5 and 92 [6-260] for other antibiotics/supportive therapy (n=16. There were 36.4% (8/22 with a poor neonatal outcome. In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81 and poor neonatal outcomes 41.8% (28/67 were high, increasing with longer FCTs (p=0.050, linear trend. Use of azithromycin was not significantly associated with improved neonatal outcomes (p=0.610.The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and

  10. Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

    Science.gov (United States)

    Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

    2016-04-15

    B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Transcriptome Profile Analysis of Mechanisms of Black and White Plumage Determination in Black-Bone Chicken

    Directory of Open Access Journals (Sweden)

    Shigang Yu

    2018-05-01

    Full Text Available Background/Aims: Melanin is a major and ubiquitous component of plumage colouration, and patterns of melanin pigmentation in birds are extremely varied. However, the molecular mechanism of pigmentation in avian plumage is still largely unknown. Methods: To elucidate the molecular mechanisms involved in the formation of black and white plumage, this study takes advantage of high-throughput sequencing technology to compare differences in the transcriptome between black and white chicken feather bulbs. In total, we constructed six cDNA libraries from black (Group B and white (Group W feather bulbs in the dorsal plumage of Muchuan black-boned chickens. Results: A comparison between Groups B and W revealed 61 differentially expressed genes, with 47 displaying higher, and 14 displaying lower, levels of expression in white feather bulbs. Our results revealed a set of candidate genes and two potential metabolic pathways involved in black-bone chicken plumage melanogenesis. These include four homeobox genes (HOXB9, HOXC8, HOXA9, and HOXC 9, two glutathione (GSH metabolism-related genes (CHAC1 and GPX3, and the transforming growth factor beta (TGF-β signalling pathway. Two known genes, TYR and MITF, were also shown to play a role in melanin formation. Conclusion: our data provide a valuable resource for discovering genes important in plumage melanin formation and will help further elucidate the molecular mechanisms for black and white plumage.

  12. Development of a murine model of acute radiation encephalopathy

    International Nuclear Information System (INIS)

    Xing Yigang; Tang Yamei; Liu Jun; Sun Ying

    2003-01-01

    Objective: To develop a murine model of acute radiation encephalopathy. Methods: A total of 40 rats were subjected to local γ-irradiation to the brain with the dosage of 7 Gy/d for 6 consecutive days. The amount of food intake, hairs and skin of irradiated field, body weight, general activities, CNS symptoms and signs were examined and recorded after irradiation. On day 3, 7, 14 and 30, the brain tissue was removed to observe histopathologic changes. Results: During the first two days after irradiation, the irradiated rats were agitated, and the amount of food intake decreased from day 2 onwards. No serious skin reaction to irradiation was observed. Survived rats had normal activities without any abnormal nervous signs. Histopathologic changes showed slight neuronal degeneration, smaller cell body, red-colored cytoplasm, disappearance of Nissl body, vacuolation, typical cell shrinkage, chromatin condensation and nuclear divergence. On the 14th and 30th days, hypochromatism, loose and reticular necrotic foci were found in some samples. Conclusion: The murine model of acute radiation encephalopathy is useful and practical in radiobiological studies

  13. JST Thesaurus Headwords and Synonyms: murine hepatitis virus [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term murine hepatitis virus 名詞 一般 * * * * マウス肝炎...ウイルス マウスカンエンウイルス マウスカンエンウイルス Thesaurus2015 200906024862381907 C LS07 UNKNOWN_2 murine hepatitis virus

  14. [Nuclear matrix organization of the chromocenters in cultured murine fibroblasts].

    Science.gov (United States)

    Sheval', E V; Poliakov, V Iu

    2010-01-01

    In the current work, the structural organization of nuclear matrix of pericentromeric heterochromatin blocks (chromocenters) inside cultured murine fibroblasts was investigated. After 2 M NaCl extraction without DNase I treatment, chromocenters were extremely swelled, and it was impossible to detect them using conventional electron microscopy. Using immunogolding with anti-topoisomerase IIalpha antibody, we demonstrated that residual chromocenters were subdivided into numerous discrete aggregates. After 2 M NaCl extraction with DNase I treatment, the residual chromocenters appeared as a dense meshwork of thin fibers, and using this feature, the residual chromocenters were easily distinguished from the rest of nuclear matrix. After extraction with dextran sulfate and heparin, the chromocenters were decondensed, and chromatin complexes having rosette organization (central core from which numerous DNA fibers radiated) were seen. Probably, the appearance of these rosettes was a consequence of incomplete chromatin extraction. Thus, the nuclear matrix of pericentromeric chromosome regions in cultured murine fibroblasts differs morphologically from the rest of nuclear matrix.

  15. Anti-tumour therapeutic efficacy of OX40L in murine tumour model.

    Science.gov (United States)

    Ali, Selman A; Ahmad, Murrium; Lynam, June; McLean, Cornelia S; Entwisle, Claire; Loudon, Peter; Choolun, Esther; McArdle, Stephanie E B; Li, Geng; Mian, Shahid; Rees, Robert C

    2004-09-09

    OX40 ligand (OX40L), a member of TNF superfamily, is a co-stimulatory molecule involved in T cell activation. Systemic administration of mOX40L fusion protein significantly inhibited the growth of experimental lung metastasis and subcutaneous (s.c.) established colon (CT26) and breast (4T1) carcinomas. Vaccination with OX40L was significantly enhanced by combination treatment with intra-tumour injection of a disabled infectious single cycle-herpes simplex virus (DISC-HSV) vector encoding murine granulocyte macrophage-colony stimulating factor (mGM-CSF). Tumour rejection in response to OX40L therapy required functional CD4+ and CD8+ T cells and correlated with splenocyte cytotoxic T lymphocytes (CTLs) activity against the AH-1 gp70 peptide of the tumour associated antigen expressed by CT26 cells. These results demonstrate the potential role of the OX40L in cancer immunotherapy.

  16. Radiobiological studies on target cell populations in murine bone marrow transplantation recipients.

    NARCIS (Netherlands)

    van Os, Ronald Peter

    1994-01-01

    The experiments presented in this thesis were designed to investigate the role of total body irradiation (TBI) in conditioning murine recipients of syngeneic and allogeneic bone marrow transplantation (BMT). ... Zie: Summary

  17. Responses of the Murine Myeloid Colony-Forming Cell to Ansamycin Antibiotics

    Science.gov (United States)

    Horoszewicz, Julius S.; Carter, William A.

    1974-01-01

    The in vitro susceptibility of murine myeloid colony-forming cells to the antiproliferative activities of three ansamycin antibiotics was determined. These cells were found to be 10- to 40-fold more susceptible than the corresponding human ones. PMID:4151701

  18. JST Thesaurus Headwords and Synonyms: murine leukemia virus [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term murine leukemia virus 名詞 一般 * * *... * マウス白血病ウイルス マウスハッケツビョウウイルス マウスハッケツビョーウイルス Thesaurus2015 200906060491156251 C LS07 UNKNOWN_2 murine leukemia virus

  19. Forced recombination of psi-modified murine leukaemia virus-based vectors with murine leukaemia-like and VL30 murine endogenous retroviruses

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Duch, M

    1999-01-01

    Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site-imp....... We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.......-impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region...... harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription...

  20. Transgene stability for three replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Duch, M.; Carrasco, M.L.; Jespersen, T.

    2004-01-01

    cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third...

  1. Evaluation of Hollow Fiber And Miniperm Bioreactors as An Alternative to Murine Ascites for Small Scale Monoclonal Antibody Production

    International Nuclear Information System (INIS)

    Abedalla, O. M.

    2007-01-01

    The objective of this study was to compare monoclonal antibody production in hollow fiber, miniPERM bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. One hybridoma cell line was grown in hollow fiber, miniPERM bioreactor systems and in groups of 5 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X10 7 hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Bioreactors were harvested three times weekly for 30 days and were monitored by cell counts, cell viability and media consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 5 mice versus the total production for the two different bioreactors (hollow fiber and miniPERM) in 30 days was as follows: cell line 2AC10E6C7 produce 158 mg vs.97.5 mg; vs 21.54 mg respectively. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, from 0.71 to 3.8 mg/ml in hollow fiber bioreactor system, and from 0.035 to 1.06 in miniPERM. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber and miniPERM bioreactor systems merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

  2. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification.

    Science.gov (United States)

    Lai, D; Ding, J; Smith, G W; Smith, G D; Takayama, S

    2015-01-01

    Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less

  3. Establishment of a method of murine obestatin RIA and its primary application

    International Nuclear Information System (INIS)

    Yan Guangtao Lin Ji; Hao Xiuhua; Xue Hui; Den Zihui; Di Dongdong

    2009-01-01

    Objective: To develop a method of murine obestatin RIA and study the role obestatin played in traumatic stress responses. Methods: New Zealand white rabbits were immunized with synthetic murine obestatin to obtain anti-serum, while chloramines-T method was used to iodinate obestatin antigen and a method of RIA for obestatin was established. A mouse sepsis model made with cecal ligation and puncture was established. Serum obestatin levels in the sacrificed models were determined with this method of RIA at 3h, 6h, 9h after the cecum puncture respectively (5 animals each time) as well as in 5 animals after sham operation. Results: Both the shape of standard curve and metrical results of the obestatin RIA were satisfactory. Serum obstatin levels at 6h after injury were significantly higher than those in animals with sham operation out the levels at 3h and 9h were not much different from those in sham operation animals. Meanwhile, the levels at 6h after injury expressed a trend to be significantly higher than that at 3h or 9h after injury. Conclusion: The established method for murine obestatin RIA is highly sensitive, simple and reliable, and it can be used to detect samples from rats and mice. Obestatin may be a traumatic stress factor participating in the modulation of homeostasis after sepsis. (authors)

  4. The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

    Science.gov (United States)

    Dickie, A P; Wilson, C E; Schreiter, K; Wehr, R; Wilson, E M; Bial, J; Scheer, N; Wilson, I D; Riley, R J

    2017-07-01

    The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08μg/mL at 0.25-0.5h post-dose with an AUC inf of 1.74±0.52μgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08μg/mL at 0.25h post-dose with an AUC inf of 1.94±0.22μgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

    Directory of Open Access Journals (Sweden)

    Patrick J Mott

    Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

  6. Noncanonical microRNAs and endogenous siRNAs in lytic infection of murine gammaherpesvirus.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available MicroRNA (miRNA and endogenous small interfering RNA (endo-siRNA are two essential classes of small noncoding RNAs (sncRNAs in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68. In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs. Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68.

  7. Limited role of murine ATM in oncogene-induced senescence and p53-dependent tumor suppression.

    Directory of Open Access Journals (Sweden)

    Alejo Efeyan

    Full Text Available Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

  8. The development of a murine model for Forcipomyia taiwana (biting midge) allergy.

    Science.gov (United States)

    Lee, Mey-Fann; Yang, Kai-Jei; Wang, Nancy M; Chiu, Yung-Tsung; Chen, Pei-Chih; Chen, Yi-Hsing

    2014-01-01

    Forcipomyia taiwana (biting midge) allergy is the most prevalent biting insect allergy in Taiwan. An animal model corresponding to the human immuno-pathologic features of midge allergy is needed for investigating the mechanisms and therapies. This study successfully developed a murine model of Forcipomyia taiwana allergy. BALB/c mice were sensitized intra-peritoneally with midge extract on days 0, 7, 14, 21 then intra-dermally on days 28, 31 and 35. Serum midge-specific IgE, IgG1, and IgG2a were measured every 14 days by indirect ELISA. The mice were challenged intradermally with midge extract at day 40 and then sacrificed. Proliferation and cytokine production of splenocytes after stimulation with midge extract were determined by MTT assay and ELISA, respectively. The cytokine mRNA expression in response to midge stimulation was analyzed by RT-PCR. Serum IgE, total IgG, and IgG1 antibody levels against midge extract were significantly higher in the midge-sensitized mice than in the control mice. After the two-step sensitization, all mice in the midge-sensitized group displayed immediate itch and plasma extravasation reactions in response to challenge with midge extract. Skin histology from midge-sensitized mice showed marked eosinophil and lymphocyte infiltrations similar to that observed in humans. Stimulation of murine splenocytes with midge extract elicited significant proliferation, IL-4, IL-10, IL-13 and IFN-γ protein production, and up-regulation of mRNA in a dose-dependent manner in the midge-sensitized group, but not in the control group. A murine model of midge bite allergy has been successfully developed using a two-step sensitization protocol. The sensitized mice have very similar clinical and immunologic reactions to challenge with midge proteins as the reactions of human to midge bites. This murine model may be a useful platform for future research and the development of treatment strategies for insect bite allergy.

  9. mEBT: multiple-matching Evidence-based Translator of Murine Genomic Responses for Human Immunity Studies.

    Science.gov (United States)

    Tae, Donghyun; Seok, Junhee

    2018-05-29

    In this paper, we introduce multiple-matching Evidence-based Translator (mEBT) to discover genomic responses from murine expression data for human immune studies, which are significant in the given condition of mice and likely have similar responses in the corresponding condition of human. mEBT is evaluated over multiple data sets and shows improved inter-species agreement. mEBT is expected to be useful for research groups who use murine models to study human immunity. http://cdal.korea.ac.kr/mebt/. jseok14@korea.ac.kr. Supplementary data are available at Bioinformatics online.

  10. Experimental myositis inducible with transfer of dendritic cells presenting a skeletal muscle C protein-derived CD8 epitope peptide.

    Science.gov (United States)

    Okiyama, Naoko; Hasegawa, Hisanori; Oida, Takatoku; Hirata, Shinya; Yokozeki, Hiroo; Fujimoto, Manabu; Miyasaka, Nobuyuki; Kohsaka, Hitoshi

    2015-07-01

    It is suggested that polymyositis, an autoimmune inflammatory myopathy, is mediated by autoaggressive CD8 T cells. Skeletal muscle C protein is a self-antigen that induces C protein-induced myositis, a murine model of polymyositis. To establish a new murine model of myositis inducible with a single CD8 T-cell epitope peptide that derives from the C protein, three internet-based prediction systems were employed to identify 24 candidate peptides of the immunogenic fragment of the C protein and bind theoretically to major histocompatibility complex class I molecules of C57BL/6 (B6) mice. RMA-S cell assay revealed that a HILIYSDV peptide, amino acid position 399-406 of the C protein, had the highest affinity to the H2-K(b) molecules. Transfer of mature bone marrow-derived dendritic cells pulsed with HILIYSDV induced myositis in naive B6 mice. This myositis was suppressed by anti-CD8-depleting antibodies but not by anti-CD4-depleting antibodies. Because this myositis model is mediated by CD8 T cells independently of CD4 T cells, it should be a useful tool to investigate pathology of polymyositis and develop therapies targeting CD8 T cells. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Trp53 activity is repressed in radio-adapted cultured murine limb bud cells

    International Nuclear Information System (INIS)

    Vares, Guillaume; Wang, Bing; Tanaka, Kaoru; Shang, Yi; Fujita, Kazuko; Hayata, Isamu; Nenoi, Mitsuru

    2011-01-01

    Understanding the effects of ionizing radiation (IR) at low dose in fetal models is of great importance, because the fetus is considered to be at the most radiosensitive stage of the development and prenatal radiation might influence subsequent development. We previously demonstrated the existence of an adaptive response (AR) in murine fetuses after pre-exposure to low doses of X-rays. Trp53-dependent apoptosis was suggested to be responsible for the teratogenic effects of IR; decreased apoptosis was observed in adapted animals. In this study, in order to investigate the role of Trp53 in AR, we developed a new model of irradiated micromass culture of fetal limb bud cells, which replicated proliferation, differentiation and response to IR in murine embryos. Murine fetuses were exposed to whole-body priming irradiation of 0.3 Gy or 0.5 Gy at embryonic day 11 (E11). Limb bud cells (collected from digital ray areas exhibiting radiation-induced apoptosis) were cultured and exposed to a challenging dose of 4 Gy at E12 equivalent. The levels of Trp53 protein and its phosphorylated form at Ser18 were investigated. Our results suggested that the induction of AR in mouse embryos was correlated with a repression of Trp53 activity. (author)

  12. Anaplasma phagocytophilum-Related Defects in CD8, NKT, and NK Lymphocyte Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Diana G. Scorpio

    2018-04-01

    Full Text Available Human granulocytic anaplasmosis, caused by the tick-transmitted Anaplasma phagocytophilum, is not controlled by innate immunity, and induces a proinflammatory disease state with innate immune cell activation. In A. phagocytophilum murine infection models, hepatic injury occurs with production of IFNγ thought to be derived from NK, NKT cells, and CD8 T lymphocytes. Specific A. phagocytophilum ligands that drive inflammation and disease are not known, but suggest a clinical and pathophysiologic basis strikingly like macrophage activation syndrome (MAS and hemophagocytic syndrome (HPS. We studied in vivo responses of NK, NKT, and CD8 T lymphocytes from infected animals for correlates of lymphocyte-mediated cytotoxicity and examined in vitro interactions with A. phagocytophilum-loaded antigen-presenting cells (APCs. Murine splenocytes were examined and found deficient in cytotoxicity as determined by CD107a expression in vitro for specific CTL effector subsets as determined by flow cytometry. Moreover, A. phagocytophilum-loaded APCs did not lead to IFNγ production among CTLs in vitro. These findings support the concept of impaired cytotoxicity with A. phagocytophilum presentation by APCs that express MHC class I and that interact with innate and adaptive immune cells with or after infection. The findings strengthen the concept of an enhanced proinflammatory phenotype, such as MAS and HPS disease states as the basis of disease and severity with A. phagocytophilum infection, and perhaps by other obligate intracellular bacteria.

  13. Protective antitumor activity induced by a fusion vaccine with murine ...

    African Journals Online (AJOL)

    Targeting angiogenesis is an effective strategy for anticancer therapy. The vascular endothelialcadherin (VE-cad) regulated angiogenesis is a potential target for anti-angiogenesis. Here, we develop a fusion vaccine plasmid DNA pSec-MBD2-VE-cad from VE-cad and murine beta defensin2 (MBD2) to induce immunity for ...

  14. Thrombopoietin inhibits murine mast cell differentiation

    Science.gov (United States)

    Martelli, Fabrizio; Ghinassi, Barbara; Lorenzini, Rodolfo; Vannucchi, Alessandro M; Rana, Rosa Alba; Nishikawa, Mitsuo; Partamian, Sandra; Migliaccio, Giovanni; Migliaccio, Anna Rita

    2009-01-01

    We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin, or addition of this growth factor to bone marrow-derived mast cell cultures, severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target anti-apoptotic gene Bcl2. PMID:18276801

  15. Human glioblastoma-associated microglia/monocytes express a distinct RNA profile compared to human control and murine samples.

    Science.gov (United States)

    Szulzewsky, Frank; Arora, Sonali; de Witte, Lot; Ulas, Thomas; Markovic, Darko; Schultze, Joachim L; Holland, Eric C; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

    2016-08-01

    Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436. © 2016 Wiley Periodicals, Inc.

  16. The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

    Directory of Open Access Journals (Sweden)

    Seim Inge

    2009-05-01

    Full Text Available Abstract Background The murine ghrelin gene (Ghrl, originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0. We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. Findings 5' RACE revealed two transcription start sites (TSSs in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR, we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. Conclusion We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1 raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

  17. Planarian homeobox genes: cloning, sequence analysis, and expression.

    Science.gov (United States)

    Garcia-Fernàndez, J; Baguñà, J; Saló, E

    1991-01-01

    Freshwater planarians (Platyhelminthes, Turbellaria, and Tricladida) are acoelomate, triploblastic, unsegmented, and bilaterally symmetrical organisms that are mainly known for their ample power to regenerate a complete organism from a small piece of their body. To identify potential pattern-control genes in planarian regeneration, we have isolated two homeobox-containing genes, Dth-1 and Dth-2 [Dugesia (Girardia) tigrina homeobox], by using degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain. Dth-1 and Dth-2 homeodomains are closely related (68% at the nucleotide level and 78% at the protein level) and show the conserved residues characteristic of the homeodomains identified to data. Similarity with most homeobox sequences is low (30-50%), except with Drosophila NK homeodomains (80-82% with NK-2) and the rodent TTF-1 homeodomain (77-87%). Some unusual amino acid residues specific to NK-2, TTF-1, Dth-1, and Dth-2 can be observed in the recognition helix (helix-3) and may define a family of homeodomains. The deduced amino acid sequences from the cDNAs contain, in addition to the homeodomain, other domains also present in various homeobox-containing genes. The expression of both genes, detected by Northern blot analysis, appear slightly higher in cephalic regions than in the rest of the intact organism, while a slight increase is detected in the central period (5 days) or regeneration. Images PMID:1714599

  18. Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor.

    Science.gov (United States)

    Riklis, I; Kletter, Y; Bleiberg, I; Fabian, I

    1989-04-01

    The effect of recombinant murine interleukin-3 (rIL-3) and recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) on in vitro murine myeloid progenitor cell (CFU-C) growth and on the function of murine resident peritoneal macrophages was investigated. Both rIL-3 and rGM-CSF are known to support the growth of CFU-C and, when combined, were found to act synergistically to induce the development of an increased number of CFU-C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM-CSF alone. Both rGM-CSF and rIL-3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM-CSF, but not rIL-3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.

  19. Detection of Xenotropic Murine Leukemia Virus-Related Virus in Prostate Biopsy Samples

    International Nuclear Information System (INIS)

    Baig, F. A.; Mirza, T.; Khanani, R.; Khan, S.

    2014-01-01

    Objective: To determine the association of Xenotropic murine leukemia virus related virus (XMRV) infection with prostate cancer and compare it with benign prostate hyperplasia. Study Design: Case control study. Place and Duration of Study: Department of Histopathology and Molecular Pathology, Dow University of Health Sciences, Karachi, from January 2009 to December 2012. Methodology: XMRV was screened in 50 prostate cancer and 50 benign prostatic hyperplasia biopsies using conventional end-point PCR. Other studied variables were family history of prostate cancer, patients age and Gleason score. Results: XMRV was detected in 4 (8%) of the 50 prostate cancer biopsy specimens compared to none in biopsies with benign prostatic hyperplasia. However, there was no significant statistical association of XMRV infection with the other variables. Conclusion: A low frequency of XMRV infection was found in this case-control study. Men, who harbor XMRV infection, may be at increased risk of prostate cancer but this needs to be investigated further at a larger scale. (author)

  20. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H

    1987-01-01

    the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native......A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form...... (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...

  1. Murine protein H is comprised of 20 repeating units, 61 amino acids in length

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Tack, B F

    1986-01-01

    A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized......, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length...

  2. Acid phosphates response in murine rhabdomyosarcoma for various tumour volumes and after different doses of neutron irradiation, alone or combined with exogenous ATP

    International Nuclear Information System (INIS)

    Szeinfeld, D.; De Villiers, N.

    1991-01-01

    Acid phosphatase activity measured in a methylocholanthrene-induced murine rhabdomyosarcoma showed a monotonically increasing relation between enzyme activity and tumour volume. This could be related to the lytic activity of the enzyme in large tumours which become more hypoxic and necrotic, and hence enhance degradation and turnover of damaged tumour cells. The tumours were also subjected to irradiation using doses of 2.0, 3.8 and 6.0 Gy from a neutron therapy facility p(66 MeV)/Be. The correlation between different doses and response of acid phosphatase activity could reflect the relation of magnitude of damage from metabolic disturbances, with dose. Furthermore exogenous ATP was shown to provide radioprotective action against neutron irradiation in two different experiments. The ATP reduced the activity of this lytic enzyme in irradiated tumours and also decreased tumour growth delay. This radioprotective role of exogenous ATP in a murine tumour could be related to physiological regulatory processes during defence mechanisms to maintain self-organisation in response to the radiation damage. (orig.) [de

  3. Tropism, Cytotoxicity, and Inflammatory Properties of Two Envelope Genes of Murine Leukemia Virus Type-Endogenous Retroviruses of C57BL/6J Mice

    Directory of Open Access Journals (Sweden)

    Young-Kwan Lee

    2011-01-01

    Full Text Available Envelope (env proteins of certain endogenous retroviruses (ERVs participate in various pathophysiological processes. In this study, we characterized pathophysiologic properties of two murine leukemia virus-type ERV (MuLV-ERV env genes cloned from the ovary of C57BL/6J mice. The two env genes (named ENVOV1 and ENVOV2, with 1,926\\,bp coding region, originated from two MuLV-ERV loci on chromosomes 8 and 18, respectively. ENVOV1 and ENVOV2 were ~75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions. Tropism trait and infectivity of ENVOV2 were similar to the polytropic env; however, ENVOV1 had very low level of infectivity. Overexpression of ENVOV2, but not ENVOV1, exerted cytotoxic effects and induced expression of COX-2, IL-1β, IL-6, and iNOS. These findings suggest that the ENVOV1 and ENVOV2 are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators.

  4. Diagnosing hypoxia in murine models of rheumatoid arthritis from reflectance multispectral images

    Science.gov (United States)

    Glinton, Sophie; Naylor, Amy J.; Claridge, Ela

    2017-07-01

    Spectra computed from multispectral images of murine models of Rheumatoid Arthritis show a characteristic decrease in reflectance within the 600-800nm region which is indicative of the reduction in blood oxygenation and is consistent with hypoxia.

  5. Evaluation of hollow fiber and mini perm bioreactors as an alternative to murine ascites for small scale monoclonal antibody production

    International Nuclear Information System (INIS)

    Abdalla, O. M.

    2006-12-01

    The objective of this study was to compare monoclonal antibody production in hollow fiber, mini perm bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. One hybridoma cell line was grown in hollow fiber, mini perm bioreactor systems and in groups of 5 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1x10 7 hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Bioreactors were harvested three times weekly for 30 days and were monitored by cell counts, cell viability and media consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 5 mice versus the total production for the two different bioreactors (hollow fiber and mini perm) in 30 days was as follows: cell line 2AC10E6C7 produce 158 mg vs.97.5 mg, vs 21.54 mg respectively. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, from 0.71 to 3.8 mg/ml in hollow fiber bioreactor system, and from 0.035 to 1.06 in mini perm. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber and mini perm bioreactor systems merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (<1mg) monoclonal antibody production.(Author)

  6. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    Science.gov (United States)

    Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

    2007-12-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (˜1×105 to 1×1010 W/m2). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

  7. Efficacy and immunological actions of FAHF-2 in a murine model of multiple food allergies.

    Science.gov (United States)

    Srivastava, Kamal D; Bardina, Ludmilla; Sampson, Hugh A; Li, Xiu-Min

    2012-05-01

    Food Allergy Herbal Formula-2 (FAHF-2) prevents anaphylaxis in a murine model of peanut allergy. Multiple food allergies (MFA) are common and associated with a higher risk of anaphylaxis. No well-characterized murine model of sensitization to multiple food allergens exists, and no satisfactory therapy for MFA is currently available. To determine the effect of FAHF-2 in a murine model of MFA. C3H/HeJ mice were orally sensitized to peanut, codfish, and egg concurrently. Oral FAHF-2 treatment commenced 1 day after completing sensitization and continued daily for 7 weeks. Mice were subsequently orally challenged with each allergen. Antibodies in sera from mice simultaneously sensitized with peanut, codfish, and egg recognized major allergens of all 3 foods, demonstrating sensitization to multiple unrelated food allergens (MFA mice). Sham-treated MFA mice exhibited anaphylactic symptoms accompanied by elevation of plasma histamine and hypothermia. In contrast, FAHF-2-treated MFA mice showed no anaphylactic symptoms, normal body temperature, and histamine levels after challenge with each allergen. Protection was accompanied by reduction in allergen-specific immunoglobulin E levels. Allergen-stimulated Th2 cytokine interleukin-4 and interleukin-13 production levels decreased, whereas the Th1 cytokine interferon-γ levels were elevated in cultured splenocytes and mesenteric lymph node cells in FAHF-2-treated mice. We established the first murine model of MFA. FAHF-2 prevents peanut, egg, and fish-induced anaphylactic reactions in this model, suggesting that FAHF-2 may have potential for treating human MFA. Copyright © 2012 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  8. The Fas-associated death domain protein/caspase-8/c-FLIP signaling pathway is involved in TNF-induced activation of ERK

    International Nuclear Information System (INIS)

    Lueschen, Silke; Falk, Markus; Scherer, Gudrun; Ussat, Sandra; Paulsen, Maren; Adam-Klages, Sabine

    2005-01-01

    The cytokine TNF activates multiple signaling pathways leading to cellular responses ranging from proliferation and survival to apoptosis. While most of these pathways have been elucidated in detail over the past few years, the molecular mechanism leading to the activation of the MAP kinases ERK remains ill defined and is controversially discussed. Therefore, we have analyzed TNF-induced ERK activation in various human and murine cell lines and show that it occurs in a cell-type-specific manner. In addition, we provide evidence for the involvement of the signaling components Fas-associated death domain protein (FADD), caspase-8, and c-FLIP in the pathway activating ERK in response to TNF. This conclusion is based on the following observations: (I) Overexpression of FADD, caspase-8, or a c-FLIP protein containing the death effector domains only leads to enhanced and prolonged ERK activation after TNF treatment. (II) TNF-induced ERK activation is strongly diminished in the absence of FADD. Interestingly, the enzymatic function of caspase-8 is not required for TNF-induced ERK activation. Additional evidence suggests a role for this pathway in the proliferative response of murine fibroblasts to TNF

  9. Effect of granulocyte colony-stimulating factor on murine thymic emigration and subsets reconstitution after a sublethal dose of irradiation

    International Nuclear Information System (INIS)

    Zhao Hongxia; Guo Mei; Sun Xuedong; Ai Huisheng

    2011-01-01

    Objective: To investigate the effects of recombinant human granulocyte colony stimulating factor (G-CSF) on murine thymic emigration and subsets reconstitution after a sublethal dose of irradiation. Methods: Female BALB/c mice were irradiated with a 6.0 Gy of γ-ray total-body irradiation and then randomly divided into GCSF group and control group. For mice in the GCSF group, recombinant human G-CSF 100 μg · kg -1 · d -1 was injected subcutaneously once daily for 14 continuous days and mice in the control group were given the same volume of phosphate buffered solution (PBS). At 7, 14, 21 and 28 days later, mice were killed and thymus mononuclear cell suspension were analyzed by flow cytometry for the percentage of the four stages of thymic CD4 - CD8 - double negative cells (DN1-4) and the CD4 + CD8 + double positive ( CD4 + CD8 + DP), CD4 + CD8 - single positive (CD4 + SP), CD4 - CD8 + single positive cells (CD8 + SP).Real-time PCR was used for detection and quantitation of murine T cell receptor rearrangement excision circles (sjTRECs) of the thymic cells of 30 and 60 d after irradiation. Results: The percentage of thymic DN1 cells in GCSF group was significantly higher than that of the control group 7 d after irradiation (t=9.59, P<0.05). 21 d later, the proportion of thymic DN3 and DN4 cells were higher than those of the control group (t=16.37, 7.6, P<0.05). The percentage of thymic CD4 + CD8 + DP cells decreased 7 d after irradiation,increased at 14 d, decreased again at 21 days,and then got a permanent recover. The percentage of thymic CD4 + CD8 + DP cells in the GCSF group recovered to normal and was significantly higher than that of the control group 28 days after irradiation (t=12.22, P<0.05). The percentage of thymic CD8 + SP cells of the GCSF group was significantly higher than that of the control group 21 d after irradiation (t=3.77, P<0.05), while G-CSF had no obvious influence on the percentage of the thymic CD4 + SP cells. The sjTRECs copies in the

  10. Mechanism of infectivity of a murine leukemia virus in adult mice

    International Nuclear Information System (INIS)

    Levy, R.L.; Barrington, M.H.; Lerner, R.A.; Dixon, F.J.

    1976-01-01

    Infection of adult BALB/c mice with murine leukemia virus (MuLV) induces typical thymic lymphomas. Expression of virus was measured by using a radioimmunoassay for murine P-30, a virion core protein. Nineteen days after injection of MuLV-S into adult mice, there were 0.3μg P-30/ml of serum. X-irradiation permitted the early expression of high levels of viremia, when given before or after MuLV-S administration, and it also hastened the development of lymphomas. Seventeen to 21 days after injection of MuLV-S into x-irradiated (600 rads) adult mice, there were 2.7 μg of P-30/ml of serum. The virus produced by infected adult mice was infectious and oncogenic when given to newborn mice. Several lines of evidence are presented that suggest the mechanism by which x-irradiation permits early expession of virion proteins and lymphomas is not immunosuppression

  11. Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Mollerup, Jens; Jørgensen, Sune T.; Hougaard, Charlotte

    2001-01-01

    We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretrea...... by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity....

  12. Clinical and Epidemiological Characteristics of Scrub Typhus and Murine Typhus among Hospitalized Patients with Acute Undifferentiated Fever in Northern Vietnam

    Science.gov (United States)

    Hamaguchi, Sugihiro; Cuong, Ngo Chi; Tra, Doan Thu; Doan, Yen Hai; Shimizu, Kenta; Tuan, Nguyen Quang; Yoshida, Lay-Myint; Mai, Le Quynh; Duc-Anh, Dang; Ando, Shuji; Arikawa, Jiro; Parry, Christopher M.; Ariyoshi, Koya; Thuy, Pham Thanh

    2015-01-01

    A descriptive study on rickettsiosis was conducted at the largest referral hospital in Hanoi, Vietnam, to identify epidemiological and clinical characteristics of specific rickettsiosis. Between March 2001 and February 2003, we enrolled 579 patients with acute undifferentiated fever (AUF), excluding patients with malaria, dengue fever, and typhoid fever, and serologically tested for Orientia tsutsugamushi and Rickettsia typhi. Of the patients, 237 (40.9%) and 193 (33.3%) had scrub and murine typhus, respectively, and 149 (25.7%) had neither of them (non–scrub and murine typhus [non-ST/MT]). The proportion of murine typhus was highest among patients living in Hanoi whereas that of scrub typhus was highest in national or regional border areas. The presence of an eschar, dyspnea, hypotension, and lymphadenopathy was significantly associated with a diagnosis of scrub typhus (OR = 46.56, 10.90, 9.01, and 7.92, respectively). Patients with murine typhus were less likely to have these findings but more likely to have myalgia, rash, and relative bradycardia (OR = 1.60, 1.56, and 1.45, respectively). Scrub typhus and murine typhus were shown to be common causes of AUF in northern Vietnam although the occurrence of spotted fever group rickettsiae was not determined. Clinical and epidemiological information may help local clinicians make clinical diagnosis of specific rickettsioses in a resource-limited setting. PMID:25778504

  13. Atomic structure of the murine norovirus protruding domain and sCD300lf receptor complex.

    Science.gov (United States)

    Kilic, Turgay; Koromyslova, Anna; Malak, Virginie; Hansman, Grant S

    2018-03-21

    Human noroviruses are the leading cause of acute gastroenteritis in human. Noroviruses also infect animals such as cow, mice, cat, and dog. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was recently identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and murine norovirus capsid-protruding domain complex at 2.05 Å resolution. We found that the sCD300lf binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. The sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on the sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that the sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf interacting residues were partially conserved in CD300ld, but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms. IMPORTANCE Noroviruses exhibit exquisite host-range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host-range restriction it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrates that noroviruses can interact with carbohydrates

  14. Internalization of Murine Norovirus 1 by Lactuca sativa during Irrigation ▿

    Science.gov (United States)

    Wei, Jie; Jin, Yan; Sims, Tom; Kniel, Kalmia E.

    2011-01-01

    Romaine lettuce (Lactuca sativa) was grown hydroponically or in soil and challenged with murine norovirus 1 (MNV) under two conditions: one mimicking a severe one-time contamination event and another mimicking a lower level of contamination occurring over time. In each condition, lettuce was challenged with MNV delivered at the roots. In the first case, contamination occurred on day one with 5 × 108 reverse transcriptase quantitative PCR (RT-qPCR) U/ml MNV in nutrient buffer, and irrigation water was replaced with virus-free buffer every day for another 4 days. In the second case, contamination with 5 × 105 RT-qPCR U/ml MNV (freshly prepared) occurred every day for 5 days. Virus had a tendency to adsorb to soil particles, with a small portion suspended in nutrient buffer; e.g., ∼8 log RT-qPCR U/g MNV was detected in soil during 5 days of challenge with virus inoculums of 5 × 108 RT-qPCR U/ml at day one, but sativa. PMID:21296944

  15. Murine model of long term obstructive jaundice

    Science.gov (United States)

    Aoki, Hiroaki; Aoki, Masayo; Yang, Jing; Katsuta, Eriko; Mukhopadhyay, Partha; Ramanathan, Rajesh; Woelfel, Ingrid A.; Wang, Xuan; Spiegel, Sarah; Zhou, Huiping; Takabe, Kazuaki

    2016-01-01

    Background With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of 3 murine models of obstructive jaundice. Methods C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. Results 70% (7/10) of tCL mice died by Day 7, whereas majority 67% (10/15) of pCL mice survived with loss of jaundice. 19% (3/16) of LMHL mice died; however, jaundice continued beyond Day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 days after ligation but jaundice rapidly decreased by Day 7. The LHML group developed portal hypertension as well as severe fibrosis by Day 14 in addition to prolonged jaundice. Conclusion The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice but long term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice. PMID:27916350

  16. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    International Nuclear Information System (INIS)

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae

    2012-01-01

    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  17. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae, E-mail: chidkim@pusan.ac.kr

    2012-04-01

    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  18. The murine retinoblastoma homolog maps to chromosome 14 near Es-10

    NARCIS (Netherlands)

    Stone, J.C.; Crosby, J.J.; Kozak, C.A.; Schievella, A.R.; Bernards, R.A.; Nadeau, J.H.

    1989-01-01

    Restriction fragment length variants have been exploited to map genetically Rb-1, the murine homolog of the human retinoblastoma gene. Rb-1 localized to mouse chromosome 14 on the basis of results from analysis of somatic cell hybrids. In an interspecific backcross involving Mus spretus, Rb-1 and

  19. Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

    Directory of Open Access Journals (Sweden)

    Lea Hiršl

    2018-05-01

    Full Text Available The development of a vaccine against human cytomegalovirus (CMV has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1 (MULT-1MCMV inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.

  20. Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

    Directory of Open Access Journals (Sweden)

    Ward Sean M

    2003-06-01

    Full Text Available Abstract Background The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. Method The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. Results Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. Conclusions These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.

  1. Efficacy of posaconazole in murine experimental sporotrichosis.

    Science.gov (United States)

    Fernández-Silva, Fabiola; Capilla, Javier; Mayayo, Emilio; Guarro, Josep

    2012-05-01

    We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology.

  2. Irradiation Design for an Experimental Murine Model

    International Nuclear Information System (INIS)

    Ballesteros-Zebadua, P.; Moreno-Jimenez, S.; Suarez-Campos, J. E.; Celis, M. A.; Larraga-Gutierrez, J. M.; Garcia-Garduno, O. A.; Rubio-Osornio, M. C.; Custodio-Ramirez, V.; Paz, C.

    2010-01-01

    In radiotherapy and stereotactic radiosurgery, small animal experimental models are frequently used, since there are still a lot of unsolved questions about the biological and biochemical effects of ionizing radiation. This work presents a method for small-animal brain radiotherapy compatible with a dedicated 6MV Linac. This rodent model is focused on the research of the inflammatory effects produced by ionizing radiation in the brain. In this work comparisons between Pencil Beam and Monte Carlo techniques, were used in order to evaluate accuracy of the calculated dose using a commercial planning system. Challenges in this murine model are discussed.

  3. Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR

    International Nuclear Information System (INIS)

    Gruschus, James M.; Ferretti, James A.

    2001-01-01

    Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant

  4. Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations.

    Science.gov (United States)

    DeStefano, A L; Cupples, L A; Arnos, K S; Asher, J H; Baldwin, C T; Blanton, S; Carey, M L; da Silva, E O; Friedman, T B; Greenberg, J; Lalwani, A K; Milunsky, A; Nance, W E; Pandya, A; Ramesar, R S; Read, A P; Tassabejhi, M; Wilcox, E R; Farrer, L A

    1998-05-01

    Waardenburg syndrome (WS) type 1 is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary abnormalities of the eye, hair, and skin, and dystopia canthorum. The phenotype is variable and affected individuals may exhibit only one or a combination of several of the associated features. To assess the relationship between phenotype and gene defect, clinical and genotype data on 48 families (271 WS individuals) collected by members of the Waardenburg Consortium were pooled. Forty-two unique mutations in the PAX3 gene, previously identified in these families, were grouped in five mutation categories: amino acid (AA) substitution in the paired domain, AA substitution in the homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser-Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects in the gene product. Association between mutation class and the presence of hearing loss, eye pigment abnormality, skin hypopigmentation, or white forelock was evaluated using generalized estimating equations, which allowed for incorporation of a correlation structure that accounts for potential similarity among members of the same family. Odds for the presence of eye pigment abnormality, white forelock, and skin hypopigmentation were 2, 8, and 5 times greater, respectively, for individuals with deletions of the homeodomain and the Pro-Ser-Thr-rich region compared to individuals with an AA substitution in the homeodomain. Odds ratios that differ significantly from 1.0 for these traits may indicate that the gene products resulting from different classes of mutations act differently in the expression of WS. Although a suggestive association was detected for hearing loss with an odds ratio of 2.6 for AA substitution in the paired domain compared with AA substitution in the homeodomain, this odds

  5. Prevention of murine cerebral malaria by low-dose cyclosporin A.

    Science.gov (United States)

    Grau, G E; Gretener, D; Lambert, P H

    1987-08-01

    The effects of cyclosporin A (CsA) were investigated in an experimental model of cerebral malaria. In this model, Plasmodium berghei ANKA-infected CBA/Ca mice develop a clinically and histologically characterized neurological syndrome which is considered to be the result of immunopathological reactions mediated by L3T4+ T cells. It was shown that CsA displayed a strong protective effect on neurological complications when given at a dose 1 mg/kg/day for 5 consecutive days (Days 4-8), which had no effect on the parasite. Paradoxically, this protection against neurological complications was not seen when parasiticidal doses were used during this limited 5-day period. A similar protective effect was observed with two CsA derivatives, C5-34 and H7-94. The mechanisms by which CsA and the two derivatives could prevent murine cerebral malaria are unknown but can be related to exquisite effects on some lymphocyte functions. In view of these results, it might be conceivable to investigate the benefits of using low doses of CsA in man, in conjunction with the classical antiparasite therapy, for the management of cerebral malaria.

  6. Bone marrow stromal elements in murine leukemia; Decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishay, Z [Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School (Israel); Barak, V [Laboratory of Immunology, Department of Oncology, Hadassah University Hospital (Israel); Shoshan, S [Faculty of Dental Medicine, Connective Tissue Research Laboratory, Hebrew University, Jerusalem (Israel); Prindull, G [Department of Pediatrics, University of Gottingen, Gottingen (Germany, F.R.)

    1990-01-01

    A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF{alpha} in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicity, reduced CSF production. (author).

  7. Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract

    Science.gov (United States)

    Becknell, Brian; Eichler, Tad; Beceiro, Susana; Li, Birong; Easterling, Robert; Carpenter, Ashley R.; James, Cindy; McHugh, Kirk M.; Hains, David S.; Partida-Sanchez, Santiago; Spencer, John David

    2014-01-01

    Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified Ribonuclease 6 (RNase 6) as the RNase A Superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are up-regulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14+ monocytes and murine bone marrow derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility. PMID:25075772

  8. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  9. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

  10. Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

    Science.gov (United States)

    Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

    2017-10-01

    We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Gastrointestinal expression and partial cDNA cloning of murine Muc2

    NARCIS (Netherlands)

    van Klinken, B. J.; Einerhand, A. W.; Duits, L. A.; Makkink, M. K.; Tytgat, K. M.; Renes, I. B.; Verburg, M.; Büller, H. A.; Dekker, J.

    1999-01-01

    To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM

  12. Erythropoietin treatment alleviates ultrastructural myelin changes induced by murine cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Staalsø, Trine

    2012-01-01

    the effects of EPO treatment in this context. METHODS: The study consisted of two groups of Plasmodium berghei-infected mice and two groups of uninfected controls that were either treated with EPO or placebo (n = 4 mice/group). In the terminal phase of murine CM the brains were removed and processed...

  13. Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Kamstrup, Søren

    2005-01-01

    In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

  14. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  15. Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

    Directory of Open Access Journals (Sweden)

    Korson Mark

    2007-04-01

    Full Text Available Abstract Background Methylmalonic acidemia (MMA, a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT. Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. Methods To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. Results Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. Conclusion These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

  16. Somatosensory BOLD fMRI reveals close link between salient blood pressure changes and the murine neuromatrix.

    Science.gov (United States)

    Reimann, Henning Matthias; Todiras, Mihail; Hodge, Russ; Huelnhagen, Till; Millward, Jason Michael; Turner, Robert; Seeliger, Erdmann; Bader, Michael; Pohlmann, Andreas; Niendorf, Thoralf

    2018-05-15

    The neuromatrix, or "pain matrix", is a network of cortical brain areas which is activated by noxious as well as salient somatosensory stimulation. This has been studied in mice and humans using blood oxygenation level-dependent (BOLD) fMRI. Here we demonstrate that BOLD effects observed in the murine neuromatrix in response to salient somatosensory stimuli are prone to reflect mean arterial blood pressure (MABP) changes, rather than neural activity. We show that a standard electrostimulus typically used in murine somatosensory fMRI can induce substantial elevations in MABP. Equivalent drug-induced MABP changes - without somatosensory stimulation - evoked BOLD patterns in the neuromatrix strikingly similar to those evoked by electrostimulation. This constitutes a serious caveat for murine fMRI. The regional specificity of these BOLD patterns can be attributed to the co-localization of the neuromatrix with large draining veins. Based on these findings we propose a cardiovascular support mechanism whereby abrupt elevations in MABP provide additional energy supply to the neuromatrix and other essential brain areas in fight-or-flight situations. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. A fluorescence model of the murine lung for optical detection of pathogenic bacteria

    Science.gov (United States)

    Durkee, Madeleine S.; Cirillo, Jeffrey D.; Maitland, Kristen C.

    2017-07-01

    We present a computer model of intravital excitation and external fluorescence detection in the murine lungs validated with a three-dimensional lung tissue phantom. The model is applied to optical detection of pulmonary tuberculosis infection.

  18. Functional imaging of murine hearts using accelerated self-gated UTE cine MRI

    NARCIS (Netherlands)

    Motaal, Abdallah G.; Noorman, Nils; de Graaf, Wolter L.; Hoerr, Verena; Florack, Luc M. J.; Nicolay, Klaas; Strijkers, Gustav J.

    2015-01-01

    We introduce a fast protocol for ultra-short echo time (UTE) Cine magnetic resonance imaging (MRI) of the beating murine heart. The sequence involves a self-gated UTE with golden-angle radial acquisition and compressed sensing reconstruction. The self-gated acquisition is performed asynchronously

  19. Scanning electron microscopy of the neuropathology of murine cerebral malaria

    Directory of Open Access Journals (Sweden)

    Brenneis Christian

    2006-11-01

    Full Text Available Abstract Background The mechanisms leading to death and functional impairments due to cerebral malaria (CM are yet not fully understood. Most of the knowledge about the pathomechanisms of CM originates from studies in animal models. Though extensive histopathological studies of the murine brain during CM are existing, alterations have not been visualized by scanning electron microscopy (SEM so far. The present study investigates the neuropathological features of murine CM by applying SEM. Methods C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. When typical symptoms of CM developed perfused brains were processed for SEM or light microscopy, respectively. Results Ultrastructural hallmarks were disruption of vessel walls, parenchymal haemorrhage, leukocyte sequestration to the endothelium, and diapedesis of macrophages and lymphocytes into the Virchow-Robin space. Villous appearance of observed lymphocytes were indicative of activated state. Cerebral oedema was evidenced by enlargement of perivascular spaces. Conclusion The results of the present study corroborate the current understanding of CM pathophysiology, further support the prominent role of the local immune system in the neuropathology of CM and might expose new perspectives for further interventional studies.

  20. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    Energy Technology Data Exchange (ETDEWEB)

    Pissuwan, Dakrong [University of Technology Sydney, Institute for Nanoscale Technology (Australia); Valenzuela, Stella M. [University of Technology Sydney, Department of Medical and Molecular Biosciences (Australia)], E-mail: stella.valenzuela@uts.edu.au; Killingsworth, Murray C. [Sydney South West Pathology Service (Australia)], E-mail: murray.killingsworth@swsahs.nsw.gov.au; Xu, Xiaoda; Cortie, Michael B. [University of Technology Sydney, Institute for Nanoscale Technology (Australia)], E-mail: michael.cortie@uts.edu.au

    2007-12-15

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation ({approx}1x10{sup 5} to 1x10{sup 10} W/m{sup 2}). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10{sup 2} W/m{sup 2} being sufficient, provided that a total fluence of {approx}30 J/cm{sup 2} is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm{sup 2} resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

  1. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

    International Nuclear Information System (INIS)

    Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

    2007-01-01

    Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (∼1x10 5 to 1x10 10 W/m 2 ). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10 2 W/m 2 being sufficient, provided that a total fluence of ∼30 J/cm 2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm 2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells

  2. Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A.

    OpenAIRE

    Fischinger, P J; Thiel, H J; Blevins, C S; Dunlop, N M

    1981-01-01

    We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow t...

  3. Assignment of the murine protein kinase gene DLK to chromosome 15 in the vicinity of the bt/Koa locus by genetic linkage analysis

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Toshio; Yanagisawa, Masahiro; Matsubara, Nobumichi [Tokyo Univ. (Japan)] [and others

    1997-03-01

    We have cloned protein kinase genes from murine primordial germ cell-derived EG cells by a PCR-based strategy using degenerate primers corresponding to the conserved sequences in the catalytic domain of protein kinases. One of these clones, designated Gek2 (germ cell kinase 2), was used as a probe for screening of a mouse brain cDNA library and obtained clones contained an entire coding sequence. Comparison of the sequence of Gek2 with those in databases revealed that it was identical to a previously reported protein kinase gene, DLK. 8 refs., 1 fig.

  4. Murine model of long-term obstructive jaundice.

    Science.gov (United States)

    Aoki, Hiroaki; Aoki, Masayo; Yang, Jing; Katsuta, Eriko; Mukhopadhyay, Partha; Ramanathan, Rajesh; Woelfel, Ingrid A; Wang, Xuan; Spiegel, Sarah; Zhou, Huiping; Takabe, Kazuaki

    2016-11-01

    With the recent emergence of conjugated bile acids as signaling molecules in cancer, a murine model of obstructive jaundice by cholestasis with long-term survival is in need. Here, we investigated the characteristics of three murine models of obstructive jaundice. C57BL/6J mice were used for total ligation of the common bile duct (tCL), partial common bile duct ligation (pCL), and ligation of left and median hepatic bile duct with gallbladder removal (LMHL) models. Survival was assessed by Kaplan-Meier method. Fibrotic change was determined by Masson-Trichrome staining and Collagen expression. Overall, 70% (7 of 10) of tCL mice died by day 7, whereas majority 67% (10 of 15) of pCL mice survived with loss of jaundice. A total of 19% (3 of 16) of LMHL mice died; however, jaundice continued beyond day 14, with survival of more than a month. Compensatory enlargement of the right lobe was observed in both pCL and LMHL models. The pCL model demonstrated acute inflammation due to obstructive jaundice 3 d after ligation but jaundice rapidly decreased by day 7. The LHML group developed portal hypertension and severe fibrosis by day 14 in addition to prolonged jaundice. The standard tCL model is too unstable with high mortality for long-term studies. pCL may be an appropriate model for acute inflammation with obstructive jaundice, but long-term survivors are no longer jaundiced. The LHML model was identified to be the most feasible model to study the effect of long-term obstructive jaundice. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Effect of fucoidan on B16 murine melanoma cell melanin formation ...

    African Journals Online (AJOL)

    Background:Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. It not only inhibits cancer cell growth but also inhibits tyrosinase in vitro. Therefore, it is of interest to investigate the effect of fucoidan on B16 murine melanoma cells as the findings may ...

  6. Effect of small dose of radiation on induction of apoptosis in murine tumors

    International Nuclear Information System (INIS)

    Seong, Jin Sil; Pyo, Hong Ryull; Chung, Eun Ji; Kim, Sung Hee; Suh, Chang Ok

    1999-01-01

    To investigate the presence of adaptive response by low dose radiation in murine tumors in relation to radiation induced apoptosis as well as related mechanism. Syngeneic murine tumors, OCa-1 and HCa-l, were given 0.05 Gy pretreatment followed by therapeutic dose of 25 Gy radiation. Induction of apoptosis was analyzed for each treatment group. Regulating molecules of apoptosis. p53, Bcl-2, Sax, Bel-X, were also analyzed by Western blotting. In 0.05 Gy pretreatment group of OCa-l, 25 Gy-induced apoptosis per 1000 cells was 229, which was estimated at 30% lower level than the expected (p<0.05). In contrast, this reduction in radiation induced apoptosis was not seen in HCa-1. In the expression of apoptosis regulating molecules, p53 increased in both tumors in response to radiation. Bcl-2 and Bax did not show significant change in both tumors however, the expression of Bcl-2 surpassed that of Bax in 0.05 Gy pretreatment group of OCa-1. Bcl-X was not expressed in OCa-1. In HCa-l, ScI-X showed increased expression even with 0.05 Gy. Adaptive response by low dose radiation is shown in one murine tumor, OCa-I, in relation to radiation induced apoptosis. Apoptosis regulating molecules including Bcl-2/Bax and Bcl-X, appear to related. This study shows an evidence that adaptive response is present, but not a generalized phenomenon in vivo

  7. Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP.

    Science.gov (United States)

    Page, Stephen H; Wright, Edward K; Gama, Lucio; Clements, Janice E

    2011-01-01

    CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.

  8. A novel method for murine intrahepatic islet transplantation via cecal vein.

    Science.gov (United States)

    Byun, Nari; Kim, Hyun-Je; Min, Byoung-Hoon; Shin, Jun-Seop; Yoon, Il-Hee; Kim, Jong-Min; Kim, Yong-Hee; Park, Chung-Gyu

    2015-12-01

    Islet transplantation is one of the most beneficial treatment modality to treat type 1 diabetic patients with frequent hypoglycemic unawareness. In clinical setting, human islets are infused via portal vein and are settled in the end-portal venules in the liver. However, mouse islets are transplanted into kidney subcapsule or liver through direct portal vein. These conventional transplantation methods have several drawbacks such as different physiological environments around the transplanted islets in kidney subcapsule from the liver and high mortality rate in direct portal vein approach. In this study, we introduced murine intrahepatic islet transplantation method via cecal vein to have the same surgical operation route in humans as well as guaranteeing low mortality rate after islet transplantation. With this protocol, consistent normoglycemia can be obtained in diabetic mice, while keeping operation-related mortality extremely low. This approach with easier accessibility and low mortality will make murine intrahepatic islet transplantation a useful model for studying immunological mechanisms such as strong innate and adaptive immune responses that occur in human islet transplantation. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. No cytotoxicity or genotoxicity of graphene and graphene oxide in murine lung epithelial FE1 cells in vitro

    DEFF Research Database (Denmark)

    Bengtson, Stefan; Kling, Kirsten; Madsen, Anne Mette

    2016-01-01

    Graphene and graphene oxide receive much attention these years, because they add attractive properties to a wide range of applications and products. Several studies have shown toxicological effects of other carbon‐based nanomaterials such as carbon black nanoparticles and carbon nanotubes in vitro...... and in vivo. Here, we report in‐depth physicochemical characterization of three commercial graphene materials, one graphene oxide (GO) and two reduced graphene oxides (rGO) and assess cytotoxicity and genotoxicity in the murine lung epithelial cell line FE1. The studied GO and rGO mainly consisted of 2......–3 graphene layers with lateral sizes of 1–2 µm. GO had almost equimolar content of C, O, and H while the two rGO materials had lower contents of oxygen with C/O and C/H ratios of 8 and 12.8, respectively. All materials had low levels of endotoxin and low levels of inorganic impurities, which were mainly...

  10. Differential Contribution of TRPA1, TRPV4 and TRPM8 to Colonic Nociception in Mice.

    Directory of Open Access Journals (Sweden)

    Sonja M Mueller-Tribbensee

    Full Text Available Various transient receptor potential (TRP channels in sensory neurons contribute to the transduction of mechanical stimuli in the colon. Recently, even the cold-sensing menthol receptor TRPM(melastatin8 was suggested to be involved in murine colonic mechano-nociception.To analyze the roles of TRPM8, TRPA1 and TRPV4 in distension-induced colonic nociception and pain, TRP-deficient mice and selective pharmacological blockers in wild-type mice (WT were used. Visceromotor responses (VMR to colorectal distension (CRD in vivo were recorded and distension/pressure-induced CGRP release from the isolated murine colon ex vivo was measured by EIA.Distension-induced colonic CGRP release was markedly reduced in TRPA1-/- and TRPV4-/- mice at 90/150 mmHg compared to WT. In TRPM8-deficient mice the reduction was only distinct at 150 mmHg. Exposure to selective pharmacological antagonists (HC030031, 100 μM; RN1734, 10 μM; AMTB, 10 μM showed corresponding effects. The unselective TRP blocker ruthenium red (RR, 10 μM was as efficient in inhibiting distension-induced CGRP release as the unselective antagonists of mechanogated DEG/ENaC (amiloride, 100 μM and stretch-activated channels (gadolinium, 50 μM. VMR to CRD revealed prominent deficits over the whole pressure range (up to 90 mmHg in TRPA1-/- and TRPV4-/- but not TRPM8-/- mice; the drug effects of the TRP antagonists were again highly consistent with the results from mice lacking the respective TRP receptor gene.TRPA1 and TRPV4 mediate colonic distension pain and CGRP release and appear to govern a wide and congruent dynamic range of distensions. The role of TRPM8 seems to be confined to signaling extreme noxious distension, at least in the healthy colon.

  11. Differential immune microenvironments and response to immune checkpoint blockade amongst molecular subtypes of murine medulloblastoma

    Science.gov (United States)

    Pham, Christina D.; Flores, Catherine; Yang, Changlin; Pinheiro, Elaine M.; Yearley, Jennifer H.; Sayour, Elias J.; Pei, Yanxin; Moore, Colin; McLendon, Roger E.; Huang, Jianping; Sampson, John H.; Wechsler-Reya, Robert; Mitchell, Duane A.

    2016-01-01

    PURPOSE Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma (MB), the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and Group 3 MB for preclinical evaluation in immunocompetent C57BL/6 mice. METHODS AND RESULTS Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid derived suppressor cells and tumor-associated macrophages in murine SHH model tumors compared with Group 3 tumors. However, murine Group 3 tumors had higher percentages of CD8+ PD-1+ T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial Group 3 tumors compared to SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1+ peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3+ T cells within the tumor microenvironment. CONCLUSIONS This is the first immunologic characterization of preclinical models of molecular subtypes of MB and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment. PMID:26405194

  12. Differential Immune Microenvironments and Response to Immune Checkpoint Blockade among Molecular Subtypes of Murine Medulloblastoma.

    Science.gov (United States)

    Pham, Christina D; Flores, Catherine; Yang, Changlin; Pinheiro, Elaine M; Yearley, Jennifer H; Sayour, Elias J; Pei, Yanxin; Moore, Colin; McLendon, Roger E; Huang, Jianping; Sampson, John H; Wechsler-Reya, Robert; Mitchell, Duane A

    2016-02-01

    Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma, the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and group 3 medulloblastoma for preclinical evaluation in immunocompetent C57BL/6 mice. Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid-derived suppressor cells, and tumor-associated macrophages in murine SHH model tumors compared with group 3 tumors. However, murine group 3 tumors had higher percentages of CD8(+) PD-1(+) T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial group 3 tumors compared with SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1(+) peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3(+) T cells within the tumor microenvironment. This is the first immunologic characterization of preclinical models of molecular subtypes of medulloblastoma and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment. ©2015 American Association for Cancer Research.

  13. Establishment of a new murine elastase-induced aneurysm model combined with transplantation.

    Directory of Open Access Journals (Sweden)

    Zuzanna Rowinska

    Full Text Available The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation.Adult male mice (n = 6-9 per group underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting.Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11 ± 0.10 mm vs. 0.75 ± 0.03 mm; p ≤ 0,001. Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts.We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals.

  14. Effectiveness of the immunomodulatory extract of Kalanchoe pinnata against murine visceral leishmaniasis.

    Science.gov (United States)

    Gomes, D C O; Muzitano, M F; Costa, S S; Rossi-Bergmann, B

    2010-04-01

    Previously, we described the protective action of the immunomodulatory extract of Kalanchoe pinnata (Kp) in murine and human cutaneous leishmaniasis. In the present study, we investigated the effectiveness of Kp against visceral leishmaniasis, using the BALB/c mouse model of infection with Leishmania chagasi. Mice receiving oral daily doses of Kp (400 mg/kg) for 30 days displayed significantly reduced hepatic and splenic parasite burden, when compared with untreated animals. Protectiveness was accompanied by a reduction in parasite-specific IgG serum levels, and impaired capacity of spleen cells to produce IL-4, but not IFN-gamma and nitric oxide upon antigen recall in vitro. The reference drug Pentostam (72 mg/kg) given by the intra-peritoneal route on alternate days produced an anti-leishmanial effect similar to oral Kp. Our findings show that the oral efficacy of Kp, seen previously in murine cutaneous leishmaniasis, extends also to visceral leishmaniasis caused by L. chagasi, a difficult to treat and lethal disease of man.

  15. Macrophage content of murine tumors: Associations with TD50 and tumor radiocurability

    International Nuclear Information System (INIS)

    Wike, J.; Hunter, N.; Volpe, J.; Milas, L.

    1987-01-01

    The experiments were designed to investigate whether the tumor-associated macrophage (TAM) content of murine solid tumors correlates with tumor response to ionizing radiation and with the clonogenic ability of tumor cells to establish s.c. tumors. Of 13 tumors studied, 6 were sarcomas and 7 were carcinomas; all tumors were of spontaneous origin in C/sub 3/Hf/Kam mice, with the exception of one sarcoma that was induced by 3-methylcholanthrene. Tumors were growing in the hind thighs of syngeneic mice, and their TAM content was determined when they were 8 mm in diameter. Their macrophage content varied greatly, ranging from 9 to 83%. Radiocurability of 8 mm tumors, determined by TCD50, ranged from 42 Gy (fibrosarcoma FSA) to > 80 Gy (hepatocarcinoma HCA-I). There was an obvious trend toward positive correlation (r = 0.43) between TAM content and reduced local tumor radiocurability. However, there was a significant negative correlation between TAM content and TD50 values, implying that cells from tumors with higher macrophage content were more clonogenic. TAM from the NFSA sarcoma, a tumor with a low TD50 value and poorly responsive to radiation, stimulated the in vitro growth of NFSA tumor cells. These observations suggest that high TAM content could be conducive to tumor cell proliferation and could be a factor in poor tumor radioresponse

  16. Thy-1+ dendritic cells in murine epidermis are bone marrow-derived

    International Nuclear Information System (INIS)

    Breathnach, S.M.; Katz, S.I.

    1984-01-01

    Thy-1+, Ly-5+ dendritic cells have recently been described as a resident cell population in murine epidermis, but their ontogeny and function are unknown. The origin and turnover of epidermal Thy-1+ cells utilizing chimeric mice were investigated. Lethally x-irradiated AKR/J (Thy-1.1+) and AKR/Cum (Thy-1.2+) mice were reconstituted with allogeneic bone marrow cells with or without thymocytes from congenic AKR/Cum or AKR/J mice, respectively. The density of residual indigenous Thy-1.1+ cells in AKR/J chimeras and Thy-1.2+ cells in AKR/Cum chimeras was substantially reduced following x-irradiation, as determined by immunofluorescence staining of epidermal sheets. Epidermal repopulation by allogeneic Thy-1+ dendritic epidermal cells was first observed at 5 weeks in AKR/J chimeras and at 7 weeks in AKR/Cum chimeras and progressed slowly. Repopulation was not enhanced by increasing the number of allogeneic bone marrow cells injected from 2 X 10(7) to 10(8) cells or by the addition of 8 X 10(7) allogeneic thymocytes to the donor inoculate. Epidermal repopulation by allogeneic Thy-1.2+ cells was not seen in AKR/J mice reconstituted with syngeneic bone marrow cells and allogeneic Thy-1.2+ AKR/Cum thymocytes. Taken together, these results indicate that Thy-1+ dendritic epidermal cells are derived from the bone marrow and suggest that they are not related to conventional peripheral T-lymphocytes

  17. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

    Science.gov (United States)

    Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

    2017-03-01

    Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

  18. Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

    Directory of Open Access Journals (Sweden)

    Selma Majerowicz

    1994-09-01

    Full Text Available Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11. Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

  19. Activation of DNA damage repair pathways by murine polyomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Katie; Nicholas, Catherine; Garcea, Robert L., E-mail: Robert.Garcea@Colorado.edu

    2016-10-15

    Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.

  20. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    International Nuclear Information System (INIS)

    Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

    2014-01-01

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  1. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Bujak, Emil [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland); Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah [Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland); Neri, Dario, E-mail: neri@pharma.ethz.ch [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

    2014-09-10

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  2. Cytogenetic study of murine rodents inhabiting in uranium-mining regions of Akmolinskaya oblast

    International Nuclear Information System (INIS)

    Kazymbet, P.; Altaeva, N.; Bakhtin, M.; Zhapbasov, R.

    2010-01-01

    Republic of Kazakhstan is ranked as the world's leading uranium ore reserves. About 25% of the world's proven uranium ore reserves occur here. Strategy of study concerning ecology effects conditioned by ionizing radiation includes as one main element an analysis of genetic processes in natural populations and ecosystems. Therefore analysis of cytogenetic effects of murine rodents inhabiting in influence zones of uranium-mining regions is one of the most important elements of radio-bio-ecological monitoring and are not completed so far. In habitat of murine rodents in influence zone of Stepnogorsk Mining-Chemical Complex tailing it is shown that gamma radiation equivalent dose rate and beta-particle flux density exceed from 6 to 15 times check measurements. In soil, plant, and water samples the activity of radionuclides like 238 U, 226 Ra, 232 Th and 210 Pb exceeds the testing level from 2 to 52 times. Dose of ionizing radiation absorbed by murine rodents inhabited in radioactive contaminated areas exceeds from 10 to 19 times the one absorbed by control animals. Big Jerboa (Allactaga major Kern) inhabited nearby of Stepnogorsk Mining-Chemical Complex tailing has rate of occurrence of cells with hypo diploidy, and hyper diploidy in hematopoietic tissue which correspondingly 1,85 and 3,5 times exceeds the control level; and this factor of Jerboa (Allactaga saltator Eversman) is correspondingly 1,7 and 4,1 times higher than control level. Rate of occurrence of cells with polyploidy in Big Jerboa (Allactaga major Kern) from radioactively contaminated areas is 2,7 times higher than in control animal; and this factor of Jerboa (Allactaga saltator Eversman) by 6,4 times exceeds control level. Levels of chromosomal rearrangements of Big Jerboa (Allactaga major Kern) from trail and control areas are 3,39±0,60% and 0,60±0,19% correspondently; and these factors of Jerboa (Allactaga saltator Eversman) are 4,63±0,91% and 1,22±0,37%, correspondently which confirms existence

  3. A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

    Science.gov (United States)

    Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M

    2017-05-01

    The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and

  4. Radioprotection by murine and human tumor-necrosis factor; Dose-dependent effects on hematopoiesis in the mouse

    Energy Technology Data Exchange (ETDEWEB)

    Sloerdal, L; Muench, M O; Warren, D J; Moore, M A.S. [James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York (USA)

    1989-01-01

    Tumor-necrosis factor (TNF) has been shown to confer significant radioprotection in murine models. Herein, we demonstrate a dose-dependent enhancement of hematological recovery when single doses of either murine or human recombinant TNF are administered prior to irradiation. In addition to its action upon leukocytes and erythocytes, TNF also alleviates radiation-induced thrombocytopenia in the mouse. These effects on circulating blood constituents are further reflected in increased numbers of both primitive (CFU-S) and more differentiated (CFU-GM, CFU-Mega) hematopoietic progenitors in TNF-treated animals. This suggests that TNF exerts it radioprotective effects on a pool of primitive multi-potential hematopoietic cells. (author).

  5. Collagen-Induced Arthritis: A model for Murine Autoimmune Arthritis

    OpenAIRE

    Pietrosimone, K. M.; Jin, M.; Poston, B.; Liu, P.

    2015-01-01

    Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund’s adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund’s adjuvant (IFA) 21 days aft...

  6. Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

    Science.gov (United States)

    Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

    2012-02-01

    Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

  7. The Probiotic Compound VSL#3 Modulates Mucosal, Peripheral, and Systemic Immunity Following Murine Broad-Spectrum Antibiotic Treatment

    Directory of Open Access Journals (Sweden)

    Ira Ekmekciu

    2017-05-01

    Full Text Available There is compelling evidence linking the commensal intestinal microbiota with host health and, in turn, antibiotic induced perturbations of microbiota composition with distinct pathologies. Despite the attractiveness of probiotic therapy as a tool to beneficially alter the intestinal microbiota, its immunological effects are still incompletely understood. The aim of the present study was to assess the efficacy of the probiotic formulation VSL#3 consisting of eight distinct bacterial species (including Streptococcus thermophilus, Bifidobacterium breve, B. longum, B. infantis, Lactobacillus acidophilus, L. plantarum, L. paracasei, and L. delbrueckii subsp. Bulgaricus in reversing immunological effects of microbiota depletion as compared to reassociation with a complex murine microbiota. To address this, conventional mice were subjected to broad-spectrum antibiotic therapy for 8 weeks and perorally reassociated with either VSL#3 bacteria or a complex murine microbiota. VSL#3 recolonization resulted in restored CD4+ and CD8+ cell numbers in the small and large intestinal lamina propria as well as in B220+ cell numbers in the former, whereas probiotic intervention was not sufficient to reverse the antibiotic induced changes of respective cell populations in the spleen. However, VSL#3 application was as efficient as complex microbiota reassociation to attenuate the frequencies of regulatory T cells, activated dendritic cells and memory/effector T cells in the small intestine, colon, mesenteric lymph nodes, and spleen. Whereas broad-spectrum antibiotic treatment resulted in decreased production of cytokines such as IFN-γ, IL-17, IL-22, and IL-10 by CD4+ cells in respective immunological compartments, VSL#3 recolonization was sufficient to completely recover the expression of the anti-inflammatory cytokine IL-10 without affecting pro-inflammatory mediators. In summary, the probiotic compound VSL#3 has an extensive impact on mucosal, peripheral, and

  8. Role of P2X7 on steroid synthesis in murine luteal cells

    Directory of Open Access Journals (Sweden)

    Chunping Zhang

    2016-03-01

    Full Text Available The extracellular adenosine triphosphate (ATP regulates different cellular functions through activating purinergic receptors as a signalling molecule or neurotransmitter. P2X7 is highly expressed in murine small luteal cells. In this study, murine luteal cells were cultured in vitro and treated with P2X7 agonists – ATP and 2′(3′-O-(4-benzoyl-benzoyl-adenosine 50-triphosphate (BzATP and with P2X7 antagonist – brilliant blue G (BBG. We found that ATP and BzATP increased the production of progesterone and had no influence on the production of estradiol. BBG reversed the effect of BzATP and ATP. Further studies demonstrated that ATP and BzATP promoted the expression of CYP11A. These results revealed that P2X7 receptor activation is involved in the steroid synthesis in corpus luteum.

  9. Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

    DEFF Research Database (Denmark)

    Christensen, Jesper; Cloos, Paul; Toftegaard, Ulla

    2005-01-01

    The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family...

  10. Murine colon proteome and characterization of the protein pathways

    Directory of Open Access Journals (Sweden)

    Magdeldin Sameh

    2012-08-01

    Full Text Available Abstract Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR I and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9 in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2, Glutathione S-transferase (Gstp1 in prostate cancer, and Cell division control protein (Cdc42, Ras-related protein (Rac1,2 in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.

  11. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    Science.gov (United States)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  12. Combination effect of cisplatin and radiation in murine solid tumors

    International Nuclear Information System (INIS)

    Egawa, Shin; Lee, Kan-ei; Ishibashi, Akira; Komiyama, Hiroki; Umezawa, Iwao.

    1986-01-01

    The combination effect of cisplatin and radiation was studied using the two different murine systems of sarcoma 180 and Ehrlich solid tumors. In sarcoma 180 solid tumor the minimal effective doses (MED) of cisplatin and radiation were 19.5 mg/kg and 10375 rad respectively whereas these doses did not show any effective antitumor activity practically. Administration of cisplatin with a doses of 9 mg/kg given 24 hours before radiation (1000 rad), however, showed synergistic antitumor activity. In Ehrlich solid tumor the MED of cisplatin and radiation were 13.8 mg/kg and 2892 rad respectively. Treatment with cisplatin, 3, 6 or 9 mg/kg, given 24 hours before radiation (1000 rad) showed also synergistic antitumor activity also. Sodium thiosulfate (STS) rescue was effective in reducing toxicity of cisplatin on combined use of the drug with radiation. Cell kinetics of sarcoma 180 solid tumor in vivo after the combined treatment was analyzed by computer aided flowcytometry. Accumulation of cells in the radiosensitive G 2 + M phase was observed 18 to 42 hours after a single intraperitoneal administration of 9 mg/kg of cisplatin. It is strongly suggested that this synchronization is one of the mechanisms of the synergism in the combination therapy. (author)

  13. microRNA-222 modulates liver fibrosis in a murine model of biliary atresia

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Wen-jun; Dong, Rui; Chen, Gong, E-mail: chengongzlp@hotmail.com; Zheng, Shan

    2014-03-28

    Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA) induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis.

  14. microRNA-222 modulates liver fibrosis in a murine model of biliary atresia

    International Nuclear Information System (INIS)

    Shen, Wen-jun; Dong, Rui; Chen, Gong; Zheng, Shan

    2014-01-01

    Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA) induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis

  15. Application of murine monoclonal antibodies to the serodiagnosis of tuberculosis

    International Nuclear Information System (INIS)

    Ivanyl, J.; Coates, A.R.M.; Krambovitis, E.

    1982-01-01

    The immune response during infectious diseases leads to a rise in antibody titre to the various different antigenic determinants of the causative organism. The response is further complicated by the fact that it is relatively unusual for one individual to respond to all antigenic components of an organism. Demonstration of the specific immune response of an infected host by serological tests is often hampered by the broad cross-reactivity between several bacterial antigens. The authors report on a serodiagnostic application of murine monoclonal antibodies (MAB), specific for a human pathogen, M. tuberculosis by a technique which is applicable in principle to the serodiagnosis of many other infectious diseases. The serum diagnostic test is based on the competitive inhibition by human sera of the binding of 125 I-labelled murine monoclonal antibodies to M. tuberculosis-coated polyvinyl plates. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of antibodies from patients' sera. When compared with healthy controls, increased titres of inhibitory antibodies were found in about 70% of patients with active tuberculosis. The diagnostic value of the individual monoclonal antibodies as well as the benefit from the use of multiple specificity probes has been qualified

  16. Neurological Disorders in a Murine Model of Chronic Renal Failure

    Directory of Open Access Journals (Sweden)

    Jean-Marc Chillon

    2014-01-01

    Full Text Available Cardiovascular disease is highly prevalent in patients with chronic renal failure (CRF. However, data on the impact of CRF on the cerebral circulatory system are scarce—despite the fact that stroke is the third most common cause of cardiovascular death in people with CRF. In the present study, we examined the impact of CRF on behavior (anxiety, recognition and ischemic stroke severity in a well-defined murine model of CRF. We did not observe any significant increases between CRF mice and non-CRF mice in terms of anxiety. In contrast, CRF mice showed lower levels of anxiety in some tests. Recognition was not impaired (vs. controls after 6 weeks of CRF but was impaired after 10 weeks of CRF. Chronic renal failure enhances the severity of ischemic stroke, as evaluated by the infarct volume size in CRF mice after 34 weeks of CRF. Furthermore, neurological test results in non-CRF mice tended to improve in the days following ischemic stroke, whereas the results in CRF mice tended to worsen. In conclusion, we showed that a murine model of CRF is suitable for evaluating uremic toxicity and the associated neurological disorders. Our data confirm the role of uremic toxicity in the genesis of neurological abnormalities (other than anxiety.

  17. Corn silk induced cyclooxygenase-2 in murine macrophages.

    Science.gov (United States)

    Kim, Kyung A; Shin, Hyun-Hee; Choi, Sang Kyu; Choi, Hye-Seon

    2005-10-01

    Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE2. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-kappaB), indicating that COX-2 induction proceeds also via the NF-kappaB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE2 elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.

  18. Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary.

    Science.gov (United States)

    van de Vrugt, H J; Cheng, N C; de Vries, Y; Rooimans, M A; de Groot, J; Scheper, R J; Zhi, Y; Hoatlin, M E; Joenje, H; Arwert, F

    2000-04-01

    Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models.

  19. Critical transition in tissue homeostasis accompanies murine lung senescence.

    Directory of Open Access Journals (Sweden)

    Carla L Calvi

    Full Text Available BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4 and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging

  20. Irradiation induced modest changes in murine cardiac function despite progressive structural damage to the myocardium and microvasculature

    International Nuclear Information System (INIS)

    Seemann, Ingar; Gabriels, Karen; Visser, Nils L.; Hoving, Saske; Poele, Johannes A. te; Pol, Jeffrey F.; Gijbels, Marion J.; Janssen, Ben J.; Leeuwen, Fijs W. van; Daemen, Mat J.; Heeneman, Sylvia; Stewart, Fiona A.

    2012-01-01

    Background: Radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiotoxicity, but the underlying mechanisms are unclear. Methods: Single doses of 2, 8, or 16 Gy were delivered to the hearts of mice and damage was evaluated at 20, 40, and 60 weeks, relative to age matched controls. Single photon emission computed tomography (SPECT/CT) and ultrasound were used to measure cardiac geometry and function, which was related to histo-morphology and microvascular damage. Results: Gated SPECT/CT and ultrasound demonstrated decreases in end diastolic and systolic volumes, while the ejection fraction was increased at 20 and 40 weeks after 2, 8, and 16 Gy. Cardiac blood volume was decreased at 20 and 60 weeks after irradiation. Histological examination revealed inflammatory changes at 20 and 40 weeks after 8 and 16 Gy. Microvascular density in the left ventricle was decreased at 40 and 60 weeks after 8 and 16 Gy, with functional damage to remaining microvasculature manifest as decreased alkaline phosphatase (2, 8, and 16 Gy), increased von Willebrand Factor and albumin leakage from vessels (8 and 16 Gy), and amyloidosis (16 Gy). 16 Gy lead to sudden death between 30 and 40 weeks in 38% of mice. Conclusions: Irradiation with 2 and 8 Gy induced modest changes in murine cardiac function within 20 weeks but this did not deteriorate further, despite progressive structural and microvascular damage. This indicates that heart function can compensate for significant structural damage, although higher doses, eventually lead to sudden death.

  1. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin

    2010-01-01

    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full......-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits......Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine...

  2. Ras-dva1 small GTPase regulates telencephalon development in Xenopus laevis embryos by controlling Fgf8 and Agr signaling at the anterior border of the neural plate

    Directory of Open Access Journals (Sweden)

    Maria B. Tereshina

    2014-07-01

    Full Text Available We previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism.

  3. Pathogenesis of Candida albicans infections in the alternative chorio-allantoic membrane chicken embryo model resembles systemic murine infections.

    Directory of Open Access Journals (Sweden)

    Ilse D Jacobsen

    Full Text Available Alternative models of microbial infections are increasingly used to screen virulence determinants of pathogens. In this study, we investigated the pathogenesis of Candida albicans and C. glabrata infections in chicken embryos infected via the chorio-allantoic membrane (CAM and analyzed the virulence of deletion mutants. The developing immune system of the host significantly influenced susceptibility: With increasing age, embryos became more resistant and mounted a more balanced immune response, characterized by lower induction of proinflammatory cytokines and increased transcription of regulatory cytokines, suggesting that immunopathology contributes to pathogenesis. While many aspects of the chicken embryo response resembled murine infections, we also observed significant differences: In contrast to systemic infections in mice, IL-10 had a beneficial effect in chicken embryos. IL-22 and IL-17A were only upregulated after the peak mortality in the chicken embryo model occurred; thus, the role of the Th17 response in this model remains unclear. Abscess formation occurs frequently in murine models, whereas the avian response was dominated by granuloma formation. Pathogenicity of the majority of 15 tested C. albicans deletion strains was comparable to the virulence in mouse models and reduced virulence was associated with significantly lower transcription of proinflammatory cytokines. However, fungal burden did not correlate with virulence and for few mutants like bcr1Δ and tec1Δ different outcomes in survival compared to murine infections were observed. C. albicans strains locked in the yeast stage disseminated significantly more often from the CAM into the embryo, supporting the hypothesis that the yeast morphology is responsible for dissemination in systemic infections. These data suggest that the pathogenesis of C. albicans infections in the chicken embryo model resembles systemic murine infections but also differs in some aspects. Despite

  4. Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach.

    Science.gov (United States)

    Wilson, Charlotte M; Loach, Diane; Lawley, Blair; Bell, Tracey; Sims, Ian M; O'Toole, Paul W; Zomer, Aldert; Tannock, Gerald W

    2014-10-01

    Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Acanthus ilicifolius plant extract prevents DNA alterations in a transplantable Ehrlich ascites carcinoma-bearing murine model.

    Science.gov (United States)

    Chakraborty, Tridib; Bhuniya, Dipak; Chatterjee, Mary; Rahaman, Mosiur; Singha, Dipak; Chatterjee, Baidya Nath; Datta, Subrata; Rana, Ajay; Samanta, Kartick; Srivastawa, Sunil; Maitra, Sankar K; Chatterjee, Malay

    2007-12-28

    To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma (EAC)-bearing murine model. Male Swiss albino mice were divided into four groups: Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal (ip) inoculations of rapidly proliferating 2 x 10(5) viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract (ALE) of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation (ALE control); and Group D was the EAC + ALE-treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein (MT) protein, sister-chromatid exchanges (SCEs), and DNA alterations. Treatment of the EAC-bearing mice with the ALE significantly (P decrement (P single-strand breaks (SSBs) by 38.53% (3.14 +/- 0.31 vs 1.93 +/- 0.23, P < 0.01) in EAC-bearing murine liver. Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

  6. Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

    Science.gov (United States)

    Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

    2017-05-31

    Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

  7. Elastin receptor (S-gal) occupancy by elastin peptides modulates T-cell response during murine emphysema.

    Science.gov (United States)

    Meghraoui-Kheddar, Aïda; Pierre, Alexandre; Sellami, Mehdi; Audonnet, Sandra; Lemaire, Flora; Le Naour, Richard

    2017-09-01

    Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4 + and CD8 + T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response. Copyright © 2017 the American Physiological Society.

  8. Development of an ex vivo BrdU labeling procedure for the murine LLNA

    Science.gov (United States)

    The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [3H]m...

  9. SEP-induced activity and its thermographic cortical representation in a murine model.

    Science.gov (United States)

    Hoffmann, Klaus-Peter; Ruff, Roman; Kirsch, Matthias

    2013-06-01

    This article is a methodical report on the generation of reproducible changes in brain activity in a murine model. Somatosensory evoked potentials (SEP) are used to generate synchronized cortical activity. After electrical stimulation of mice forelimbs, the potentials were recorded with a flexible thin-film polyimide electrode structure directly from the cortex. Every registration included a simultaneous recording from both hemispheres that repeated four times to reproduce and compare the results. The SEPs in the murine model were shown to generate a very stable signal. The latency of the second positive wave (P2 wave) ranged between 16 and 19 ms, and the N1-P2 amplitude ranged between 39 and 48 µV. In addition, the temperature distribution of the cortex was acquired using infrared thermography. Surface cortical temperature changed during electrical stimulation without a clear hemispheric correlation. These initial results could be a step toward a better understanding of the different synchronized cortical activities and basic methods of evaluation of various mathematical algorithms to detect them.

  10. Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models

    Directory of Open Access Journals (Sweden)

    Soledad eMac Keon

    2015-05-01

    Full Text Available Dendritic cells (DCs play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel T there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts towards an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.

  11. Activation of farnesoid X receptor downregulates monocyte chemoattractant protein-1 in murine macrophage

    Energy Technology Data Exchange (ETDEWEB)

    Li, Liangpeng; Zhang, Qian; Peng, Jiahe; Jiang, Chanjui; Zhang, Yan; Shen, Lili; Dong, Jinyu; Wang, Yongchao; Jiang, Yu, E-mail: yujiang0207@163.com

    2015-11-27

    Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily, which plays important roles in bile acids/lipid homeostasis and inflammation. Monocyte chemoattractant protein-1 (MCP-1) contributes to macrophage infiltration into body tissues during inflammation. Here we investigated whether FXR can regulate MCP-1 expression in murine macrophage. FXR activation down regulate MCP-1 mRNA and protein levels in ANA-1 and Raw264.7 cells. Luciferase reporter assay, Gel shift and Chromatin immunoprecipitation assays have revealed that the activated FXR bind to the FXR element located in −738 bp ∼  −723 bp in MCP-1 promoter. These results suggested that FXR may serve as a novel target for regulating MCP-1 levels for the inflammation related diseases therapies. - Highlights: • FXR is expressed in murine macrophage cell line. • FXR down regulates MCP-1 expression. • FXR binds to the DR4 in MCP-1 promoter.

  12. Endogenous murine tau promotes neurofibrillary tangles in 3xTg-AD mice without affecting cognition.

    Science.gov (United States)

    Baglietto-Vargas, David; Kitazawa, Masashi; Le, Elaine J; Estrada-Hernandez, Tatiana; Rodriguez-Ortiz, Carlos J; Medeiros, Rodrigo; Green, Kim N; LaFerla, Frank M

    2014-02-01

    Recent studies on tauopathy animal models suggest that the concomitant expression of the endogenous murine tau delays the pathological accumulation of human tau, and interferes with the disease progression. To elucidate the role of endogenous murine tau in a model with both plaques and tangles, we developed a novel transgenic mouse model by crossing 3xTg-AD with mtauKO mice (referred to as 3xTg-AD/mtauKO mice). Therefore, this new model allows us to determine the pathological consequences of the murine tau. Here, we show that 3xTg-AD/mtauKO mice have lower tau loads in both soluble and insoluble fractions, and lower tau hyperphosphorylation level in the soluble fraction relative to 3xTg-AD mice. In the 3xTg-AD model endogenous mouse tau is hyperphosphorylated and significantly co-aggregates with human tau. Despite the deletion of the endogenous tau gene in 3xTg-AD/mtauKO mice, cognitive dysfunction was equivalent to 3xTg-AD mice, as there was no additional impairment on a spatial memory task, and thus despite increased tau phosphorylation, accumulation and NFTs in 3xTg-AD mice no further effects on cognition are seen. These findings provide better understanding about the role of endogenous tau to Alzheimer's disease (AD) pathology and for developing new AD models. © 2013.

  13. β-Arrestin2 mediates progression of murine primary myelofibrosis.

    Science.gov (United States)

    Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

    2017-12-21

    Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

  14. Hox gene clusters in the Indonesian coelacanth, Latimeria menadoensis

    Science.gov (United States)

    Koh, Esther G. L.; Lam, Kevin; Christoffels, Alan; Erdmann, Mark V.; Brenner, Sydney; Venkatesh, Byrappa

    2003-01-01

    The Hox genes encode transcription factors that play a key role in specifying body plans of metazoans. They are organized into clusters that contain up to 13 paralogue group members. The complex morphology of vertebrates has been attributed to the duplication of Hox clusters during vertebrate evolution. In contrast to the single Hox cluster in the amphioxus (Branchiostoma floridae), an invertebrate-chordate, mammals have four clusters containing 39 Hox genes. Ray-finned fishes (Actinopterygii) such as zebrafish and fugu possess more than four Hox clusters. The coelacanth occupies a basal phylogenetic position among lobe-finned fishes (Sarcopterygii), which gave rise to the tetrapod lineage. The lobe fins of sarcopterygians are considered to be the evolutionary precursors of tetrapod limbs. Thus, the characterization of Hox genes in the coelacanth should provide insights into the origin of tetrapod limbs. We have cloned the complete second exon of 33 Hox genes from the Indonesian coelacanth, Latimeria menadoensis, by extensive PCR survey and genome walking. Phylogenetic analysis shows that 32 of these genes have orthologs in the four mammalian HOX clusters, including three genes (HoxA6, D1, and D8) that are absent in ray-finned fishes. The remaining coelacanth gene is an ortholog of hoxc1 found in zebrafish but absent in mammals. Our results suggest that coelacanths have four Hox clusters bearing a gene complement more similar to mammals than to ray-finned fishes, but with an additional gene, HoxC1, which has been lost during the evolution of mammals from lobe-finned fishes. PMID:12547909

  15. H4 histamine receptors mediate cell cycle arrest in growth factor-induced murine and human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Anne-France Petit-Bertron

    Full Text Available The most recently characterized H4 histamine receptor (H4R is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs.

  16. Meta-analysis of genome-wide association studies identifies 8 novel loci involved in shape variation of human head hair

    Science.gov (United States)

    Liu, Fan; Chen, Yan; Zhu, Gu; Hysi, Pirro G; Wu, Sijie; Adhikari, Kaustubh; Breslin, Krystal; Pośpiech, Ewelina; Hamer, Merel A; Peng, Fuduan; Muralidharan, Charanya; Acuna-Alonzo, Victor; Canizales-Quinteros, Samuel; Bedoya, Gabriel; Gallo, Carla; Poletti, Giovanni; Rothhammer, Francisco; Bortolini, Maria Catira; Gonzalez-Jose, Rolando; Zeng, Changqing; Xu, Shuhua; Jin, Li; Uitterlinden, André G; Ikram, M Arfan; van Duijn, Cornelia M; Nijsten, Tamar; Walsh, Susan; Branicki, Wojciech; Wang, Sijia; Ruiz-Linares, Andrés; Spector, Timothy D; Martin, Nicholas G; Medland, Sarah E; Kayser, Manfred

    2018-01-01

    Abstract Shape variation of human head hair shows striking variation within and between human populations, while its genetic basis is far from being understood. We performed a series of genome-wide association studies (GWASs) and replication studies in a total of 28 964 subjects from 9 cohorts from multiple geographic origins. A meta-analysis of three European GWASs identified 8 novel loci (1p36.23 ERRFI1/SLC45A1, 1p36.22 PEX14, 1p36.13 PADI3, 2p13.3 TGFA, 11p14.1 LGR4, 12q13.13 HOXC13, 17q21.2 KRTAP, and 20q13.33 PTK6), and confirmed 4 previously known ones (1q21.3 TCHH/TCHHL1/LCE3E, 2q35 WNT10A, 4q21.21 FRAS1, and 10p14 LINC00708/GATA3), all showing genome-wide significant association with hair shape (P < 5e-8). All except one (1p36.22 PEX14) were replicated with nominal significance in at least one of the 6 additional cohorts of European, Native American and East Asian origins. Three additional previously known genes (EDAR, OFCC1, and PRSS53) were confirmed at the nominal significance level. A multivariable regression model revealed that 14 SNPs from different genes significantly and independently contribute to hair shape variation, reaching a cross-validated AUC value of 0.66 (95% CI: 0.62–0.70) and an AUC value of 0.64 in an independent validation cohort, providing an improved accuracy compared with a previous model. Prediction outcomes of 2504 individuals from a multiethnic sample were largely consistent with general knowledge on the global distribution of hair shape variation. Our study thus delivers target genes and DNA variants for future functional studies to further evaluate the molecular basis of hair shape in humans. PMID:29220522

  17. Meta-analysis of genome-wide association studies identifies 8 novel loci involved in shape variation of human head hair.

    Science.gov (United States)

    Liu, Fan; Chen, Yan; Zhu, Gu; Hysi, Pirro G; Wu, Sijie; Adhikari, Kaustubh; Breslin, Krystal; Pospiech, Ewelina; Hamer, Merel A; Peng, Fuduan; Muralidharan, Charanya; Acuna-Alonzo, Victor; Canizales-Quinteros, Samuel; Bedoya, Gabriel; Gallo, Carla; Poletti, Giovanni; Rothhammer, Francisco; Bortolini, Maria Catira; Gonzalez-Jose, Rolando; Zeng, Changqing; Xu, Shuhua; Jin, Li; Uitterlinden, André G; Ikram, M Arfan; van Duijn, Cornelia M; Nijsten, Tamar; Walsh, Susan; Branicki, Wojciech; Wang, Sijia; Ruiz-Linares, Andrés; Spector, Timothy D; Martin, Nicholas G; Medland, Sarah E; Kayser, Manfred

    2018-02-01

    Shape variation of human head hair shows striking variation within and between human populations, while its genetic basis is far from being understood. We performed a series of genome-wide association studies (GWASs) and replication studies in a total of 28 964 subjects from 9 cohorts from multiple geographic origins. A meta-analysis of three European GWASs identified 8 novel loci (1p36.23 ERRFI1/SLC45A1, 1p36.22 PEX14, 1p36.13 PADI3, 2p13.3 TGFA, 11p14.1 LGR4, 12q13.13 HOXC13, 17q21.2 KRTAP, and 20q13.33 PTK6), and confirmed 4 previously known ones (1q21.3 TCHH/TCHHL1/LCE3E, 2q35 WNT10A, 4q21.21 FRAS1, and 10p14 LINC00708/GATA3), all showing genome-wide significant association with hair shape (P 5e-8). All except one (1p36.22 PEX14) were replicated with nominal significance in at least one of the 6 additional cohorts of European, Native American and East Asian origins. Three additional previously known genes (EDAR, OFCC1, and PRSS53) were confirmed at the nominal significance level. A multivariable regression model revealed that 14 SNPs from different genes significantly and independently contribute to hair shape variation, reaching a cross-validated AUC value of 0.66 (95% CI: 0.62-0.70) and an AUC value of 0.64 in an independent validation cohort, providing an improved accuracy compared with a previous model. Prediction outcomes of 2504 individuals from a multiethnic sample were largely consistent with general knowledge on the global distribution of hair shape variation. Our study thus delivers target genes and DNA variants for future functional studies to further evaluate the molecular basis of hair shape in humans. © The Author(s) 2017. Published by Oxford University Press.

  18. Antioxidative effects in vivo and colonization of Lactobacillus plantarum MA2 in the murine intestinal tract.

    Science.gov (United States)

    Tang, Wei; Xing, Zhuqing; Hu, Wei; Li, Chao; Wang, Jinju; Wang, Yanping

    2016-08-01

    Lactobacillus plantarum MA2 was isolated from traditional Chinese Tibet kefir grains, which possess several excellent properties and functions. We previously demonstrated the antioxidant activities of this bacterium in vitro. However, the maintenance and survival of L. plantarum MA2 inside the murine intestinal tract, where it exerts its probiotic properties, and whether its effects are elicited directly on the host remain unknown. Therefore, this study investigated the mechanisms of L. plantarum MA2 in aging mice following D-galactose administration. The levels of malondialdehyde decreased significantly in the L. plantarum MA2 groups after oral ingestion compared to the D-galactose model group, and total antioxidant capacity and glutathione peroxidase and superoxide dismutase activities increased significantly in the serum and liver. We combined fluorescein isothiocyanate labeling and green fluorescent protein expression to dynamically monitor the colonization and distribution of L. plantarum MA2 in the murine intestinal tract. The results indicated that L. plantarum MA2 was detected in the ileum, colon, and feces after single and continuous oral administration at day 21 and was maintained at 10(4)-10(5) CFU/g. These results suggest that L. plantarum MA2 colonizes and survives in the murine intestinal tract to exert its antioxidative effects.

  19. Significance of fractionation regimens in radiation and combined hyperthermia using a murine fibrosarcoma

    International Nuclear Information System (INIS)

    Hahn, E.W.; Alfieri, A.A.; Kim, J.H.

    1978-01-01

    The significance of time--dose ralationships in the use of local tumor hyperthermia (LTH) when combined with radiation (RAD) was studied in a murine fibrosarcoma. RAD, either alone or combined with LTH, was delivered in four equal fractions (total doses, 1.8 to 4.2 krad) separated by 1 to 4 days. LTH (43.1 C +- .05 C for 15 minutes, water bath) was applied immediately after RAD. In this tumor system, RAD was most effective when delivered every 2nd or 3rd day, by a factor of 1.25 over the response achieved when the four fractions were delivered every 1 or 4 days. At all levels studied, RAD + LTH produced a superior tumor response compared to RAD alone. The ratio of the RAD + LTH/RAD doses to achieve an isobiological response ranged from 1.7 to 2.5. Most significant was the finding that the RAD + LTH treatment response was independent of the fractionation scheme used and more dependent on the total RAD dose delivered

  20. Cdh11 Acts as a Tumor Suppressor in a Murine Retinoblastoma Model by Facilitating Tumor Cell Death

    Science.gov (United States)

    Marchong, Mellone N.; Yurkowski, Christine; Ma, Clement; Spencer, Clarellen; Pajovic, Sanja; Gallie, Brenda L.

    2010-01-01

    CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers), while proliferation in vivo remained unaffected (p = 0.121). Activated caspase-3 was significantly decreased and β-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death. PMID:20421947

  1. Cdh11 acts as a tumor suppressor in a murine retinoblastoma model by facilitating tumor cell death.

    Directory of Open Access Journals (Sweden)

    Mellone N Marchong

    2010-04-01

    Full Text Available CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01. Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8 (p = 0.01 and had fewer multifocal tumors at PND28 (p = 0.016, compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003. In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers, while proliferation in vivo remained unaffected (p = 0.121. Activated caspase-3 was significantly decreased and beta-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death.

  2. Murine cell glycolipids customization by modular expression of glycosyltransferases.

    Science.gov (United States)

    Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

    2013-01-01

    Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

  3. Acute and chronic in vivo effects of exposure to nicotine and propylene glycol from an E-cigarette on mucociliary clearance in a murine model.

    Science.gov (United States)

    Laube, Beth L; Afshar-Mohajer, Nima; Koehler, Kirsten; Chen, Gang; Lazarus, Philip; Collaco, Joseph M; McGrath-Morrow, Sharon A

    2017-04-01

    To determine the effect of an acute (1 week) and chronic (3 weeks) exposure to E-cigarette (E-cig) emissions on mucociliary clearance (MCC) in murine lungs. C57BL/6 male mice (age 10.5 ± 2.4 weeks) were exposed for 20 min/day to E-cigarette aerosol generated by a Joyetech 510-T ® E-cig containing either 0% nicotine (N)/propylene glycol (PG) for 1 week (n = 6), or 3 weeks (n = 9), or 2.4% N/PG for one week (n = 6), or 3 weeks (n = 9), followed by measurement of MCC. Control mice (n = 15) were not exposed to PG alone, or N/PG. MCC was assessed by gamma camera following aspiration of 99m technetium aerosol and was expressed as the amount of radioactivity removed from both lungs over 6 hours (MCC6hrs). Venous blood was assayed for cotinine levels in control mice and in mice exposed for 3-weeks to PG alone and N/PG. MCC6hrs in control mice and in mice acutely exposed to PG alone and N/PG was similar, averaging (±1 standard deviation) 8.6 ± 5.2%, 7.5 ± 2.8% and 11.2 ± 5.9%, respectively. In contrast, chronic exposure to PG alone stimulated MCC6hrs (17.2 ± 8.0)% and this stimulation was significantly blunted following chronic exposure to N/PG (8.7 ± 4.6)% (p < .05). Serum cotinine levels were <0.5 ng/ml in control mice and in mice exposed to PG alone, whereas, N/PG exposed mice averaged 14.6 ± 12.0 ng/ml. In this murine model, a chronic, daily, 20 min-exposure to N/PG, but not an acute exposure, slowed MCC, compared to exposure to PG alone and led to systemic absorption of nicotine.

  4. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  5. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  6. Alpha 4 integrin directs virus-activated CD8+ T cells to sites of infection

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Andersson, E C; Scheynius, A

    1995-01-01

    This article examines the role of VLA-4 in directing lymphocytes to sites of viral infection using the murine lymphocytic choriomeningitis virus infection (LCMV) as the model system. This virus by itself induces little or no inflammation, but in most mouse/virus strain combinations a potent T cell...... response is induced, which is associated with marked CD8+ cell-mediated inflammation. Two expressions of LCMV-induced inflammation were studied: meningitis induced by intracerebral infection and adoptive transfer of virus-specific delayed-type hypersensitivity. Our previous studies have shown that LCMV...

  7. Gene expression in IFN-g-activated murine macrophages

    Directory of Open Access Journals (Sweden)

    Pereira C.A.

    2004-01-01

    Full Text Available Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated or BALB/c (297 and 58 genes, respectively, up- and down-regulated mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

  8. NetH2pan: A Computational Tool to Guide MHC peptide prediction on Murine Tumors

    DEFF Research Database (Denmark)

    DeVette, Christa I; Andreatta, Massimo; Bardet, Wilfried

    2018-01-01

    With the advancement of personalized cancer immunotherapies, new tools are needed to identify tumor antigens and evaluate T-cell responses in model systems, specifically those that exhibit clinically relevant tumor progression. Key transgenic mouse models of breast cancer are generated and mainta......With the advancement of personalized cancer immunotherapies, new tools are needed to identify tumor antigens and evaluate T-cell responses in model systems, specifically those that exhibit clinically relevant tumor progression. Key transgenic mouse models of breast cancer are generated...... for evaluating antigen specificity in the murine FVB strain. Our study provides the first detailed molecular and immunoproteomic characterization of the FVB H-2q MHC Class I alleles, including >8500 unique peptide ligands, a multi-allele murine MHC peptide prediction tool, and in vivo validation of these data...

  9. Lipid peroxidation in microsomes of murine bone marrow after low-dose γ-irradiation

    International Nuclear Information System (INIS)

    Schwenke, K.; Coslar, S.; Muehlensiepen, H.; Altman, K.I.; Feinendegen, L.E.

    1994-01-01

    The principal aim of the study was to investigate the effect of low-dose γ-irradiation on lipid peroxidation (LPO) in murine bone marrow. To this end, the degree of LPO in suspensions of microsomes of murine bone marrow cells (BMC) was determined in terms of malondialdehyde (MDA) formation after whole-body or in vitro exposure to various doses of γ-radiation. These effects were compared to some extent with similar effects in liver and spleen preparations. As to the effect of γ-irradiation on LPO in BMC, the response depends on the dose level and on whether whole-body or in vitro exposures are involved. Whole-body irradiation did not result in an increase in LPO in BMC microsomes, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of BMC microsomes with 0.1, 10 and 50 Gy brought about an increase in LPO. This increase was already significant (P < 0.05) at 0.1 Gy following a post-irradiation incubation and substantial at 50 Gy, even without subsequent incubation. The results show that low doses of γ-irradiation are able to induce an elevation of LPO in murine BMC microsomes, but only after in vitro irradiation. In the case of whole-body irradiation cellular radical scavengers and other metabolic reactions may prevent a measurable increase in LPO. This is partly illustrated by the case of vitamin-E deficiency, where a substantial increase in LPO in BMC microsomes is observed even without γ-irradiation in comparison with euvitaminotic mice because normally occurring radicals are not scavenged sufficiently. (orig.)

  10. Vitrification by Ultra-fast Cooling at a Low Concentration of Cryoprotectants in a Quartz Microcapillary: A Study Using Murine Embryonic Stem Cells

    Science.gov (United States)

    He, Xiaoming; Park, Eric Y.H.; Fowler, Alex; Yarmush, Martin L.; Toner, Mehmet

    2009-01-01

    Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells. PMID:18462712

  11. An Archaeosome-Adjuvanted Vaccine and Checkpoint Inhibitor Therapy Combination Significantly Enhances Protection from Murine Melanoma

    Directory of Open Access Journals (Sweden)

    Felicity C. Stark

    2017-10-01

    Full Text Available Archaeosomes constitute archaeal lipid vesicle vaccine adjuvants that evoke a strong CD8+ T cell response to antigenic cargo. Therapeutic treatment of murine B16-ovalbumin (B16-OVA melanoma with archaeosome-OVA eliminates small subcutaneous solid tumors; however, they eventually resurge despite an increased frequency of circulating and tumor infiltrating OVA-CD8+ T cells. Herein, a number of different approaches were evaluated to improve responses, including dose number, interval, and the combination of vaccine with checkpoint inhibitors. Firstly, we found that tumor protection could not be enhanced by repetitive and/or delayed boosting to maximize the CD8+ T cell number and/or phenotype. The in vivo cytotoxicity of vaccine-induced OVA-CD8+ T cells was impaired in tumor-bearing mice. Additionally, tumor-infiltrating OVA-CD8+ T cells had an increased expression of programmed cell death protein-1 (PD-1 compared to other organ compartments, suggesting impaired function. Combination therapy of tumor-bearing mice with the vaccine archaeosome-OVA, and α-CTLA-4 administered concurrently as well as α-PD-1 and an α-PD-L1 antibody administered starting 9 days after tumor challenge given on a Q3Dx4 schedule (days 9, 12, 15 and 18, significantly enhanced survival. Following multi-combination therapy ~70% of mice had rapid tumor recession, with no detectable tumor mass after >80 days in comparison to a median survival of 17–22 days for untreated or experimental groups receiving single therapies. Overall, archaeosomes offer a powerful platform for delivering cancer antigens when used in combination with checkpoint inhibitor immunotherapies.

  12. Pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3.

    Science.gov (United States)

    Chen, Wei-Sheng; Cao, Zhiyi; Sugaya, Satoshi; Lopez, Maria J; Sendra, Victor G; Laver, Nora; Leffler, Hakon; Nilsson, Ulf J; Fu, Jianxin; Song, Jianhua; Xia, Lijun; Hamrah, Pedram; Panjwani, Noorjahan

    2016-04-12

    Lymphangiogenesis plays a pivotal role in diverse pathological conditions. Here, we demonstrate that a carbohydrate-binding protein, galectin-8, promotes pathological lymphangiogenesis. Galectin-8 is markedly upregulated in inflamed human and mouse corneas, and galectin-8 inhibitors reduce inflammatory lymphangiogenesis. In the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection. Further, in the murine model of herpes simplex virus keratitis, corneal pathology and lymphangiogenesis are ameliorated in Lgals8(-/-) mice. Mechanistically, VEGF-C-induced lymphangiogenesis is significantly reduced in the Lgals8(-/-) and Pdpn(-/-) mice; likewise, galectin-8-induced lymphangiogenesis is reduced in Pdpn(-/-) mice. Interestingly, knockdown of VEGFR-3 does not affect galectin-8-mediated lymphatic endothelial cell (LEC) sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtails both galectin-8- and VEGF-C-mediated LEC sprouting. Together, this study uncovers a unique molecular mechanism of lymphangiogenesis in which galectin-8-dependent crosstalk among VEGF-C, podoplanin and integrin pathways plays a key role.

  13. A natural human IgM that binds to gangliosides is therapeutic in murine models of amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Xiaohua Xu

    2015-08-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating, fatal neurological disease that primarily affects spinal cord anterior horn cells and their axons for which there is no treatment. Here we report the use of a recombinant natural human IgM that binds to the surface of neurons and supports neurite extension, rHIgM12, as a therapeutic strategy in murine models of human ALS. A single 200 µg intraperitoneal dose of rHIgM12 increases survival in two independent genetic-based mutant SOD1 mouse strains (SOD1G86R and SOD1G93A by 8 and 10 days, delays the onset of neurological deficits by 16 days, delays the onset of weight loss by 5 days, and preserves spinal cord axons and anterior horn neurons. Immuno-overlay of thin layer chromatography and surface plasmon resonance show that rHIgM12 binds with high affinity to the complex gangliosides GD1a and GT1b. Addition of rHIgM12 to neurons in culture increases α-tubulin tyrosination levels, suggesting an alteration of microtubule dynamics. We previously reported that a single peripheral dose of rHIgM12 preserved neurological function in a murine model of demyelination with axon loss. Because rHIgM12 improves three different models of neurological disease, we propose that the IgM might act late in the cascade of neuronal stress and/or death by a broad mechanism.

  14. The virucidal effects against murine norovirus and feline calicivirus F4 as surrogates for human norovirus by the different additive concentrations of ethanol-based sanitizers.

    Science.gov (United States)

    Akasaka, Tempei; Shimizu-Onda, Yuko; Hayakawa, Satoshi; Ushijima, Hiroshi

    2016-03-01

    Since human norovirus is non-cultivable, murine norovirus and feline calicivirus have been used as surrogates. In this study, the virucidal effects of ethanol-based sanitizers with different concentrations of additives (malic acid/sodium malate, glycerin-fatty acid ester) against murine norovirus and feline calicivirus F4 were examined. The ethanol-based sanitizers at pH 7 showed sufficient virucidal effects, but glycerin-fatty acid ester included in ethanol-based sanitizers at pH 4 or 6 reduced the virucidal effects against murine norovirus. The ethanol-based sanitizers containing malic acid/sodium malate inactivated feline calicivirus F4 in shorter time, but there is no difference between ethanol-based sanitizers with and without glycerin-fatty acid ester. Traditionally, feline calicivirus has been used for long time as a surrogate virus for human norovirus. However, this study suggested that murine norovirus and feline calicivirus F4 had different sensitivity with the additive components of ethanol-based sanitizers. Therefore, using feline calicivirus alone as a surrogate for human norovirus may not be sufficient to evaluate the virucidal effect of sanitizers on food-borne infections caused by human norovirus. Sanitizers having virucidal effects against at least both murine norovirus and feline calicivirus may be more suitable to inactivate human norovirus. Copyright © 2015. Published by Elsevier Ltd.

  15. In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

    NARCIS (Netherlands)

    De Jonge, F; De Laet, A; Van Nassauw, L; Miller, HRP; van Bogaert, PP; Timmermans, JP; Kroese, ABA

    Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close

  16. Similar [DE]XXXL[LI] motifs differentially target GLUT8 and GLUT12 in Chinese Hamster Ovary Cells

    OpenAIRE

    Flessner, Lauren B.; Moley, Kelle H.

    2008-01-01

    The transport of glucose across cell membranes is mediated by facilitative glucose transporters. The recently identified Class III glucose transporter GLUT12 is predominantly expressed in insulin-sensitive tissues such as heart, fat, and skeletal muscle. We examined the subcellular localization of GLUT12 in CHO and HEK293 cells stably expressing murine GLUT12. We have previously shown that another Class III glucose transporter, GLUT8, contains a [DE]XXXL[LI] motif that directs it to late endo...

  17. A murine model of graft-versus-host disease induced by allogeneic bone marrow transplantation

    International Nuclear Information System (INIS)

    Hu Jiangwei; Jin Jiangang; Ning Hongmei; Yu Liquan; Feng Kai; Chen Hu; Wang Lisha

    2007-01-01

    Objective: To establish the model of graft-versus-host disease (GVHD) in mice with allogeneic bone marrow transplantation. Methods: Bone marrow cells were combined with spleen cells of male donor C57BL/6 mice according to different proportions, then were transfused into female postradiation recipient BALB/c mice. General state, life span and histopathology of the recipient mice and detected chimera were observed. Results and Conclusion:The recipient mice groups which accepted above 5 x 10 6 donor spleen cells developed acute GVHD after different peroids of time. The GVHD model in mice after allo-BMT was successfully established. The transfusion of 5 x 10 6 -5 x 10 7 spleen cells may be adequate to establish the murine model of GVHD for the prevention and treatment of GVHD. The number of murine spleen cells can be chosen according to the experimental requirement. (authors)

  18. Application and evaluation of a molecular approach for detection of the schistosomicidal effect of Mirazid® (myrrh in the murine model

    Directory of Open Access Journals (Sweden)

    Wael M. Lotfy

    2013-11-01

    Full Text Available The conventional PCR technique was used for studying the schistosomicidal effect of Mirazid® in the murine model. Results of the molecular study were compared with the parasitological results (ova and worm count. The used PCR technique was more sensitive than the Kato-Katz thick smears. Mirazid® showed some schistosomicidal effects against murine Schistosoma mansoni. However, it was not efficient enough to cure any of the studied mice.

  19. Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model

    DEFF Research Database (Denmark)

    Cavanagh, Jorunn Pauline; Granslo, Hildegunn Norbakken; Fredheim, Elizabeth Aarag

    2013-01-01

    Objectives Biofilm-forming Staphylococcus epidermidis is a prevalent cause of peritonitis during peritoneal dialysis. We compared the efficacy of a synthetic antimicrobial peptidomimetic (Ltx21) versus vancomycin in a murine model mimicking a device-related peritonitis. Methods Silicone implants......, pre-colonized with an S. epidermidis biofilm, were inserted into the peritoneal cavity of BALB/c mice. Three groups (36 mice in each) with pre-colonized implants received intraperitoneal treatment with Ltx21, vancomycin or placebo. Mice were euthanized on day 3 (n = 12), day 6 (n = 12) or day 8 (n...... = 12) post-implantation. Controls were mice with sterile implants (n = 18) and mice without surgery (n = 6). Bacterial reductions in cfu were analysed from implants and peritoneal fluid (PF). Inflammatory responses in serum and PF were measured. Results Vancomycin resulted in a stronger reduction...

  20. Adiponectin and plant-derived mammalian adiponectin homolog exert a protective effect in murine colitis

    KAUST Repository

    Arsenescu, Violeta

    2011-04-11

    Background: Hypoadiponectinemia has been associated with states of chronic inflammation in humans. Mesenteric fat hypertrophy and low adiponectin have been described in patients with Crohn\\'s disease. We investigated whether adiponectin and the plant-derived homolog, osmotin, are beneficial in a murine model of colitis. Methods: C57BL/6 mice were injected (i.v.) with an adenoviral construct encoding the full-length murine adiponectin gene (AN+DSS) or a reporter-LacZ (Ctr and V+DSS groups) prior to DSS colitis protocol. In another experiment, mice with DSS colitis received either osmotin (Osm+DSS) or saline (DSS) via osmotic pumps. Disease progression and severity were evaluated using body weight, stool consistency, rectal bleeding, colon lengths, and histology. In vitro experiments were carried out in bone marrow-derived dendritic cells. Results: Mice overexpressing adiponectin had lower expression of proinflammatory cytokines (TNF, IL-1β), adipokines (angiotensin, osteopontin), and cellular stress and apoptosis markers. These mice had higher levels of IL-10, alternative macrophage marker, arginase 1, and leukoprotease inhibitor. The plant adiponectin homolog osmotin similarly improved colitis outcome and induced robust IL-10 secretion. LPS induced a state of adiponectin resistance in dendritic cells that was reversed by treatment with PPARγ agonist and retinoic acid. Conclusion: Adiponectin exerted protective effects during murine DSS colitis. It had a broad activity that encompassed cytokines, chemotactic factors as well as processes that assure cell viability during stressful conditions. Reducing adiponectin resistance or using plant-derived adiponectin homologs may become therapeutic options in inflammatory bowel disease. © 2011 Springer Science+Business Media, LLC.

  1. Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

    Science.gov (United States)

    Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

    2017-08-01

    Designing CD8 + T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 + T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1 + CD8 + T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 + T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 + T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 + T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Gender and dose dependent ovalbumin induced hypersensitivity responses in murine model of food allergy

    Science.gov (United States)

    While federal regulations mandate the labeling of major food allergens, allowable food allergen thresholds have yet to be determined. Therefore the aim of this project was to identify the lowest egg allergen ovalbumin (OVA) dose causing hypersensitization using a validated murine model. Mice were or...

  3. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    Science.gov (United States)

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. © 2015 Wiley Periodicals, Inc.

  4. Rediscovering peritoneal macrophages in a murine endometriosis model.

    Science.gov (United States)

    Yuan, Ming; Li, Dong; An, Min; Li, Qiuju; Zhang, Lu; Wang, Guoyun

    2017-01-01

    What are the features of peritoneal macrophage subgroups and T helper cells in the development of murine endometriosis? During the development of endometriosis in a murine model, large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs) are polarized into M1 and M2 cells, respectively, and the proportions of T helper (Th) 1, Th17 and T regulatory (T reg ) cells are increased. Numerous studies investigating the etiology and pathogenesis of endometriosis have focused on the polarization states of peritoneal macrophages in endometriosis models and patients, but the results are inconclusive. Further studies indicate that peritoneal macrophages are composed of two distinct subsets: LPMs and SPMs, although their roles in endometriosis are unknown. This study involves a prospective and randomized experiment. Fifty C57BL/6 female mice were randomly allocated to five control and five experimental groups (n = 5/group) according to the presence or absence of transplantation. The transplant periods are 0.25, 3, 14, 28 and 42 days. C57BL/6 mice were utilized to establish an endometriosis model by i.p. injection of allogeneic endometrial segments. Dynamic changes of peritoneal macrophage subsets and polarization profiles were evaluated by flow cytometry (FCM). Macrophage morphology and density were assessed by cell counting under a microscope. Dynamic changes of Th1, Th2, Th17 and T reg cells were estimated by FCM. Peritoneal macrophages are composed of two distinct subsets: LPMs and SPMs. The proportion of SPMs increased immediately after peritoneal injection of endometrial tissues, whereas LPMs showed an opposite trend. Peritoneal macrophages differentiated into both M1 and M2 macrophages. The bidirectional polarization of macrophages was caused by the inverse trends of polarization of LPMs and SPMs. Consistently, the proportions of Th1, Th17 and T reg cells were all increased in mice with endometriosis. N/A. In this study, detection was only performed in a

  5. Tofacitinib Ameliorates Murine Lupus and Its Associated Vascular Dysfunction.

    Science.gov (United States)

    Furumoto, Yasuko; Smith, Carolyne K; Blanco, Luz; Zhao, Wenpu; Brooks, Stephen R; Thacker, Seth G; Abdalrahman, Zarzour; Sciumè, Giuseppe; Tsai, Wanxia L; Trier, Anna M; Nunez, Leti; Mast, Laurel; Hoffmann, Victoria; Remaley, Alan T; O'Shea, John J; Kaplan, Mariana J; Gadina, Massimo

    2017-01-01

    Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized. MRL/lpr lupus-prone mice were administered tofacitinib or vehicle by gavage for 6 weeks (therapeutic arm) or 8 weeks (preventive arm). Nephritis, skin inflammation, serum levels of autoantibodies and cytokines, mononuclear cell phenotype and gene expression, neutrophil extracellular traps (NETs) release, endothelium-dependent vasorelaxation, and endothelial differentiation were compared in treated and untreated mice. Treatment with tofacitinib led to significant improvement in measures of disease activity, including nephritis, skin inflammation, and autoantibody production. In addition, tofacitinib treatment reduced serum levels of proinflammatory cytokines and interferon responses in splenocytes and kidney tissue. Tofacitinib also modulated the formation of NETs and significantly increased endothelium-dependent vasorelaxation and endothelial differentiation. The drug was effective in both preventive and therapeutic strategies. Tofacitinib modulates the innate and adaptive immune responses, ameliorates murine lupus, and improves vascular function. These results indicate that JAK inhibitors have the potential to be beneficial in SLE and its associated vascular damage. © 2016, American College of Rheumatology.

  6. Murine leukemia viruses: objects and organisms.

    Science.gov (United States)

    Rein, Alan

    2011-01-01

    Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes-protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.

  7. S100A8/A9 is not involved in host defense against murine urinary tract infection.

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    Mark C Dessing

    Full Text Available BACKGROUND: Inflammation is commonly followed by the release of endogenous proteins called danger associated molecular patterns (DAMPs that are able to warn the host for eminent danger. S100A8/A9 subunits are DAMPs that belong to the S100 family of calcium binding proteins. S100A8/A9 complexes induce an inflammatory response and their expression correlates with disease severity in several inflammatory disorders. S100A8/A9 promote endotoxin- and Escherichia (E. coli-induced sepsis showing its contribution in systemic infection. The role of S100A8/A9 during a local infection of the urinary tract system caused by E. coli remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the contribution of S100A8/A9 in acute urinary tract infection (UTI by instilling 2 different doses of uropathogenic E. coli transurethrally in wild type (WT and S100A9 knockout (KO mice. Subsequently, we determined bacterial outgrowth, neutrophilic infiltrate and inflammatory mediators in bladder and kidney 24 and 48 hours later. UTI resulted in a substantial increase of S100A8/A9 protein in bladder and kidney tissue of WT mice. S100A9 KO mice displayed similar bacterial load in bladder or kidney homogenate compared to WT mice using 2 different doses at 2 different time points. S100A9 deficiency had little effect on the inflammatory responses to E. Coli-induced UTI infection, as assessed by myeloperoxidase activity in bladder and kidneys, histopathologic analysis, and renal and bladder cytokine concentrations. CONCLUSIONS: We show that despite high S100A8/A9 expression in bladder and kidney tissue upon UTI, S100A8/A9 does not contribute to an effective host response against E. Coli in the urinary tract system.

  8. A microculture system for the measurement of antigen-induced murine lymphocyte proliferation: advantages of 5% horse serum and 5 X 10(-5) M mercaptoethanol.

    Science.gov (United States)

    Brummer, E; Vris, T W; Lawrence, H S

    1977-01-01

    Short term microculture systems which measure murine lymphocyte proliferative responses to mitogens are well established. We demonstrate here that these microculture methods are not suitable for antigen-induced responses because of the high levels of murine lymphocyte proliferation in control cultures associated with the use of fetal calf serum or human serum. We also show that this problem can be eliminated with the use of a combination of 5% horse serum and 5 X 10(-5) M mercaptoethanol. We describe an antigen-induced murine lymphocyte proliferation microculture system in which good stimulation indices are achieved and the lymphocyte proliferation in control cultures remain at a low level throughout the 7 day culture period.

  9. Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

    Science.gov (United States)

    Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

    2008-03-01

    We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

  10. L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

    Science.gov (United States)

    Dewannieux, Marie; Heidmann, Thierry

    2005-06-03

    SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

  11. Receptor for advanced glycation end-products and World Trade Center particulate induced lung function loss: A case-cohort study and murine model of acute particulate exposure.

    Science.gov (United States)

    Caraher, Erin J; Kwon, Sophia; Haider, Syed H; Crowley, George; Lee, Audrey; Ebrahim, Minah; Zhang, Liqun; Chen, Lung-Chi; Gordon, Terry; Liu, Mengling; Prezant, David J; Schmidt, Ann Marie; Nolan, Anna

    2017-01-01

    World Trade Center-particulate matter(WTC-PM) exposure and metabolic-risk are associated with WTC-Lung Injury(WTC-LI). The receptor for advanced glycation end-products (RAGE) is most highly expressed in the lung, mediates metabolic risk, and single-nucleotide polymorphisms at the AGER-locus predict forced expiratory volume(FEV). Our objectives were to test the hypotheses that RAGE is a biomarker of WTC-LI in the FDNY-cohort and that loss of RAGE in a murine model would protect against acute PM-induced lung disease. We know from previous work that early intense exposure at the time of the WTC collapse was most predictive of WTC-LI therefore we utilized a murine model of intense acute PM-exposure to determine if loss of RAGE is protective and to identify signaling/cytokine intermediates. This study builds on a continuing effort to identify serum biomarkers that predict the development of WTC-LI. A case-cohort design was used to analyze a focused cohort of male never-smokers with normal pre-9/11 lung function. Odds of developing WTC-LI increased by 1.2, 1.8 and 1.0 in firefighters with soluble RAGE (sRAGE)≥97pg/mL, CRP≥2.4mg/L, and MMP-9≤397ng/mL, respectively, assessed in a multivariate logistic regression model (ROCAUC of 0.72). Wild type(WT) and RAGE-deficient(Ager-/-) mice were exposed to PM or PBS-control by oropharyngeal aspiration. Lung function, airway hyperreactivity, bronchoalveolar lavage, histology, transcription factors and plasma/BAL cytokines were quantified. WT-PM mice had decreased FEV and compliance, and increased airway resistance and methacholine reactivity after 24-hours. Decreased IFN-γ and increased LPA were observed in WT-PM mice; similar findings have been reported for firefighters who eventually develop WTC-LI. In the murine model, lack of RAGE was protective from loss of lung function and airway hyperreactivity and was associated with modulation of MAP kinases. We conclude that in a multivariate adjusted model increased sRAGE is

  12. Receptor for advanced glycation end-products and World Trade Center particulate induced lung function loss: A case-cohort study and murine model of acute particulate exposure.

    Directory of Open Access Journals (Sweden)

    Erin J Caraher

    Full Text Available World Trade Center-particulate matter(WTC-PM exposure and metabolic-risk are associated with WTC-Lung Injury(WTC-LI. The receptor for advanced glycation end-products (RAGE is most highly expressed in the lung, mediates metabolic risk, and single-nucleotide polymorphisms at the AGER-locus predict forced expiratory volume(FEV. Our objectives were to test the hypotheses that RAGE is a biomarker of WTC-LI in the FDNY-cohort and that loss of RAGE in a murine model would protect against acute PM-induced lung disease. We know from previous work that early intense exposure at the time of the WTC collapse was most predictive of WTC-LI therefore we utilized a murine model of intense acute PM-exposure to determine if loss of RAGE is protective and to identify signaling/cytokine intermediates. This study builds on a continuing effort to identify serum biomarkers that predict the development of WTC-LI. A case-cohort design was used to analyze a focused cohort of male never-smokers with normal pre-9/11 lung function. Odds of developing WTC-LI increased by 1.2, 1.8 and 1.0 in firefighters with soluble RAGE (sRAGE≥97pg/mL, CRP≥2.4mg/L, and MMP-9≤397ng/mL, respectively, assessed in a multivariate logistic regression model (ROCAUC of 0.72. Wild type(WT and RAGE-deficient(Ager-/- mice were exposed to PM or PBS-control by oropharyngeal aspiration. Lung function, airway hyperreactivity, bronchoalveolar lavage, histology, transcription factors and plasma/BAL cytokines were quantified. WT-PM mice had decreased FEV and compliance, and increased airway resistance and methacholine reactivity after 24-hours. Decreased IFN-γ and increased LPA were observed in WT-PM mice; similar findings have been reported for firefighters who eventually develop WTC-LI. In the murine model, lack of RAGE was protective from loss of lung function and airway hyperreactivity and was associated with modulation of MAP kinases. We conclude that in a multivariate adjusted model increased s

  13. Oxaliplatin but Not Irinotecan Impairs Posthepatectomy Liver Regeneration in a Murine Model

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    Perry A. Soriano

    2011-01-01

    Full Text Available Introduction. We examined the murine hepatectomy model of liver regeneration (LR in the setting of neoadjuvant chemotherapy. Methods. C57BL/6 mice were randomized to receive neoadjuvant intraperitoneal (IP injections of a control, oxaliplatin (15 mg/kg, or irinotecan (100 mg/Kg or 250 mg/Kg solution. Hepatectomy (70% was performed 14 days after the final IP treatment. Animals were sacrificed at postoperative day (D 0, 1, 2, 3, and 7. Liver remnants and serum were collected for analysis. -tests for independent samples were used for statistical comparisons. Results. For oxaliplatin, percent LR did not differ at D1 or D2 but was significantly less at D3 (89.0% versus 70.0%, =0.048 with no difference on D7 (=0.21. Irinotecan-treated mice at both dose levels (100 mg/Kg and 250 mg/Kg showed no significant differences in LR. BrdU incorporation was significantly decreased in oxaliplatin-treated animals (D1,2,3. Conclusions. Neoadjuvant oxaliplatin but not irinotecan impairs early LR in a posthepatectomy murine model which correlates with decreased DNA synthesis.

  14. Oxaliplatin but Not Irinotecan Impairs Posthepatectomy Liver Regeneration in a Murine Model

    Science.gov (United States)

    Soriano, Perry A.; Liu, Nian; Castillo, Erick; Foster, Brock; Artinyan, Avo; Kim, Joseph; Huang, Wendong; Wagman, Lawrence D.

    2011-01-01

    Introduction. We examined the murine hepatectomy model of liver regeneration (LR) in the setting of neoadjuvant chemotherapy. Methods. C57BL/6 mice were randomized to receive neoadjuvant intraperitoneal (IP) injections of a control, oxaliplatin (15 mg/kg), or irinotecan (100 mg/Kg or 250 mg/Kg) solution. Hepatectomy (70%) was performed 14 days after the final IP treatment. Animals were sacrificed at postoperative day (D) 0, 1, 2, 3, and 7. Liver remnants and serum were collected for analysis. T-tests for independent samples were used for statistical comparisons. Results. For oxaliplatin, percent LR did not differ at D1 or D2 but was significantly less at D3 (89.0% versus 70.0%, P = 0.048) with no difference on D7 (P = 0.21). Irinotecan-treated mice at both dose levels (100 mg/Kg and 250 mg/Kg) showed no significant differences in LR. BrdU incorporation was significantly decreased in oxaliplatin-treated animals (D1,2,3). Conclusions. Neoadjuvant oxaliplatin but not irinotecan impairs early LR in a posthepatectomy murine model which correlates with decreased DNA synthesis. PMID:22164336

  15. Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples.

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    Li Ling Lee

    Full Text Available Gammaretroviruses related to murine leukemia virus (MLV have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

  16. The use of in situ proteolysis in the crystallization of murine CstF-77

    International Nuclear Information System (INIS)

    Bai, Yun; Auperin, Thierry C.; Tong, Liang

    2007-01-01

    In situ proteolysis with fungal protease or subtilisin is crucial for the crystallization of murine CstF-77. The cleavage-stimulation factor (CstF) is required for the cleavage of the 3′-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF-77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF-77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality

  17. Volume measurement variability in three-dimensional high-frequency ultrasound images of murine liver metastases

    International Nuclear Information System (INIS)

    Wirtzfeld, L A; Graham, K C; Groom, A C; MacDonald, I C; Chambers, A F; Fenster, A; Lacefield, J C

    2006-01-01

    The identification and quantification of tumour volume measurement variability is imperative for proper study design of longitudinal non-invasive imaging of pre-clinical mouse models of cancer. Measurement variability will dictate the minimum detectable volume change, which in turn influences the scheduling of imaging sessions and the interpretation of observed changes in tumour volume. In this paper, variability is quantified for tumour volume measurements from 3D high-frequency ultrasound images of murine liver metastases. Experimental B16F1 liver metastases were analysed in different size ranges including less than 1 mm 3 , 1-4 mm 3 , 4-8 mm 3 and 8-70 mm 3 . The intra- and inter-observer repeatability was high over a large range of tumour volumes, but the coefficients of variation (COV) varied over the volume ranges. The minimum and maximum intra-observer COV were 4% and 14% for the 1-4 mm 3 and 3 tumours, respectively. For tumour volumes measured by segmenting parallel planes, the maximum inter-slice distance that maintained acceptable measurement variability increased from 100 to 600 μm as tumour volume increased. Comparison of free breathing versus ventilated animals demonstrated that respiratory motion did not significantly change the measured volume. These results enable design of more efficient imaging studies by using the measured variability to estimate the time required to observe a significant change in tumour volume

  18. Effect of in vitro and in vivo UV irradiation on the production of ETAF activity by human and murine keratinocytes

    International Nuclear Information System (INIS)

    Ansel, J.C.; Luger, T.A.; Green, I.

    1983-01-01

    Cultured epidermal cells and keratinocytes produce a potent hormone-like factor called epidermal cell-derived thymocyte-activating factor (ETAF). ETAF appears to be similar if not identical to a monocyte-derived lymphokine, known as interleukin 1 (IL-1). These two cytokines are able to amplify a diverse number of proliferative and inflammatory processes. Several recent investigations have suggested that UV-induced immunosuppression may be due in part to the inhibition of IL-1/ETAF production by monocytes and keratinocytes, respectively. We therefore decided to directly study the effects of various doses of in vitro and in vivo UV radiation (UVR) on the production of ETAF by normal murine epidermal cells and a murine (Pam 212) and a human (SCC) keratinocyte cell line. Our results surprisingly demonstrated an increase in both the extracellular and the intracellular ETAF activity of the murine epidermal, Pam 212, and SCC after sublethal amounts of in vitro UVR. Likewise, increased ETAF activity of murine epidermal cells was detected after sublethal doses of in vivo UVR. The UV-induced ETAF activity was cycloheximide-sensitive, suggesting that de novo synthesis of ETAF rather than cell membrane leakage was responsible for the increased ETAF activity. The fact that UV irradiation can increase ETAF activity by keratinocytes could have important local and systemic consequences for the host and may provide an efficient, contaminant-free method for generating ETAF activity for further biochemical and immunologic studies

  19. Expression, refolding and crystallization of murine MHC class I H-2Db in complex with human β2-microglobulin

    International Nuclear Information System (INIS)

    Sandalova, Tatyana; Michaëlsson, Jakob; Harris, Robert A.; Ljunggren, Hans-Gustaf; Kärre, Klas; Schneider, Gunter; Achour, Adnane

    2005-01-01

    Mouse MHC class I H-2Db in complex with human β2m and the LCMV-derived peptide gp33 has been produced and crystallized. Resolution of the structure of this complex combined with the structural comparison with the previously solved crystal structure of H-2Db/mβ2m/gp33 should lead to a better understanding of how the β2m subunit affects the overall conformation of MHC complexes as well as the stability of the presented peptides. β 2 -Microglobulin (β 2 m) is non-covalently linked to the major histocompatibility (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I can bind human β 2 m (hβ 2 m) and such hybrid molecules are often used in structural and functional studies. The replacement of mouse β 2 m (mβ 2 m) by hβ 2 m has important functional consequences for MHC class I complex stability and specificity, but the structural basis for this is unknown. To investigate the impact of species-specific β 2 m subunits on MHC class I conformation, murine MHC class I H-2D b in complex with hβ 2 m and the peptide gp33 derived from lymphocytic choriomeningitis virus (LCMV) has been expressed, refolded in vitro and crystallized. Crystals containing two complexes per asymmetric unit and belonging to the space group P2 1 , with unit-cell parameters a = 68.1, b = 65.2, c = 101.9 Å, β = 102.4°, were obtained

  20. Co-expression of the transcription factors CEH-14 and TTX-1 regulates AFD neuron-specific genes gcy-8 and gcy-18 in C. elegans.

    Science.gov (United States)

    Kagoshima, Hiroshi; Kohara, Yuji

    2015-03-15

    A wide variety of cells are generated by the expression of characteristic sets of genes, primarily those regulated by cell-specific transcription. To elucidate the mechanism regulating cell-specific gene expression in a highly specialized cell, AFD thermosensory neuron in Caenorhabditis elegans, we analyzed the promoter sequences of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. In this study, we showed that AFD-specific expression of gcy-8 and gcy-18 requires the co-expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1. We observed that mutation of ttx-1 or ceh-14 caused a reduction in the expression of gcy-8 and gcy-18 and that the expression was completely lost in double mutants. This synergy effect was also observed with other AFD marker genes, such as ntc-1, nlp-21and cng-3. Electrophoretic mobility shift assays revealed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters in vitro. The binding sites of CEH-14 and TTX-1 proteins were confirmed to be essential for AFD-specific expression of gcy-8 and gcy-18 in vivo. We also demonstrated that forced expression of CEH-14 and TTX-1 in AWB chemosensory neurons induced ectopic expression of gcy-8 and gcy-18 reporters in this neuron. Finally, we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Taken together, ceh-14 and ttx-1 expression determines the fate of AFD as terminal selector genes at the final step of cell specification. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture.

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    Yeojun Yun

    Full Text Available Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.

  2. DGCR8 Promotes Neural Progenitor Expansion and Represses Neurogenesis in the Mouse Embryonic Neocortex

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    Nadin Hoffmann

    2018-04-01

    Full Text Available DGCR8 and DROSHA are the minimal functional core of the Microprocessor complex essential for biogenesis of canonical microRNAs and for the processing of other RNAs. Conditional deletion of Dgcr8 and Drosha in the murine telencephalon indicated that these proteins exert crucial functions in corticogenesis. The identification of mechanisms of DGCR8- or DROSHA-dependent regulation of gene expression in conditional knockout mice are often complicated by massive apoptosis. Here, to investigate DGCR8 functions on amplification/differentiation of neural progenitors cells (NPCs in corticogenesis, we overexpress Dgcr8 in the mouse telencephalon, by in utero electroporation (IUEp. We find that DGCR8 promotes the expansion of NPC pools and represses neurogenesis, in absence of apoptosis, thus overcoming the usual limitations of Dgcr8 knockout-based approach. Interestingly, DGCR8 selectively promotes basal progenitor amplification at later developmental stages, entailing intriguing implications for neocortical expansion in evolution. Finally, despite a 3- to 5-fold increase of DGCR8 level in the mouse telencephalon, the composition, target preference and function of the DROSHA-dependent Microprocessor complex remain unaltered. Thus, we propose that DGCR8-dependent modulation of gene expression in corticogenesis is more complex than previously known, and possibly DROSHA-independent.

  3. Amphipathic DNA polymers exhibit antiviral activity against systemic Murine Cytomegalovirus infection

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    Juteau Jean-Marc

    2009-12-01

    Full Text Available Abstract Background Phosphorothioated oligonucleotides (PS-ONs have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV infections in vitro and in vivo was therefore investigated. Results In vitro, a 40 mer degenerate AP (REP 9 inhibited both murine CMV (MCMV and guinea pig CMV (GPCMV with an IC50 of 0.045 μM and 0.16 μM, respectively, and a 40 mer poly C AP (REP 9C inhibited MCMV with an IC50 of 0.05 μM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers. Conclusion These studies indicate that APs exhibit potent, well tolerated

  4. Differential chemokine responses in the murine brain following lyssavirus infection.

    Science.gov (United States)

    Hicks, D J; Núñez, A; Banyard, A C; Williams, A; Ortiz-Pelaez, A; Fooks, A R; Johnson, N

    2013-11-01

    The hallmark of lyssavirus infection is lethal encephalomyelitis. Previous studies have reported distinct lyssavirus isolate-related differences in severity of cellular recruitment into the encephalon in a murine model of infection following peripheral inoculation with rabies virus (RABV) and European bat lyssavirus (EBLV)-1 and -2. In order to understand the role of chemokines in this process, comparative studies of the chemokine pattern, distribution and production in response to infection with these lyssaviruses were undertaken. Expression of CCL2, CCL5 and CXCL10 was observed throughout the murine brain with a distinct caudal bias in distribution, similar to both inflammatory changes and virus antigen distribution. CCL2 immunolabelling was localized to neuronal and astroglial populations. CCL5 immunolabelling was only detected in the astroglia, while CXCL10 labelling, although present in the astroglia, was more prominent in neurons. Isolate-dependent differences in the amount of chemokine immunolabelling in specific brain regions and chemokine production by neurons in vitro were observed, with a greater expression of CCL5 in vivo and CXCL10 production in vitro after EBLV infection. Additionally, strong positive associations between chemokine immunolabelling and perivascular cuffing and, to a lesser extent, virus antigen score were also observed. These differences in chemokine expression may explain the variation in severity of encephalitic changes observed in animals infected with different lyssavirus isolates. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  5. Coagulation and inflammation in scrub typhus and murine typhusu-a prospective comparative study from Laos

    NARCIS (Netherlands)

    Paris, D. H.; Chansamouth, V.; Nawtaisong, P.; Löwenberg, E. C.; Phetsouvanh, R.; Blacksell, S. D.; Lee, S. J.; Dondorp, A. M.; van der Poll, T.; Newton, P. N.; Levi, M. [=Marcel M.; Day, N. P. J.

    2012-01-01

    Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited.

  6. Impact of neutrophil-secreted myeloid related proteins 8 and 14 (MRP 8/14) on leishmaniasis progression.

    Science.gov (United States)

    Contreras, Irazú; Shio, Marina T; Cesaro, Annabelle; Tessier, Philippe A; Olivier, Martin

    2013-01-01

    The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.

  7. Murine nephrotoxic nephritis as a model of chronic kidney disease

    DEFF Research Database (Denmark)

    Ougaard, M. K.E.; Kvist, P. H.; Jensen, H. E.

    2018-01-01

    Using the nonaccelerated murine nephrotoxic nephritis (NTN) as a model of chronic kidney disease (CKD) could provide an easily inducible model that enables a rapid test of treatments. Originally, the NTN model was developed as an acute model of glomerulonephritis, but in this study we evaluate...... progressive mesangial expansion and significant renal fibrosis within three weeks suggesting CKD development. CD1 and C57BL/6 females showed a similar disease progression, but female mice seemed more susceptible to NTS compared to male mice. The presence of albuminuria, GFR decline, mesangial expansion...

  8. Anticonvulsive evaluation of THIP in the murine pentylenetetrazole kindling model

    DEFF Research Database (Denmark)

    Simonsen, Charlotte; Boddum, Kim; von Schoubye, Nadia L

    2017-01-01

    . Evaluation of THIP as a potential anticonvulsant has given contradictory results in different animal models and for this reason, we reevaluated the anticonvulsive properties of THIP in the murine pentylenetetrazole (PTZ) kindling model. As loss of δ-GABAA R in the dentate gyrus has been associated...... the observed upregulation of δ-GABAA Rs. Even in the demonstrated presence of functional δ-GABAA Rs, THIP (0.5-4 mg/kg) showed no anticonvulsive effect in the PTZ kindling model using a comprehensive in vivo evaluation of the anticonvulsive properties....

  9. Production of antibodies which recognize opiate receptors on murine leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

    1988-01-01

    An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

  10. Adrenaline influences the release of interleukin-6 from murine pituicytes

    DEFF Research Database (Denmark)

    Christensen, J D; Hansen, E W; Frederiksen, C

    1999-01-01

    In this study, we examined the effect of adrenaline and interleukin-1beta on interleukin-6 secretion from cultured murine neurohypophyseal cells. Cells were cultured from neurohypophyses of 3- to 5-week-old mice and experiments were performed after 13 days in culture. Interleukin-6 was measured...... in 24-h samples using a sandwich fluoroimmunoassay. Unstimulated cells released 19+/-3 fmol interleukin-6/neurohypophysis/24 h (mean +/- S.E.M., n = 42). Adrenaline and interleukin-1beta increased the release of interleukin-6 from the cells in a concentration-dependent manner. Incubation with adrenaline...

  11. Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

    International Nuclear Information System (INIS)

    Liu Junyan; Qiu Hong; Morisseau, Christophe; Hwang, Sung Hee; Tsai, Hsing-Ju; Ulu, Arzu; Chiamvimonvat, Nipavan; Hammock, Bruce D.

    2011-01-01

    The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs. - Graphical abstract: Display Omitted Research Highlights: → Anti-microbial triclocarban (TCC) is anti-inflammatory in a murine model. → TCC significantly shifted the oxylipin profile in vivo as expected from a sEHI. → TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. → TCC significantly repressed LPS-induced increased release of inflammatory cytokines.

  12. IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

    Directory of Open Access Journals (Sweden)

    Babban Jee

    2017-07-01

    Full Text Available Mycobacterium tuberculosis (MTB adopts a special survival strategy to overcome the killing mechanism(s of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3 of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ and recombinant murine interleukin-10 (rmIL-10 on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

  13. Resolvin E1 (RX-10001) reduces corneal epithelial barrier disruption and protects against goblet cell loss in a murine model of dry eye.

    Science.gov (United States)

    de Paiva, Cintia S; Schwartz, C Eric; Gjörstrup, Per; Pflugfelder, Stephen C

    2012-11-01

    Resolvin E1 (RvE1; RX-10001) belongs to a new class of endogenous immunoregulating mediators, originally identified as a metabolite of the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid. Based on its proven efficacy in models of chronic inflammation, this study investigated the efficacy of resolvin E1 in a murine model of dry eye. C57/B6 mice, aged 6 to 8 weeks, were treated with systemic scopolamine and exposed to air draft and low humidity for 16 hours/day for 5 days and allocated to the following groups: unexposed controls, disease controls, treatment with vehicle or RvE1 delivered topically as its methyl ester prodrug, RX-10005, to enhance corneal surface penetration. Treatment was initiated at the time of desiccating stress induction. Treatment efficacy was assessed by corneal permeability using Oregon Green Dextran and by conjunctival goblet cell density using periodic acid-Schiff reagent. RvE1 reduced the increase in corneal staining by 80% compared with untreated disease controls. Goblet cell density was reduced by 20% in disease controls but fully maintained in the group receiving RvE1. RvE1, delivered as its methyl ester prodrug, improved the outcome measures of corneal staining and goblet cell density in this murine model of dry eye, indicating the potential utility of endogenous resolvins and resolvin analogues in the treatment of dry eye.

  14. Generation of a conditional knockout of murine glucocerebrosidase: utility for the study of Gaucher disease.

    Science.gov (United States)

    Sinclair, Graham B; Jevon, Gareth; Colobong, Karen E; Randall, Derrick R; Choy, Francis Y M; Clarke, Lorne A

    2007-02-01

    Gaucher disease is a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal hydrolase glucocerebrosidase. Affected individuals present with a spectrum of clinical symptoms ranging from hepatosplenomegaly, haematological abnormalities, and bone pain in type 1 disease, to severe neurodegeneration and premature death in types 2 and 3 disease. Although the basic biochemical defect is well characterized, there remains a poor understanding of the underlying pathophysiology of disease. In vitro studies suggest that macrophage glucocerebroside storage leads to tissue dysfunction through complex mechanisms involving altered intracellular calcium homeostasis and apoptosis. In order to study the pathogenic roles of these complex interactions, a viable animal model for Gaucher disease is needed. The complexity of this single gene disorder has been emphasized by the varied results of previous murine Gaucher models, ranging from perinatal lethality to phenotypically and biochemically asymptomatic animals. Recognizing the need to modulate the biochemical phenotype in mice to produce a relevant model, we have created a murine strain with key exons of the glucocerebrosidase gene flanked by loxP sites. We show that expression of Cre-recombinase in cells of hematopoietic and endothelial origin results in deficiency of glucocerebrosidase in the liver, spleen, bone marrow, and peripheral white cells. Glucocerebroside storage in this model leads to progressive splenomegaly with Gaucher cell infiltration and modest storage in the liver by 26 weeks of age. These results indicate the utility of this loxP GBA targeted murine strain for understanding the complex pathophysiology of Gaucher disease.

  15. Efficacy of irreversible electroporation in human pancreatic adenocarcinoma: advanced murine model

    Directory of Open Access Journals (Sweden)

    Prejesh Philips

    Full Text Available Irreversible electroporation (IRE is a promising cell membrane ablative modality for pancreatic cancer. There have been recent concerns regarding local recurrence and the potential use of IRE as a debulking (partial ablation modality. We hypothesize that incomplete ablation leads to early recurrence and a more aggressive biology. We created the first ever heterotopic murine model by inoculating BALB/c nude mice in the hindlimb with a subcutaneous injection of Panc-1 cells, an immortalized human pancreatic adenocarcinoma cell line. Tumors were allowed to grow from 0.75 to 1.5 cm and then treated with the goal of complete ablation or partial ablation using standard IRE settings. Animals were recovered and survived for 2 days (n = 6, 7 (n = 6, 14 (n = 6, 21 (n = 6, 30 (n = 8, and 60 (n = 8 days. All 40 animals/tumors underwent successful IRE under general anesthesia with muscle paralysis. The mean tumor volume of the animals undergoing ablation was 1,447.6 mm3 ± 884. Histologically, in the 14-, 21-, 30-, and 60-day survival groups the entire tumor was nonviable, with a persistent tumor nodule completely replaced fibrosis. In the group treated with partial ablation, incomplete electroporation/recurrences (N = 10 animals were seen, of which 66% had confluent tumors and this was a significant predictor of recurrence (P < 0.001. Recurrent tumors were also significantly larger (mean 4,578 mm3 ± SD 877 versus completed electroporated tumors 925.8 ± 277, P < 0.001. Recurrent tumors had a steeper growth curve (slope = 0.73 compared with primary tumors (0.60, P = 0.02. Recurrent tumors also had a significantly higher percentage of EpCAM expression, suggestive of stem cell activation. Tumors that recur after incomplete electroporation demonstrate a biologically aggressive tumor that could be more resistant to standard of care chemotherapy. Clinical correlation of this data is limited, but should be considered when IRE of pancreatic cancer is being

  16. T-cell help permits memory CD8(+) T-cell inflation during cytomegalovirus latency.

    Science.gov (United States)

    Walton, Senta M; Torti, Nicole; Mandaric, Sanja; Oxenius, Annette

    2011-08-01

    CD4(+) T cells are implied to sustain CD8(+) T-cell responses during persistent infections. As CD4(+) T cells are often themselves antiviral effectors, they might shape CD8(+) T-cell responses via help or via controlling antigen load. We used persistent murine CMV (MCMV) infection to dissect the impact of CD4(+) T cells on virus-specific CD8(+) T cells, distinguishing between increased viral load in the absence of CD4(+) T cells and CD4(+) T-cell-mediated helper mechanisms. Absence of T-helper cells was associated with sustained lytic MCMV replication and led to a slow and gradual reduction of the size and function of the MCMV-specific CD8(+) T-cell pool. However, when virus replication was controlled in the absence of CD4(+) T cells, CD8(+) T-cell function was comparably impaired, but in addition CD8(+) T-cell inflation, a hallmark of CMV infection, was completely abolished. Thus, CD8(+) T-cell inflation during latent CMV infection is strongly dependent on CD4(+) T-cell helper functions, which can partially be compensated by ongoing lytic viral replication in the absence of CD4(+) T cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Immunohistochemical Characterization of S100A6 in the Murine Ovary

    International Nuclear Information System (INIS)

    Hanaue, Mayu; Miwa, Naofumi; Takamatsu, Ken

    2012-01-01

    S100 proteins comprise a large family of Ca 2+ -binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions

  18. Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation

    International Nuclear Information System (INIS)

    Pascale, E.; Valle, E.; Furano, A.V.

    1990-01-01

    Each mammalian genus examined so far contains 50,000-100,000 members of an L1 (LINE 1) family of long interspersed repeated DNA elements. Current knowledge on the evolution of L1 families presents a paradox because, although L1 families have been in mammalian genomes since before the mammalian radiation ∼80 million years ago, most members of the L1 families are only a few million years old. Accordingly it has been suggested either that the extensive amplification that characterizes present-day L1 families did not occur in the past or that old members were removed as new one were generated. However, the authors show here that an ancestral rodent L1 family was extensively amplified ∼10 million years ago and that the relics of this amplification have persisted in modern murine genomes. This amplification occurred just before the divergence of modern murine genera from their common ancestor and identifies the murine node in the lineage of modern muroid rodents The results suggest that repeated amplification of L1 elements is a feature of the evaluation of mammalian genomes and that ancestral amplification events could provide a useful tool for determining mammalian lineages

  19. Modulation of fibronectin-mediated Bacillus Calmette-Guérin attachment to murine bladder mucosa by drugs influencing the coagulation pathways.

    Science.gov (United States)

    Hudson, M A; Brown, E J; Ritchey, J K; Ratliff, T L

    1991-07-15

    Adjuvant intravesical Bacillus Calmette-Guérin (BCG) has proved to be an effective treatment for superficial bladder cancer. Intraluminal attachment of BCG organisms via binding to the extracellular matrix protein, fibronectin (FN), appears to be required for expression of the antitumor efficacy of BCG against a murine bladder tumor. Initial studies demonstrated that radiolabeled FN localized to the acutely injured urothelium but not to intact urothelium. These studies also demonstrated that exogenous administration of FN enhanced BCG attachment to the injured but not to the intact urothelium. Because FN has been shown to be an integral part of clot formation at sites of urothelial injury, drugs known to affect fibrin clot formation were tested for their effects on BCG attachment and antitumor efficacy in a murine bladder tumor model. A stabilizer of fibrin clot formation was shown to enhance both BCG attachment and antitumor efficacy in the same model. An increased number of BCG organisms were also retained in the lymph nodes and spleens of mice receiving fibrin clot stabilizers, suggesting indirectly that immunological mechanisms are involved in the antitumor efficacy of BCG. The data presented herein provide further support for the hypothesis that BCG attachment to the injured bladder is mediated by FN. Furthermore, modulation of BCG-FN attachment is demonstrated to be possible with drugs influencing the coagulation pathway. This attachment is shown to be required for the antitumor efficacy in a murine bladder tumor model, and thus modulation of BCG-FN attachment appears to have significant influence on the antitumor efficacy of BCG in the murine bladder tumor model.

  20. NCBI nr-aa BLAST: CBRC-PVAM-01-1002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PVAM-01-1002 sp|Q86UP3|ZFHX4_HUMAN RecName: Full=Zinc finger homeobox protein ...4; AltName: Full=Zinc finger homeodomain protein 4; Short=ZFH-4 gb|AAP20225.1| zinc finger homeodomain 4 protein [Homo sapiens] Q86UP3 0.0 86% ...

  1. NCBI nr-aa BLAST: CBRC-OPRI-01-1361 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-1361 sp|Q86UP3|ZFHX4_HUMAN RecName: Full=Zinc finger homeobox protein ...4; AltName: Full=Zinc finger homeodomain protein 4; Short=ZFH-4 gb|AAP20225.1| zinc finger homeodomain 4 protein [Homo sapiens] Q86UP3 0.0 93% ...

  2. NCBI nr-aa BLAST: CBRC-TTRU-01-1120 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1120 sp|Q86UP3|ZFHX4_HUMAN RecName: Full=Zinc finger homeobox protein ...4; AltName: Full=Zinc finger homeodomain protein 4; Short=ZFH-4 gb|AAP20225.1| zinc finger homeodomain 4 protein [Homo sapiens] Q86UP3 0.0 87% ...

  3. NCBI nr-aa BLAST: CBRC-MDOM-03-0053 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-03-0053 sp|Q86UP3|ZFHX4_HUMAN RecName: Full=Zinc finger homeobox protein ...4; AltName: Full=Zinc finger homeodomain protein 4; Short=ZFH-4 gb|AAP20225.1| zinc finger homeodomain 4 protein [Homo sapiens] Q86UP3 0.0 94% ...

  4. NCBI nr-aa BLAST: CBRC-MDOM-03-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-03-0052 sp|Q86UP3|ZFHX4_HUMAN RecName: Full=Zinc finger homeobox protein ...4; AltName: Full=Zinc finger homeodomain protein 4; Short=ZFH-4 gb|AAP20225.1| zinc finger homeodomain 4 protein [Homo sapiens] Q86UP3 0.0 91% ...

  5. Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved

    DEFF Research Database (Denmark)

    Møller, Cathrine Laustrup; Raun, Kirsten; Jacobsen, Marianne Lambert

    2011-01-01

    hormone (a-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal...

  6. Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

    Czech Academy of Sciences Publication Activity Database

    Zjablovskaja, Polina; Daněk, Petr; Kardošová, Miroslava; Alberich-Jorda, Meritxell

    č. 132 (2018), č. článku e57033. ISSN 1940-087X R&D Projects: GA ČR GA15-03796S Institutional support: RVO:68378050 Keywords : 32D/G-CSF-R cells * murine myeloid precursor cells * liquid culture * differentiation * neutrophils * proliferation * cytokines * IL-3 * G-CSF Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.232, year: 2016

  7. The effect of prenatal pravastatin treatment on altered fetal programming of postnatal growth and metabolic function in a preeclampsia-like murine model.

    Science.gov (United States)

    McDonnold, Mollie; Tamayo, Esther; Kechichian, Talar; Gamble, Phyllis; Longo, Monica; Hankins, Gary D V; Saade, George R; Costantine, Maged M

    2014-06-01

    Preeclampsia alters fetal programming and results in long-term metabolic consequences in the offspring. Pravastatin has been shown to prevent preeclampsia in animal models. Our aim was to characterize the effects of preeclampsia on fetal programming of adult growth and metabolic function, and evaluate the role of preventive pravastatin therapy, using a well characterized murine model. CD-1 mice were injected through the tail vein with adenovirus carrying soluble fms-like tyrosine kinase 1 (sFlt-1) and randomly allocated to pravastatin (5 mg/kg/day; sFlt-1/prav, n = 7) or water (sFlt-1, n = 6) until weaning. A control group was injected with adenovirus carrying the murine immunoglobulin G2α Fc fragment (mFc, n = 8). Male and female offspring (6-8/group) were weighed every month until 6 months of age. Intraperitoneal glucose tolerance testing was performed after 16 hours of fasting at 3 and 6 months of age; glucose and insulin responses were measured. sFlt-1 offspring weight was lower than mFc control (P < .001) until 2 months of age for females and 5 months of age for males (P < .001). There were no differences in postnatal growth between mFc and sFlt-1/prav offspring. At 3 and 6 months, female sFlt-1 offspring had higher glucose response compared with mFc and sFlt-1/prav. Three-month-old male sFlt-1 had lower insulin response compared with mFc offspring. Preeclampsia alters postnatal growth and metabolic function in the adult offspring in this animal model. Maternal therapy with prav prevents some of these alterations in the offspring. Copyright © 2014 Mosby, Inc. All rights reserved.

  8. The Antimalarial Chloroquine Suppresses LPS-Induced NLRP3 Inflammasome Activation and Confers Protection against Murine Endotoxic Shock

    Directory of Open Access Journals (Sweden)

    Xiaoli Chen

    2017-01-01

    Full Text Available Activation of the NLRP3 inflammasome, which catalyzes maturation of proinflammatory cytokines like IL-1β and IL-18, is implicated and essentially involved in many kinds of inflammatory disorders. Chloroquine (CQ is a traditional antimalarial drug and also possesses an anti-inflammatory property. In this study, we investigated whether CQ suppresses NLRP3 inflammasome activation and thereby confers protection against murine endotoxic shock. CQ attenuated NF-κB and MAPK activation and prohibited expression of IL-1β, IL-18, and Nlrp3 in LPS treated murine bone marrow-derived macrophages (BMDMs, demonstrating its inhibitory effect on the priming signal of NLRP3 activation. Then, CQ was shown to inhibit caspase-1 activation and ASC specks formation in BMDMs, which indicates that CQ also suppresses inflammasome assembly, the second signal for NLRP3 inflammasome activation. In a murine endotoxic shock model, CQ effectively improved survival and markedly reduced IL-1β and IL-18 production in serum, peritoneal fluid, and lung tissues. Moreover, CQ reduced protein levels of NLRP3 and caspases-1 p10 in lung homogenates of mice with endotoxic shock, which may possibly explain its anti-inflammatory activity and life protection efficacy in vivo. Overall, our results demonstrate a new role of CQ that facilitates negative regulation on NLRP3 inflammasome, which thereby confers protection against lethal endotoxic shock.

  9. Adam8 Limits the Development of Allergic Airway Inflammation in Mice

    Science.gov (United States)

    Knolle, Martin D.; Nakajima, Takahiro; Hergrueter, Anja; Gupta, Kushagra; Polverino, Francesca; Craig, Vanessa J.; Fyfe, Susanne E.; Zahid, Muhammad; Permaul, Perdita; Cernadas, Manuela; Montano, Gilbert; Tesfaigzi, Yohannes; Sholl, Lynette; Kobzik, Lester; Israel, Elliot; Owen, Caroline A.

    2013-01-01

    To determine whether a disintegrin and a metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyper-responsiveness (AHR), we compared AAI and AHR in wild type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with ovalbumin (OVA) or house dust mite protein extract (HDM). OVA- and HDM-treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some TH2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma. PMID:23670189

  10. First transplantation of isolated murine follicles in alginate.

    Science.gov (United States)

    Vanacker, Julie; Dolmans, Marie-Madeleine; Luyckx, Valérie; Donnez, Jacques; Amorim, Christiani A

    2014-01-01

    Our aim is to develop an artificial ovary allowing survival and growth of isolated follicles and ovarian cells, to restore fertility in women diagnosed with pathologies at high risk of ovarian involvement. For this, alginate beads containing isolated preantral follicles and ovarian cells were autografted to immunocompetent mice. One week after grafting, the beads were invaded by proliferating murine cells (12.1%) and capillaries. The recovery rate of follicles per graft ranged from 0% to 35.5%. Of the analyzed follicles, 77% were Ki67-positive and 81%, TUNEL-negative. Three antral follicles were also identified, evidencing their ability to grow in the matrix. Our results suggest that an artificial ovary is now conceivable, opening new perspectives to restore fertility in women.

  11. An effective in vitro and in vivo antileishmanial activity and mechanism of action of 8-hydroxyquinoline against Leishmania species causing visceral and tegumentary leishmaniasis.

    Science.gov (United States)

    Costa Duarte, Mariana; dos Reis Lage, Letícia Martins; Lage, Daniela Pagliara; Mesquita, Juliana Tonini; Salles, Beatriz Cristina Silveira; Lavorato, Stefânia Neiva; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Alves, Ricardo José; Tavares, Carlos Alberto Pereira; Tempone, André Gustavo; Coelho, Eduardo Antonio Ferraz

    2016-02-15

    The development of new therapeutic strategies to treat leishmaniasis has become a priority. In the present study, the antileishmanial activity of 8-hydroxyquinoline (8-HQN) was investigated against in vitro promastigotes and in vivo intra-macrophage amastigotes of three Leishmania species: Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis. Studies were performed to establish the 50% Leishmania inhibitory concentration (IC50) of 8-HQN, as well as its 50% cytotoxic concentration (CC50) on murine macrophages and in human red blood cells. The inhibition of macrophages infection was also evaluated using parasites that were pre-treated with 8-HQN. The effects of this compound on nitric oxide (NO) production and in the mitochondrial membrane potential were also evaluated. Finally, the therapeutic efficacy of 8-HQN was assessed in a known murine model, L. amazonensis-chronically infected BALB/c mice. Our results showed that 8-HQN was effective against promastigote and amastigote stages of all tested Leishmania species, presenting a selectivity index of 328.0, 62.0 and 47.0 for L. amazonensis, L. infantum and L. braziliensis, respectively. It was effective in treating infected macrophages, as well as in preventing the infection of these cells using pre-treated parasites. In addition, 8-HQN caused an alteration in the mitochondrial membrane potential of the parasites. When administered at 10mg/kg body weight/day by subcutaneous route, this product was effective in reducing the lesion diameter, as well as the parasite load in evaluated tissues and organs of infected animals. The results showed the in vitro and in vivo efficacy of 8-HQN against three different Leishmania species causing tegumentary and/or visceral leishmaniasis, and it could well be used for future therapeutic optimization studies to treat leishmaniasis. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. S100A8/A9 Is Not Involved in Host Defense against Murine Urinary Tract Infection

    NARCIS (Netherlands)

    Dessing, M.C.; Butter, L.M.; Teske, G.J.; Claessen, N.; van der Loos, C.M.; Vogl, T.; Roth, J.; van der Poll, T.; Florquin, S.; Leemans, J.C.

    2010-01-01

    Background: Inflammation is commonly followed by the release of endogenous proteins called danger associated molecular patterns (DAMPs) that are able to warn the host for eminent danger. S100A8/A9 subunits are DAMPs that belong to the S100 family of calcium binding proteins. S100A8/A9 complexes

  13. Modeling of Chronic Myeloid Leukemia : An Overview of In Vivo Murine and Human Xenograft Models

    NARCIS (Netherlands)

    Sontakke, Pallavi; Jaques, Jenny; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    Over the past years, a wide variety of in vivo mouse models have been generated in order to unravel the molecular pathology of Chronic Myeloid Leukemia (CML) and to develop and improve therapeutic approaches. These models range from (conditional) transgenic models, knock-in models, and murine bone

  14. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    NARCIS (Netherlands)

    C. Buecker (Christa); H.H. Chen; J.M. Polo (Jose); L. Daheron (Laurence); L. Bu (Lei); T.S. Barakat (Tahsin Stefan); P. Okwieka (Patricia); A. Porter (Andrew); J.H. Gribnau (Joost); K. Hochedlinger (Konrad); N. Geijsen (Niels)

    2010-01-01

    textabstractMurine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human

  15. Noninvasive murine glioma detection improved following photobleaching of skin PpIX fluorescence

    Science.gov (United States)

    Gibbs-Strauss, Summer L.; Davis, Scott C.; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

    2008-02-01

    Aminolevulinic Acid (ALA) is a prodrug which can be administered to cells, animals or patients after which it is transformed via the Heme synthesis pathway into the fluorescent molecule Protoporphyrin IX (PpIX). PpIX has been shown to be useful as both a photosensitizer for photodynamic therapy (PDT) and as a fluorescence imaging contrast agent. The ALA-PpIX system not only provides contrast for fluorescence imaging but also gives information about the metabolic activity of the imaged tissue and thus could be useful for monitoring cancer therapy. In the current study skin photobleaching was examined to determine if PpIX fluorescence contrast in malignant brain tumors could be better visualized noninvasively. Red light photobleaching decreased skin PpIX fluorescence and increased the ability to noninvasively quantify PpIX fluorescence in murine gliomas, as in vivo measurements of mean PpIX fluorescence more closely matched ex vivo quantification following skin photobleaching. Three doses of blue light photobleaching (4 J/cm2, 8 J/cm2 and 12 J/cm2) were tested and determined to give similar levels of skin photobleaching as well as a similar window of decreased skin PpIX fluorescence for noninvasive fluorescence imaging following the photobleaching dose administration.

  16. Murine strain differences and the effects of zinc on cadmium concentrations in tissues after acute cadmium exposure

    Energy Technology Data Exchange (ETDEWEB)

    King, L.M. [ARS USDA, Germplasm and Gamete Physiology Lab., Beltsville, MD (United States); Anderson, M.B. [Dept. of Anatomy, Tulane Univ. School of Medicine, New Orleans, LA (United States); Sikka, S.C. [Dept. of Urology, Tulane Univ. School of Medicine, New Orleans, LA (United States); George, W.J. [Dept. of Pharmacology, Tulane Univ. School of Medicine, New Orleans, LA (United States)

    1998-10-01

    The role of strain differences in cadmium tissue distribution was studied using sensitive (129/J) and resistant (A/J) mice. These murine strains have previously been shown to differ in their susceptibility to cadmium-induced testicular toxicity. Cadmium concentration was measured in testis, epididymis, seminal vesicle, liver, and kidney at 24 h after cadmium chloride exposure (4, 10, and 20 {mu}mol/kg CdCl{sub 2}). The 129/J mice exhibited a significant increase in cadmium concentration in testis, epididymis, and seminal vesicle at all cadmium doses used, compared to A/J mice. However, cadmium concentrations in liver and kidney were not different between the strains, at any dose, indicating that cadmium uptake is similar in these organs at 24 h. These murine strains demonstrate similar hepatic and renal cadmium uptake but significantly different cadmium accumulation in the reproductive organs at 24 h. The mechanism of the protective effect of zinc on cadmium toxicity was studied by assessing the impact of zinc acetate (ZnAc) treatment on cadmium concentrations in 129/J mice after 24 h. Zinc pretreatment (250 {mu}mol/kg ZnAc), given 24 h prior to 20 {mu}mol/kg CdCl{sub 2} administration, significantly decreased the amount of cadmium in the testis, epididymis, and seminal vesicle of 129/J mice, and significantly increased the cadmium content of the liver after 24 h. Cadmium levels in the kidney were unaffected at this time. Zinc pretreatment also prevented the cadmium-induced decrease in testicular sperm concentration and epididymal sperm motility seen in 129/J mice. These findings suggest that the differences in the two murine strains may be attributed partly to the differential accumulation of cadmium in murine gonads. This may be caused by strain differences in the specificity of cadmium transport mechanisms. The protective role of zinc in cadmium-induced testicular toxicity in the sensitive strain may be due to an interference in the cadmium uptake by susceptible

  17. CXCL10/CXCR3-Dependent Mobilization of Herpes Simplex Virus-Specific CD8+ TEM and CD8+ TRM Cells within Infected Tissues Allows Efficient Protection against Recurrent Herpesvirus Infection and Disease.

    Science.gov (United States)

    Srivastava, Ruchi; Khan, Arif A; Chilukuri, Sravya; Syed, Sabrina A; Tran, Tien T; Furness, Julie; Bahraoui, Elmostafa; BenMohamed, Lbachir

    2017-07-15

    Herpes simplex virus 1 (HSV-1) establishes latency within the sensory neurons of the trigeminal ganglia (TG). HSV-specific memory CD8 + T cells play a critical role in preventing HSV-1 reactivation from TG and subsequent virus shedding in tears that trigger recurrent corneal herpetic disease. The CXC chemokine ligand 10 (CXCL10)/CXC chemokine receptor 3 (CXCR3) chemokine pathway promotes T cell immunity to many viral pathogens, but its importance in CD8 + T cell immunity to recurrent herpes has been poorly elucidated. In this study, we determined how the CXCL10/CXCR3 pathway affects TG- and cornea-resident CD8 + T cell responses to recurrent ocular herpesvirus infection and disease using a well-established murine model in which HSV-1 reactivation was induced from latently infected TG by UV-B light. Following UV-B-induced HSV-1 reactivation, a significant increase in both the number and function of HSV-specific CXCR3 + CD8 + T cells was detected in TG and corneas of protected C57BL/6 (B6) mice, but not in TG and corneas of nonprotected CXCL10 -/- or CXCR3 -/- deficient mice. This increase was associated with a significant reduction in both virus shedding and recurrent corneal herpetic disease. Furthermore, delivery of exogenous CXCL10 chemokine in TG of CXCL10 -/- mice, using the neurotropic adeno-associated virus type 8 (AAV8) vector, boosted the number and function of effector memory CD8 + T cells (T EM ) and tissue-resident memory CD8 + T cells (T RM ), but not of central memory CD8 + T cells (T CM ), locally within TG, and improved protection against recurrent herpesvirus infection and disease in CXCL10 -/- deficient mice. These findings demonstrate that the CXCL10/CXCR3 chemokine pathway is critical in shaping CD8 + T cell immunity, locally within latently infected tissues, which protects against recurrent herpesvirus infection and disease. IMPORTANCE We determined how the CXCL10/CXCR3 pathway affects CD8 + T cell responses to recurrent ocular herpesvirus

  18. Scanning laser optical tomography for in toto imaging of the murine cochlea.

    Directory of Open Access Journals (Sweden)

    Lena Nolte

    Full Text Available The mammalian cochlea is a complex macroscopic structure due to its helical shape and the microscopic arrangements of the individual layers of cells. To improve the outcomes of hearing restoration in deaf patients, it is important to understand the anatomic structure and composition of the cochlea ex vivo. Hitherto, only one histological technique based on confocal laser scanning microscopy and optical clearing has been developed for in toto optical imaging of the murine cochlea. However, with a growing size of the specimen, e.g., human cochlea, this technique reaches its limitations. Here, we demonstrate scanning laser optical tomography (SLOT as a valuable imaging technique to visualize the murine cochlea in toto without any physical slicing. This technique can also be applied in larger specimens up to cm3 such as the human cochlea. Furthermore, immunolabeling allows visualization of inner hair cells (otoferlin or spiral ganglion cells (neurofilament within the whole cochlea. After image reconstruction, the 3D dataset was used for digital segmentation of the labeled region. As a result, quantitative analysis of position, length and curvature of the labeled region was possible. This is of high interest in order to understand the interaction of cochlear implants (CI and cells in more detail.

  19. A Murine Model of Candida glabrata Vaginitis Shows No Evidence of an Inflammatory Immunopathogenic Response.

    Directory of Open Access Journals (Sweden)

    Evelyn E Nash

    Full Text Available Candida glabrata is the second most common organism isolated from women with vulvovaginal candidiasis (VVC, particularly in women with uncontrolled diabetes mellitus. However, mechanisms involved in the pathogenesis of C. glabrata-associated VVC are unknown and have not been studied at any depth in animal models. The objective of this study was to evaluate host responses to infection following efforts to optimize a murine model of C. glabrata VVC. For this, various designs were evaluated for consistent experimental vaginal colonization (i.e., type 1 and type 2 diabetic mice, exogenous estrogen, varying inocula, and co-infection with C. albicans. Upon model optimization, vaginal fungal burden and polymorphonuclear neutrophil (PMN recruitment were assessed longitudinally over 21 days post-inoculation, together with vaginal concentrations of IL-1β, S100A8 alarmin, lactate dehydrogenase (LDH, and in vivo biofilm formation. Consistent and sustained vaginal colonization with C. glabrata was achieved in estrogenized streptozotocin-induced type 1 diabetic mice. Vaginal PMN infiltration was consistently low, with IL-1β, S100A8, and LDH concentrations similar to uninoculated mice. Biofilm formation was not detected in vivo, and co-infection with C. albicans did not induce synergistic immunopathogenic effects. This data suggests that experimental vaginal colonization of C. glabrata is not associated with an inflammatory immunopathogenic response or biofilm formation.

  20. A Murine Model of Candida glabrata Vaginitis Shows No Evidence of an Inflammatory Immunopathogenic Response.

    Science.gov (United States)

    Nash, Evelyn E; Peters, Brian M; Lilly, Elizabeth A; Noverr, Mairi C; Fidel, Paul L

    2016-01-01

    Candida glabrata is the second most common organism isolated from women with vulvovaginal candidiasis (VVC), particularly in women with uncontrolled diabetes mellitus. However, mechanisms involved in the pathogenesis of C. glabrata-associated VVC are unknown and have not been studied at any depth in animal models. The objective of this study was to evaluate host responses to infection following efforts to optimize a murine model of C. glabrata VVC. For this, various designs were evaluated for consistent experimental vaginal colonization (i.e., type 1 and type 2 diabetic mice, exogenous estrogen, varying inocula, and co-infection with C. albicans). Upon model optimization, vaginal fungal burden and polymorphonuclear neutrophil (PMN) recruitment were assessed longitudinally over 21 days post-inoculation, together with vaginal concentrations of IL-1β, S100A8 alarmin, lactate dehydrogenase (LDH), and in vivo biofilm formation. Consistent and sustained vaginal colonization with C. glabrata was achieved in estrogenized streptozotocin-induced type 1 diabetic mice. Vaginal PMN infiltration was consistently low, with IL-1β, S100A8, and LDH concentrations similar to uninoculated mice. Biofilm formation was not detected in vivo, and co-infection with C. albicans did not induce synergistic immunopathogenic effects. This data suggests that experimental vaginal colonization of C. glabrata is not associated with an inflammatory immunopathogenic response or biofilm formation.

  1. Immune tolerance induction using fetal directed placental injection in rodent models: a murine model.

    Directory of Open Access Journals (Sweden)

    Kei Takahashi

    Full Text Available Induction of the immune response is a major problem in replacement therapies for inherited protein deficiencies. Tolerance created in utero can facilitate postnatal treatment. In this study, we aimed to induce immune tolerance towards a foreign protein with early gestational cell transplantation into the chorionic villi under ultrasound guidance in the murine model.Pregnant C57BL/6 (B6 mice on day 10 of gestation were anesthetized and imaged by high resolution ultrasound. Murine embryos and their placenta were positioned to get a clear view in B-mode with power mode of the labyrinth, which is the equivalent of chorionic villi in the human. Bone marrow cells (BMCs from B6-Green Fluorescence Protein (B6GFP transgenic mice were injected into the fetal side of the placenta which includes the labyrinth with glass microcapillary pipettes. Each fetal mouse received 2 x 105 viable GFP-BMCs. After birth, we evaluated the humoral and cell-mediated immune response against GFP.Bone marrow transfer into fetal side of placenta efficiently distributed donor cells to the fetal mice. The survival rate of this procedure was 13.5%(5 out of 37. Successful engraftment of the B6-GFP donor skin grafts was observed in all recipient (5 out of 5 mice 6 weeks after birth. Induction of anti-GFP antibodies was completely inhibited. Cytotoxic immune reactivity of thymic cells against cells harboring GFP was suppressed by ELISPOT assay.In this study, we utilized early gestational placental injection targeting the murine fetus, to transfer donor cells carrying a foreign protein into the fetal circulation. This approach is sufficient to induce both humoral and cell-mediated immune tolerance against the foreign protein.

  2. Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Akporiaye, E T; Stewart, S; Stewart, C C

    1984-01-01

    Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. The authors report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc ..gamma.. receptors, and had esterase activity. 23 references, 1 figure, 1 table.

  3. Toxicity and Immunogenicity in Murine Melanoma following Exposure to Physical Plasma-Derived Oxidants

    Directory of Open Access Journals (Sweden)

    Sander Bekeschus

    2017-01-01

    Full Text Available Metastatic melanoma is an aggressive and deadly disease. Therapeutic advance has been achieved by antitumor chemo- and radiotherapy. These modalities involve the generation of reactive oxygen and nitrogen species, affecting cellular viability, migration, and immunogenicity. Such species are also created by cold physical plasma, an ionized gas capable of redox modulating cells and tissues without thermal damage. Cold plasma has been suggested for anticancer therapy. Here, melanoma cell toxicity, motility, and immunogenicity of murine metastatic melanoma cells were investigated following plasma exposure in vitro. Cells were oxidized by plasma, leading to decreased metabolic activity and cell death. Moreover, plasma decelerated melanoma cell growth, viability, and cell cycling. This was accompanied by increased cellular stiffness and upregulation of zonula occludens 1 protein in the cell membrane. Importantly, expression levels of immunogenic cell surface molecules such as major histocompatibility complex I, calreticulin, and melanocortin receptor 1 were significantly increased in response to plasma. Finally, plasma treatment significantly decreased the release of vascular endothelial growth factor, a molecule with importance in angiogenesis. Altogether, these results suggest beneficial toxicity of cold plasma in murine melanomas with a concomitant immunogenicity of potential interest in oncology.

  4. Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

    International Nuclear Information System (INIS)

    Smith, L.I.; Bodwell, J.E.; Mendel, D.B.; Ciardelli, T.; North, W.G.; Munck, A.

    1988-01-01

    Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [ 3 H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [ 3 H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [ 3 H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [ 3 H]dexamethasone 21-mesylate and [ 35 S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [ 3 H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells

  5. Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity

    International Nuclear Information System (INIS)

    Sedlar, Ales; Dolinsek, Tanja; Markelc, Bostjan; Prosen, Lara; Kranjc, Simona; Bosnjak, Masa; Blagus, Tanja; Cemazar, Maja; Sersa, Gregor

    2012-01-01

    Electrochemotherapy provides good local tumor control but requires adjuvant treatment for increased local response and action on distant metastasis. In relation to this, intramuscular interleukin-12 (IL-12) gene electro-transfer, which provides systemic shedding of IL-12, was combined with local electrochemotherapy with cisplatin. Furthermore, the dependence on tumor immunogenicity and immunocompetence of the host on combined treatment response was evaluated. Sensitivity of SA-1 sarcoma and TS/A carcinoma cells to electrochemotherapy with cisplatin was tested in vitro. In vivo, intratumoral electrochemotherapy with cisplatin (day 1) was combined with a single (day 0) or multiple (days 0, 2, 4) intramuscular murine IL-12 (mIL-12) gene electrotransfer. The antitumor effectiveness of combined treatment was evaluated on immunogenic murine SA-1 sarcoma in A/J mice and moderately immunogenic murine TS/A carcinoma, in immunocompetent BALB/c and immunodeficient SCID mice. Electrochemotherapy in vitro resulted in a similar IC 50 values for both sarcoma and carcinoma cell lines. However, in vivo electrochemotherapy was more effective in the treatment of sarcoma, the more immunogenic of the tumors, resulting in a higher log cell kill, longer specific tumor growth delay, and also 17% tumor cures compared to carcinoma where no tumor cures were observed. Adjuvant intramuscular mIL-12 gene electrotransfer increased the log cell kill in both tumor models, potentiating the specific tumor growth delay by a factor of 1.8-2 and increasing tumor cure rate by approximately 20%. In sarcoma tumors, the potentiation of the response by intramuscular mIL-12 gene electrotransfer was dose-dependent and also resulted in a faster onset of tumor cures. Comparison of the carcinoma response to the combined treatment modality in immunocompetent and immunodeficient mice demonstrated that the immune system is needed both for increased cell kill and for attaining tumor cures. Based on the comparison of

  6. Murine models of osteosarcoma: A piece of the translational puzzle.

    Science.gov (United States)

    Walia, Mannu K; Castillo-Tandazo, Wilson; Mutsaers, Anthony J; Martin, Thomas John; Walkley, Carl R

    2018-06-01

    Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome. © 2017 Wiley Periodicals, Inc.

  7. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    International Nuclear Information System (INIS)

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2015-01-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

  8. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Energy Technology Data Exchange (ETDEWEB)

    Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: t-takai@juntendo.ac.jp [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  9. Expression of mink cell focus-forming murine leukemia virus-related transcripts in AKR mice

    International Nuclear Information System (INIS)

    Khan, A.S.; Laigret, F.; Rodi, C.P.

    1987-01-01

    The authors used a synthetic 16-base-pair mink cell focus-forming (MCF) env-specific oligomer as radiolabeled probe to study MCF murine leukemia virus (MuLV)-related transcripts in brain, kidney, liver, spleen, and thymus tissues of AKR mice ranging from 5 weeks to 6 months (mo) of age. Tissue-specific expression of poly(A) + RNAs was seen. In addition, all the tissues tested contained 3.0-kb messages. The transcription of these MCF-related mRNAs was independent of the presence of ecotropic and xenotropic MuLVs. In general, expression of the MCF env-related transcripts appeared to peak at 2 mo of age; these messages were barely detectable in brain, kidney, liver, and spleen tissues after 2 mo and in thymus tissue after 4 mo of age. All of the subgenomic MCF env-related mRNAs appeared to contain the 190-base-pair cellular DNA insert, characteristic of the long terminal repeats associated with endogenous MCF env-related proviruses. No genomic-size (8.4-kb) transcripts corresponding to endogenous MCF-related proviruses were detected. An 8.4-kb MCF env-related mRNA was first seen at 3 mo of age, exclusively in thymus tissue. This species most likely represents the first appearance of a recombinant MCF-related MuLV genome. The transcripts which were detected in thymus tissue might be involved in the generation of leukemogenic MCF viruses

  10. Bacterial Clearance and Cytokine Profiles in a Murine Model of Postsurgical Nosocomial Pneumonia

    OpenAIRE

    Manderscheid, Patricia A.; Bodkin, Ryan P.; Davidson, Bruce A.; Jensen, Erik; Russo, Thomas A.; Knight, Paul R.

    2004-01-01

    The development of a nosocomial pneumonia is facilitated by alterations in host innate pulmonary antibacterial defenses following surgical trauma, which can result in decreased pulmonary bacterial clearance and increased morbidity and mortality. In a murine model of postoperative nosocomial infection, surgical stress (laparotomy) decreased Escherichia coli clearance from the lungs of animals that underwent surgery. Consistent with previous studies, (i) pulmonary levels of tumor necrosis facto...

  11. Murine but not human basophil undergoes cell-specific proteolysis of a major endoplasmic reticulum chaperone.

    Directory of Open Access Journals (Sweden)

    Bei Liu

    Full Text Available Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown.We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils.We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils.Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function.

  12. Murine but not human basophil undergoes cell-specific proteolysis of a major endoplasmic reticulum chaperone.

    Science.gov (United States)

    Liu, Bei; Staron, Matthew; Li, Zihai

    2012-01-01

    Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown. We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils. We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s) in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils. Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function.

  13. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Fitzpatrick, D.R.; Zamb, T.; Parker, M.D.; van Drunen Littel-van den Hurk, S.; Babiuk, L.A.; Lawman, M.J.P.

    1988-01-01

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK - cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  14. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1

    International Nuclear Information System (INIS)

    Plant, Kathryn E.; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G. Gordon; Lyon, Jon; Plant, Nick

    2009-01-01

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism

  15. Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

    NARCIS (Netherlands)

    Hovius, Joppe W. R.; Li, Xin; Ramamoorthi, Nandhini; van Dam, Alje P.; Barthold, Stephen W.; van der Poll, Tom; Speelman, Peter; Fikrig, Erol

    2007-01-01

    Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the

  16. Molecular and functional characterization of Kv7 K+ channel in murine gastrointestinal smooth muscles

    DEFF Research Database (Denmark)

    Jepps, Thomas Andrew; Greenwood, Iain A; Moffatt, James D

    2009-01-01

    that K(v)7.x especially K(v)7.4 and K(v)7.5 are expressed in different regions of the murine gastrointestinal tract and blockers of K(v)7 channels augment inherent contractile activity. Drugs that selectively block K(v)7.4/7.5 might be promising therapeutics for the treatment of motility disorders...

  17. Multivariate modelling with 1H NMR of pleural effusion in murine cerebral malaria

    Directory of Open Access Journals (Sweden)

    Ghosh Soumita

    2011-11-01

    Full Text Available Abstract Background Cerebral malaria is a clinical manifestation of Plasmodium falciparum infection. Although brain damage is the predominant pathophysiological complication of cerebral malaria (CM, respiratory distress, acute lung injury, hydrothorax/pleural effusion are also observed in several cases. Immunological parameters have been assessed in pleural fluid in murine models; however there are no reports of characterization of metabolites present in pleural effusion. Methods 1H NMR of the sera and the pleural effusion of cerebral malaria infected mice were analyzed using principal component analysis, orthogonal partial least square analysis, multiway principal component analysis, and multivariate curve resolution. Results It has been observed that there was 100% occurrence of pleural effusion (PE in the mice affected with CM, as opposed to those are non-cerebral and succumbing to hyperparasitaemia (NCM/HP. An analysis of 1H NMR and SDS-PAGE profile of PE and serum samples of each of the CM mice exhibited a similar profile in terms of constituents. Multivariate analysis on these two classes of biofluids was performed and significant differences were detected in concentrations of metabolites. Glucose, creatine and glutamine contents were high in the PE and lipids being high in the sera. Multivariate curve resolution between sera and pleural effusion showed that changes in PE co-varied with that of serum in CM mice. The increase of glucose in PE is negatively correlated to the glucose in serum in CM as obtained from the result of multiway principal component analysis. Conclusions This study reports for the first time, the characterization of metabolites in pleural effusion formed during murine cerebral malaria. The study indicates that the origin of PE metabolites in murine CM may be the serum. The loss of the components like glucose, glutamine and creatine into the PE may worsen the situation of patients, in conjunction with the enhanced

  18. Efficacy of paracetamol on patent ductus arteriosus closure may be dose dependent: evidence from human and murine studies.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif

    2014-09-01

    We evaluated the clinical effectiveness of variable courses of paracetamol on patent ductus arteriosus (PDA) closure and examined its effect on the in vitro term and preterm murine ductus arteriosus (DA).

  19. Proliferative capacity of murine hematopoietic stem cells

    International Nuclear Information System (INIS)

    Hellman, S.; Botnick, L.E.; Hannon, E.C.; Vigneulle, R.M.

    1978-01-01

    The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferatively quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cells progress in the continuum in one direction and such progression is not reversible

  20. Major differences between human atopic dermatitis and murine models as determined by global transcriptomic profiling

    DEFF Research Database (Denmark)

    Ewald, David Adrian; Noda, Shinji; Oliva, Margeaux

    2017-01-01

    , and a comparison of these models with the human AD transcriptomic fingerprint is lacking. We sought to evaluate the transcriptomic profiles of six common murine models and determine how they relate to human AD skin. Transcriptomic profiling was performed using microarrays and qRT-PCR on biopsies from NC/Nga, flaky...

  1. Gp96 Peptide Antagonist gp96-II Confers Therapeutic Effects in Murine Intestinal Inflammation

    Directory of Open Access Journals (Sweden)

    Claudia A. Nold-Petry

    2017-12-01

    Full Text Available BackgroundThe expression of heat shock protein gp96 is strongly correlated with the degree of tissue inflammation in ulcerative colitis and Crohn’s disease, thereby leading us to the hypothesis that inhibition of expression via gp96-II peptide prevents intestinal inflammation.MethodsWe employed daily injections of gp96-II peptide in two murine models of intestinal inflammation, the first resulting from five daily injections of IL-12/IL-18, the second via a single intrarectal application of TNBS (2,4,6-trinitrobenzenesulfonic acid. We also assessed the effectiveness of gp96-II peptide in murine and human primary cell culture.ResultsIn the IL-12/IL-18 model, all gp96-II peptide-treated animals survived until day 5, whereas 80% of placebo-injected animals died. gp96-II peptide reduced IL-12/IL-18-induced plasma IFNγ by 89%, IL-1β by 63%, IL-6 by 43% and tumor necrosis factor (TNF by 70% compared to controls. The clinical assessment Disease Activity Index of intestinal inflammation severity was found to be significantly lower in the gp96-II-treated animals when compared to vehicle-injected mice. gp96-II peptide treatment in the TNBS model limited weight loss to 5% on day 7 compared with prednisolone treatment, whereas placebo-treated animals suffered a 20% weight loss. Histological disease severity was reduced equally by prednisolone (by 40% and gp96-II peptide (35%. Mice treated with either gp96-II peptide or prednisolone exhibited improved endoscopic scores compared with vehicle-treated control mice: vascularity, fibrin, granularity, and translucency scores were reduced by up to 49% by prednisolone and by up to 30% by gp96-II peptide. In vitro, gp96-II peptide reduced TLR2-, TLR4- and IL-12/IL-18-induced cytokine expression in murine splenocytes, with declines in constitutive IL-6 (54%, lipopolysaccharide-induced TNF (48%, IL-6 (81% and in Staphylococcus epidermidis-induced TNF (67% and IL-6 (81%, as well as IL-12/IL-18-induced IFNγ (75%. gp

  2. Improvement of Outcome Measures of Dry Eye by a Novel Integrin Antagonist in the Murine Desiccating Stress Model.

    Science.gov (United States)

    Krauss, Achim H; Corrales, Rosa M; Pelegrino, Flavia S A; Tukler-Henriksson, Johanna; Pflugfelder, Stephen C; de Paiva, Cintia S

    2015-09-01

    We investigated the effects of GW559090, a novel, competitive, and high-affinity α4 integrin antagonist, in a murine model of dry eye. Through interaction with vascular cell adhesion molecule 1 (VCAM-1) and fibronectin α4β1 integrin is involved in leukocyte trafficking and activation. Female C57BL/6 mice, aged 6 to 8 weeks, were subjected to desiccating stress (DS). Bilateral topical twice daily treatment with GW559090 was compared to vehicle-treated controls. Treatment was initiated at the time of DS induction. Treatment effects were assessed on corneal staining with Oregon Green Dextran (OGD) and expression of inflammatory markers in ocular surface tissues by real time PCR. Dendritic cell activation was measured in draining cervical lymph nodes (CLN) by flow cytometry. Separate groups of mice received GW559090 subcutaneously to evaluate the effects of systemic administration on corneal staining and cells in CLN. Topical GW559090 significantly reduced corneal uptake of OGD compared to vehicle-treated disease controls in a dose-dependent manner (1, 3, 10, and 30 mg/mL) with 30 mg/mL showing the greatest reduction in OGD staining. When administered topically, corneal expression of IL-1α, matrix metalloproteinase (MMP)-9, chemokine ligand 9 (CXCL9), and TGF-β1 was reduced in GW559090-treated eyes. Topical treatment with GW559090 decreased dendritic cell activation in lymph nodes. The effects on corneal staining and cellular composition in CLN were not reproduced by systemic administration of GW559090, suggestive of a local role for integrin antagonism in the treatment of dry eye. The novel α4 integrin antagonist, GW559090, improved outcome measures of corneal staining and ocular surface inflammation in this murine model of dry eye. These results indicate the potential of this novel agent for the treatment of dry eye disease.

  3. Dual Role of Host Par2 in a Murine Model of Spontaneous Metastatic B16 Melanoma

    Czech Academy of Sciences Publication Activity Database

    Olejár, Tomáš; Větvička, D.; Zadinová, M.; Poučková, P.; Kukal, J.; Ježek, Petr; Matěj, R.

    2014-01-01

    Roč. 34, č. 7 (2014), s. 3511-3515 ISSN 0250-7005 R&D Projects: GA ČR(CZ) GAP302/10/0346 Institutional support: RVO:67985823 Keywords : PAR2 * melanoma * metastasis * murine model Subject RIV: EA - Cell Biology Impact factor: 1.826, year: 2014

  4. Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions

    NARCIS (Netherlands)

    Nagao, Keisuke; Ginhoux, Florent; Leitner, Wolfgang W.; Motegi, Sei-Ichiro; Bennett, Clare L.; Clausen, Björn E.; Merad, Miriam; Udey, Mark C.

    2009-01-01

    A new langerin(+) DC subset has recently been identified in murine dermis (langerin(+) dDC), but the lineage and functional relationships between these cells and langerin(+) epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC

  5. A novel inexpensive murine model of oral chronic digitalization.

    Science.gov (United States)

    Helber, Izo; Kanashiro, Rosemeire M; Alarcon, Ernesto A; Antonio, Ednei L; Tucci, Paulo J F

    2004-01-01

    A novel inexpensive murine model of oral administration of digitoxin (100 micro g/kg per day) added to routine chow is described. Serum digitoxin levels achieved after oral (n = 5; 116 +/- 14 ng/mL) and subcutaneous (n = 5; 124 +/- 11 ng/mL) administration were similar. A significant increase in the maximal left ventricular pressure rise of treated (n = 9) compared with control (n = 6) rats (dP/dt: 8956 +/- 233 vs 7980 +/- 234 mmHg/s, respectively; P = 0.01) characterized the positive inotropic action of digitoxin. In addition, no differences were observed in treated compared with control rats with regard to the electrocardiogram and systolic and diastolic left ventricular pressures.

  6. Local expression of vaginal Th1 and Th2 cytokines in murine vaginal candidiasis under different immunity conditions.

    Science.gov (United States)

    Chen, Shanjuan; Li, Shaohua; Wu, Yan; Liu, Zhixiang; Li, Jiawen

    2008-08-01

    To investigate the expression of vaginal Th1 and Th2 cytokines in rats with experimental vaginal candidiasis under different immune conditions, ICR murine vaginal candidiasis model was established and immno-suppressed murine models of vaginal cadidiasis were established in estrogen-treated mice. Non-estrogen-treated mice were used as controls. The mRNA level of Th1 (IL-2)/Th2 (IL-4, IL-10, TGF-beta1) cytokines in murine vaginal tissues was determined by RT-PCR. The cykotine in local tissues was increased to different extent under normal immune condition. IL-2 mRNA was increased during early stage of infection, while IL-10 was increased transiently during late stage of infection. TGF-beta1 production was found to be increased persistently. At same time, the expression of IL-2 mRNA was suppressed in immno-suppressed group, and the level of IL-4, IL-10, and TGF-beta1 were higher than the normal immunity group to different degree during infection. The high level of IL-2 mRNA during early stage of infection was associated with clearance of mucosal Candidia albicans (C. albicans), and its expression suppressed leading to decreased clearance of mucosal C. albican in immuno-suppression. The over-expression of IL-4 and IL-10 could significantly enhance the susceptibility to C. albicans infection in mice.

  7. Experimental parameters differentially affect the humoral response of the cholera-toxin-based murine model of food allergy

    DEFF Research Database (Denmark)

    Kroghsbo, S.; Christensen, Hanne Risager; Frøkiær, Hanne

    2003-01-01

    Background: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle...... interested in characterizing the individual effects of the parameters in the CT-based model: CT dose, antigen type and dose, and number of immunizations. Methods: BALB/c mice were orally sensitized weekly for 3 or 7 weeks with graded doses of CT and various food antigens (soy-trypsin inhibitor, ovalbumin...... of the antibody response depended on the type of antigen and number of immunizations. Conclusions: The critical parameters of the CT-based murine allergy model differentially control the intensity and kinetics of the developing immune response. Adjustment of these parameters could be a key tool for tailoring...

  8. Functional analysis of a Wheat Homeodomain protein, TaR1, reveals that host chromatin remodelling influences the dynamics of the switch to necrotrophic growth in the phytopathogenic fungus Zymoseptoria tritici.

    Science.gov (United States)

    Lee, Jack; Orosa, Beatriz; Millyard, Linda; Edwards, Martin; Kanyuka, Kostya; Gatehouse, Angharad; Rudd, Jason; Hammond-Kosack, Kim; Pain, Naomi; Sadanandom, Ari

    2015-04-01

    A distinguishing feature of Septoria leaf blotch disease in wheat is the long symptomless growth of the fungus amongst host cells followed by a rapid transition to necrotrophic growth resulting in disease lesions. Global reprogramming of host transcription marks this switch to necrotrophic growth. However no information exists on the components that bring about host transcriptional reprogramming. Gene-silencing, confocal-imaging and protein-protein interaction assays where employed to identify a plant homeodomain (PHD) protein, TaR1 in wheat that plays a critical role during the transition from symptomless to necrotrophic growth of Septoria. TaR1-silenced wheat show earlier symptom development upon Septoria infection but reduced fungal sporulation indicating that TaR1 is key for prolonging the symptomless phase and facilitating Septoria asexual reproduction. TaR1 is localized to the nucleus and binds to wheat Histone 3. Trimethylation of Histone 3 at lysine 4 (H3K4) and lysine 36 (H3K36) are found on open chromatin with actively transcribed genes, whereas methylation of H3K27 and H3K9 are associated with repressed loci. TaR1 specifically recognizes dimethylated and trimethylated H3K4 peptides suggesting that it regulates transcriptional activation at open chromatin. We conclude that TaR1 is an important component for the pathogen life cycle in wheat that promotes successful colonization by Septoria. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  9. [Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms].

    Science.gov (United States)

    Zhou, Yu-feng; Fang, Feng; Dong, Yong-sui; Zhou, Hua; Zhen, Hong; Liu, Jin; Li, Ge

    2006-07-01

    Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR. NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased

  10. A murine model for developmental dysplasia of the hip: ablation of CX3CR1 affects acetabular morphology and gait

    Directory of Open Access Journals (Sweden)

    George Feldman

    2017-11-01

    Full Text Available Abstract Background Developmental dysplasia of the hip (DDH is a debilitating condition whose distinguishing signs include incomplete formation of the acetabulum leading to dislocation of the femur, accelerated wear of the articular cartilage and joint laxity resulting in osteoarthritis. It is a complex disorder having environmental and genetic causes. Existing techniques fail to detect milder forms of DDH in newborns leading to hip osteoarthritis in young adults. A sensitive, specific and cost effective test would allow identification of newborns that could be non-invasively corrected by the use of a Pavlik harness. Previously, we identified a 2.5 MB candidate region on human chromosome 3 by using linkage analysis of a 4 generation, 72 member family. Whole exome sequencing of the DNA of 4 severely affected members revealed a single nucleotide polymorphism variant, rs3732378 co-inherited by all 11 affected family members. This variant causes a threonine to methionine amino acid change in the coding sequence of the CX3CR1 chemokine receptor and is predicted to be harmful to the function of the protein To gain further insight into the function of this mutation we examined the effect of CX3CR1 ablation on the architecture of the mouse acetabulum and on the murine gait. Methods The hips of 5 and 8 weeks old wild type and CX3CR1 KO mice were analyzed using micro-CT to measure acetabular diameter and ten additional dimensional parameters. Eight week old mice were gait tested using an inclined treadmill with and without load and then underwent micro-CT analysis. Results (1 KO mice showed larger a 5–17% larger diameter left acetabula than WT mice at both ages. (2 At 8 weeks the normalized area of space (i.e. size discrepancy between the femur head and acetabulum is significantly larger [38% (p = 0.001–21% (p = 0.037] in the KO mice. (3 At 8 weeks gait analysis of these same mice shows several metrics that are consistent with impairment in

  11. 1,3,5,8-tetrahydroxyxanthone regulates ANGPTL3-LPL pathway to lessen the ketosis in mice.

    Science.gov (United States)

    Xiao, Hong-Bo; Sun, Zhi-Liang; Zhou, Nan

    2012-05-12

    Ketosis is a metabolic disorder closely associated with both lipid and carbohydrate metabolism. Recent studies show that angiopoietin-like protein 3 (ANGPTL3) contributes to the development of metabolic disorder. The objective of this study was to explore the inhibitory effect of 1,3,5,8-tetrahydroxyxanthone (Xan), a naturally occurring flavonoid compound, on ketosis and the mechanisms involved in this regulation. After 4weeks, Xan (10 or 30mg/kg, intragastrically) treatment decreased plasma total ketone bodies, malondialdehyde, 8-isoprostane, triglyceride, total cholesterol levels, and hepatic ANGPTL3 expression concomitantly with increased plasma glucose concentration and adipose lipoprotein lipase (LPL) expression in ketosis murine. The present results suggest that Xan regulates ANGPTL3-LPL pathway to lessen the ketosis in mice. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Delayed contrast enhancement imaging of a murine model for ischemia reperfusion with carbon nanotube micro-CT.

    Directory of Open Access Journals (Sweden)

    Laurel M Burk

    Full Text Available We aim to demonstrate the application of free-breathing prospectively gated carbon nanotube (CNT micro-CT by evaluating a myocardial infarction model with a delayed contrast enhancement technique. Evaluation of murine cardiac models using micro-CT imaging has historically been limited by extreme imaging requirements. Newly-developed CNT-based x-ray sources offer precise temporal resolution, allowing elimination of physiological motion through prospective gating. Using free-breathing, cardiac-gated CNT micro-CT, a myocardial infarction model can be studied non-invasively and with high resolution. Myocardial infarction was induced in eight male C57BL/6 mice aged 8-12 weeks. The ischemia reperfusion model was achieved by surgically occluding the LAD artery for 30 minutes followed by 24 hours of reperfusion. Tail vein catheters were placed for contrast administration. Iohexol 300 mgI/mL was administered followed by images obtained in diastole. Iodinated lipid blood pool contrast agent was then administered, followed with images at systole and diastole. Respiratory and cardiac signals were monitored externally and used to gate the scans of free-breathing subjects. Seven control animals were scanned using the same imaging protocol. After imaging, the heart was harvested, cut into 1mm slices and stained with TTC. Post-processing analysis was performed using ITK-Snap and MATLAB. All animals demonstrated obvious delayed contrast enhancement in the left ventricular wall following the Iohexol injection. The blood pool contrast agent revealed significant changes in cardiac function quantified by 3-D volume ejection fractions. All subjects demonstrated areas of myocardial infarct in the LAD distribution on both TTC staining and micro-CT imaging. The CNT micro-CT system aids straightforward, free-breathing, prospectively-gated 3-D murine cardiac imaging. Delayed contrast enhancement allows identification of infarcted myocardium after a myocardial ischemic

  13. Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry.

    Science.gov (United States)

    Rhein, Bethany A; Brouillette, Rachel B; Schaack, Grace A; Chiorini, John A; Maury, Wendy

    2016-07-01

    Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses

  14. Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes

    DEFF Research Database (Denmark)

    Petrovic, Natasa; Walden, Tomas B; Shabalina, Irina G

    2009-01-01

    The recent insight that brown adipocytes and muscle cells share a common origin and in this respect are distinct from white adipocytes has spurred questions concerning the origin and molecular characteristics of the UCP1-expressing cells observed in classic white adipose tissue depots under certain...... physiological or pharmacological conditions. Examining precursors from the purest white adipose tissue depot (epididymal), we report here that chronic treatment with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone promotes not only the expression of PGC-1alpha and mitochondriogenesis...... associated with classic brown adipocytes (Zic1, Lhx8, Meox2, and characteristically PRDM16) or for myocyte-associated genes (myogenin and myomirs (muscle-specific microRNAs)) and retain white fat characteristics such as Hoxc9 expression. Co-culture experiments verify that the UCP1-expressing cells...

  15. Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

    International Nuclear Information System (INIS)

    Kleczkowska, H.E.; Szumiel, I.; Althaus, F.R.

    1990-01-01

    We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD(+)-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells

  16. Murine Ileocolic Bowel Resection with Primary Anastomosis

    Science.gov (United States)

    Perry, Troy; Borowiec, Anna; Dicken, Bryan; Fedorak, Richard; Madsen, Karen

    2014-01-01

    Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn’s disease and colon cancer being two common examples. Despite the frequency of these procedures, a significant knowledge gap remains in describing the inherent effects of intestinal resection on host physiology and disease pathophysiology. This article provides detailed instructions for an ileocolic resection with primary end-to-end anastomosis in mice, as well as essential aspects of peri-operative care to maximize post-operative success. When followed closely, this procedure yields a 95% long-term survival rate, no failure to thrive, and minimizes post-operative complications of bowel obstruction and anastomotic leak. The technical challenges of performing the procedure in mice are a barrier to its wide spread use in research. The skills described in this article can be acquired without previous surgical experience. Once mastered, the murine ileocolic resection procedure will provide a reproducible tool for studying the effects of intestinal resection in models of human disease. PMID:25406841

  17. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene

    DEFF Research Database (Denmark)

    Xu, H; Wu, X R; Wewer, U M

    1994-01-01

    The classic murine muscular dystrophy strain, dy, was first described almost 40 years ago. We have identified the molecular basis of an allele of dy, called dy2J, by detecting a mutation in the laminin alpha 2 chain gene--the first identified mutation in laminin-2. The G to A mutation in a splice...

  18. The role of adrenergic activation on murine luteal cell viability and progesterone production.

    Science.gov (United States)

    Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

    2016-09-15

    Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Efficacy of Eosin B as a New Antimalarial Drug in a Murine Model

    Directory of Open Access Journals (Sweden)

    Zahra Zamani

    2012-01-01

    Full Text Available The initial success of any adopted anti-infective strategy to malaria is followed by a descent due to the emergence of resistance to it. The search for new drugs and drug targets is a consistent demand in this disease. Eosin B, a common laboratory dye, is reported to have good antiparasitic properties in vitro. It was studied for its antiparasitic effect in vivo on chloroquine-sensitive Plasmodium berghei murine malaria. Eosin B was administered in 2 different doses by either the oral or parenteral route, once or twice daily to mice infected with Plasmodium berghei. Both the doses of eosin B 400 mg/kg and 800 mg/kg gave better results than the controls which were 40 mg/kg chloroquine and 100 mg/kg of arteether with P<0.005 significance. Percentage suppressive activity by Peter’s test of eosin B was better, though at a higher dose than both the controls. Survival rate of mice receiving the higher dose of eosin B was longer than that of the controls. When administered twice daily, the mice were fully cured after 4 days. Eosin B seems to be a promising drug exhibiting good antimalarial effects in the murine model of the disease.

  20. Experimental murine fascioliasis derives early immune suppression with increased levels of TGF-β and IL-4.

    Science.gov (United States)

    Chung, Joon-Yong; Bae, Young-An; Yun, Doo-Hee; Yang, Hyun-Jong; Kong, Yoon

    2012-12-01

    In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19(+) B cells was observed as early as week 1 post-infection while CD4(+)/CD8(+) T cells were down-regulated. Accumulation of Mac1(+) cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1β expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-β were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-β and IL-4 during the early stages of infection.