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Article ; Online: Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium.

Sato, Masahiro / Sato-Yamamoto, Nami / Wakita, Ai / Haraguchi, Misako / Shimonishi, Manabu / Okuno, Hiroyuki

International journal of molecular sciences

2022 Volume 23, Issue 9

Abstract: Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently ... ...

Abstract	Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system-a modern genome editing technology-AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed "ex vivo handling of embryos", requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [
MeSH term(s)	Animals ; Blastocyst ; Dependovirus/genetics ; Epithelium ; Fallopian Tubes ; Female ; Gene Editing/methods ; Genetic Vectors/genetics ; Humans ; Mice ; Oviducts/metabolism ; Pregnancy
Language	English
Publishing date	2022-04-28
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms23094897
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Article ; Online: Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium

Masahiro Sato / Nami Sato-Yamamoto / Ai Wakita / Misako Haraguchi / Manabu Shimonishi / Hiroyuki Okuno

International Journal of Molecular Sciences, Vol 23, Iss 4897, p

2022 Volume 4897

Abstract	Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system—a modern genome editing technology—AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed “ex vivo handling of embryos”, requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [ EGFP ] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken β-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.
Keywords	adeno-associated virus ; serotype 6 ; preimplantation embryo ; intra-oviductal injection ; enhanced green fluorescent protein ; GONAD ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	616
Language	English
Publishing date	2022-04-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: E-cadherin loss in RMG-1 cells inhibits cell migration and its regulation by Rho GTPases.

Haraguchi, Misako / Fukushige, Tomoko / Kanekura, Takuro / Ozawa, Masayuki

Biochemistry and biophysics reports

2019 Volume 18, Page(s) 100650

Abstract: E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial-mesenchymal transition (EMT). However, recent ... ...

Abstract	E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial-mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell-extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased β-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for β-catenin expression and β-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration.
Language	English
Publishing date	2019-05-14
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2831046-9
ISSN	2405-5808 ; 2405-5808
ISSN (online)	2405-5808
ISSN	2405-5808
DOI	10.1016/j.bbrep.2019.100650
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Article ; Online: The role of the transcriptional regulator snail in cell detachment, reattachment and migration.

Haraguchi, Misako

Cell adhesion & migration

2009 Volume 3, Issue 3, Page(s) 259–263

Abstract: In order to metastasize, cancer cells must first detach from the primary tumor, migrate, invade through tissues and attach to a second site. The transcription factor snail is an important mediator of epithelial-mesenchymal transitions and is involved in ... ...

Abstract	In order to metastasize, cancer cells must first detach from the primary tumor, migrate, invade through tissues and attach to a second site. The transcription factor snail is an important mediator of epithelial-mesenchymal transitions and is involved in tumor progression. Recent data have provided evidence for a requirement for snail expression in metastatic dissemination. Although very little is known about the molecular mechanisms governing metastatic dissemination, we review the possible roles of snail expression in this process. We also review the regulation of snail expression.
MeSH term(s)	Apoptosis ; Cell Adhesion/genetics ; Cell Movement ; Extracellular Matrix/metabolism ; Gene Expression Regulation ; Humans ; Snail Family Transcription Factors ; Transcription Factors/metabolism
Chemical Substances	Snail Family Transcription Factors ; Transcription Factors
Language	English
Publishing date	2009-07-23
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2268518-2
ISSN	1933-6926 ; 1933-6918
ISSN (online)	1933-6926
ISSN	1933-6918
DOI	10.4161/cam.3.3.8259
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Article ; Online: E-cadherin loss in RMG-1 cells inhibits cell migration and its regulation by Rho GTPases

Misako Haraguchi / Tomoko Fukushige / Takuro Kanekura / Masayuki Ozawa

Biochemistry and Biophysics Reports, Vol 18, Iss , Pp - (2019)

2019

Abstract: E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial–mesenchymal transition (EMT). However, recent ... ...

Abstract	E-cadherin is an adherens junction protein that forms intercellular contacts in epithelial cells. Downregulation of E-cadherin is frequently observed in epithelial tumors and it is a hallmark of epithelial–mesenchymal transition (EMT). However, recent findings suggest that E-cadherin plays a more complex role in certain types of cancers. Previous studies investigating the role of E-cadherin mainly used gene-knockdown systems; therefore, we used the CRISPR/Cas9n system to develop E-cadherin-knockout (EcadKO) ovarian cancer RMG-1 cell to clarify the role of E-cadherin in RMG-1 cells. EcadKO RMG-1 cells demonstrated a complete loss of the adherens junctions and failed to form cell clusters. Cell–extracellular matrix (ECM) interactions were increased in EcadKO RMG-1 cells. Upregulation of integrin beta1 and downregulation of collagen 4 were confirmed. EcadKO RMG-1 cells showed decreased β-catenin levels and decreased expression of its transcriptional target cyclin D1. Surprisingly, a marked decrease in the migratory ability of EcadKO RMG-1 cells was observed and the cellular response to Rho GTPase inhibitors was diminished. Thus, we demonstrated that E-cadherin in RMG-1 cells is indispensable for β-catenin expression and β-catenin mediated transcription and Rho GTPase-regulated directionally persistent cell migration. Keywords: E-cadherin, CRISPR/Cas9n, Cell migration, RhoGTPse, β-catenin, Dispase
Keywords	Biology (General) ; QH301-705.5 ; Biochemistry ; QD415-436
Subject code	570
Language	English
Publishing date	2019-07-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: The Utility of Automated ASPECTS in Acute Ischemic Stroke for Intravenous Recombinant Tissue Plasminogen Activator (IV-rtPA) Therapy.

Shibata, Soichiro / Sakurai, Kenzo / Tachikawa, Keiji / Ko, Riyoko / Hino, Sakae / Fukano, Takayuki / Isahaya, Kenji / Haraguchi, Takafumi / Yamauchi, Junji / Tanabe, Kenichiro / Nagasaka, Misako / Hagiwara, Yuta / Shimizu, Takahiro / Akiyama, Hisanao / Kobayashi, Yasuyuki / Hasegawa, Yasuhiro / Yamano, Yoshihisa

Neurology international

2022 Volume 14, Issue 4, Page(s) 981–990

Abstract: Purpose: This study aimed to investigate the accuracy and clinical significance of an artificial intelligence (AI)-based automated Alberta Stroke Program Early Computed Tomography (ASPECT) scoring software of head CT for the indication of intravenous ... ...

Abstract	Purpose: This study aimed to investigate the accuracy and clinical significance of an artificial intelligence (AI)-based automated Alberta Stroke Program Early Computed Tomography (ASPECT) scoring software of head CT for the indication of intravenous recombinant tissue plasminogen activator (rt-PA) therapy. Methods: This study included two populations of acute ischemic stroke: one comprised patients who had undergone head CT within 48 h of presentation (Population #1, Results: The reading accuracy of AI software was comparable to that of the board-certified vascular neurologists. The detection rate of cardiogenic cerebral embolism was better than that of atherothrombotic cerebral infarction. By excluding extensive infarction, AI-software showed a higher specificity and equivalent sensitivity compared to those of experts. Conclusions: The AI software for ASPECTS showed convincing agreement with expert evaluation and would be supportive in determining the indications of intravenous rt-PA therapy.
Language	English
Publishing date	2022-11-21
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2514727-4
ISSN	2035-8377 ; 2035-8385
ISSN (online)	2035-8377
ISSN	2035-8385
DOI	10.3390/neurolint14040077
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Article ; Online: CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells.

Haraguchi, Misako / Sato, Masahiro / Ozawa, Masayuki

PloS one

2015 Volume 10, Issue 7, Page(s) e0132260

Abstract: Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E- ... ...

Abstract	Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.
MeSH term(s)	Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Adhesion/drug effects ; Cell Communication/drug effects ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Movement/genetics ; Cell Shape/drug effects ; Cytoskeleton/metabolism ; Female ; Gene Deletion ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Molecular Sequence Data ; Ovarian Neoplasms/genetics ; Ovarian Neoplasms/pathology ; Snail Family Transcription Factors ; Transcription Factors/metabolism ; Transforming Growth Factor beta/pharmacology
Chemical Substances	SNAI1 protein, human ; Snail Family Transcription Factors ; Transcription Factors ; Transforming Growth Factor beta
Language	English
Publishing date	2015
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0132260
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Article ; Online: Risk factors for falls in terms of attention during gait in community-dwelling older adults.

Inoue, Tadatoshi / Kamijo, Kenji / Haraguchi, Kenzo / Suzuki, Akihiro / Noto, Misako / Yamashita, Yuh / Nakamura, Takashi

Geriatrics & gerontology international

2018 Volume 18, Issue 8, Page(s) 1267–1271

Abstract: Aim: We examined factors related to conditions of life function and falls, including eye movements and gait variability, in community-dwelling older adults in Japan.: Methods: Participants were 82 older adults (21 men, 61 women, mean age 76.1 years). ...

Abstract	Aim: We examined factors related to conditions of life function and falls, including eye movements and gait variability, in community-dwelling older adults in Japan. Methods: Participants were 82 older adults (21 men, 61 women, mean age 76.1 years). We measured eye movements and gait variability during walking, and cognitive, attentional and life function. We compared two groups according to their fall history, and used a multiple logistic regression analysis to determine its relevance. Results: Fixation time, which was estimated from eye movements during obstacle crossing, and gait variability (vertical) were significantly associated with falls. There was also a significant correlation between fixation time and gait variability during obstacle crossing. In other words, the higher the gait variability in older adults, the higher the risk of falls, which was due to reduced attention estimated from eye movements during obstacle crossing that required obstacle avoidance. Conclusions: These results show that poor attention during gait is a critical risk factor for falls in community-dwelling older adults. For fall prevention, it is necessary to promote exercises for attention, and to maintain an older adult-friendly pedestrian environment. Geriatr Gerontol Int 2018; 18: 1267-1271.
MeSH term(s)	Accidental Falls/prevention & control ; Accidental Falls/statistics & numerical data ; Age Distribution ; Aged ; Aged, 80 and over ; Attention/physiology ; Cohort Studies ; Eye Movements/physiology ; Female ; Gait/physiology ; Geriatric Assessment/methods ; Humans ; Incidence ; Independent Living ; Japan ; Logistic Models ; Male ; Multivariate Analysis ; Postural Balance/physiology ; Prospective Studies ; Risk Assessment ; Sex Distribution
Language	English
Publishing date	2018-06-27
Publishing country	Japan
Document type	Comparative Study ; Journal Article
ZDB-ID	2113849-7
ISSN	1447-0594 ; 1444-1586
ISSN (online)	1447-0594
ISSN	1444-1586
DOI	10.1111/ggi.13462
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Article ; Online: CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1) Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1) Cells.

Misako Haraguchi / Masahiro Sato / Masayuki Ozawa

PLoS ONE, Vol 10, Iss 7, p e

2015 Volume 0132260

Abstract	Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO) by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.
Keywords	Medicine ; R ; Science ; Q
Subject code	612
Language	English
Publishing date	2015-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
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Article ; Online: The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition.

Izawa, Genya / Kobayashi, Wakako / Haraguchi, Misako / Sudo, Akiharu / Ozawa, Masayuki

International journal of molecular medicine

2015 Volume 36, Issue 1, Page(s) 166–172

Abstract: Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise ... ...

Abstract	Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail‑induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the downregulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.
MeSH term(s)	Animals ; Cadherins/biosynthesis ; Cadherins/genetics ; Cattle ; Cell Adhesion/physiology ; Cell Aggregation/physiology ; Cell Line ; DNA Methylation ; Desmogleins/biosynthesis ; Dogs ; Epithelial-Mesenchymal Transition/physiology ; Gene Expression Regulation, Neoplastic ; Homeodomain Proteins/biosynthesis ; Madin Darby Canine Kidney Cells ; Promoter Regions, Genetic/genetics ; Snail Family Transcription Factors ; Transcription Factors/biosynthesis ; Zinc Fingers/physiology
Chemical Substances	Cadherins ; Desmogleins ; Homeodomain Proteins ; SNAI1 protein, human ; Snail Family Transcription Factors ; Transcription Factors
Language	English
Publishing date	2015-07
Publishing country	Greece
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1444428-8
ISSN	1791-244X ; 1107-3756
ISSN (online)	1791-244X
ISSN	1107-3756
DOI	10.3892/ijmm.2015.2215
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