Establishment of a cell line (XTC-2) from the South African clawed toad,Xenopus laevis

Pudney, Mary; Varma, M. G. R.; Leake, C. J.

doi:10.1007/bf01926785

Cited by 120 publications

(56 citation statements)

References 7 publications

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“…COS-7 (ATCC, CRL1651), F9, and TKO cell lines were grown as a monolayer in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Xenopus XTC cells (28) were grown in 67% L15 medium (Sigma) supplemented with 10% fetal bovine serum. Drosophila S2 cells (29) were grown in Schneider's insect medium (Biomedia) supplemented with 10% fetal bovine serum or 10% Prolifix S6 (Biomedia) during protein expression.…”

Section: Methodsmentioning

confidence: 99%

ADAM13 Disintegrin and Cysteine-rich Domains Bind to the Second Heparin-binding Domain of Fibronectin

Gaultier¹,

Cousin²,

Darribère³

et al. 2002

Journal of Biological Chemistry

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ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR 3 PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via ␤ 1 integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and ␤ 1 integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteinerich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins. ADAMs1 are transmembrane glycoproteins that contain a disintegrin and a metalloprotease domain (1). To date, 33 ADAMs have been identified in worms, insects, amphibians, and mammals (2). They can be classified in two major subgroups depending on the presence or absence of a putative active site (HEXGHXXGXHD) in the metalloprotease domain (3). ADAM proteins have been implicated in many biological processes including fertilization (4, 5), myogenesis (6), neurogenesis (7), axon extension and pathfinding (8), and neural crest cell migration (9). Although the number of potential functions involving ADAMs increases, the function of individual ADAM protein domains remains poorly understood.The ADAM metalloprotease domain appears to be involved mostly in shedding cell surface molecules (10). Among these molecules are growth factors (tumor necrosis factor-␣, transforming growth factor-␣, heparin binding-epidermal growth factor), other signaling molecules (Notch, Delta, ephrin), and cell adhesion molecules (selectin, L1-Cam). These cleavages allow cells to send and/or receive a signal as well as change their adhesion to the surrounding cells or substrate. Recently, ADAM13 was shown to cleave fibronectin, one of the major components of the extracellular matrix (ECM) that lies in all cell migration pathways (9).The disintegrin and cysteine-rich domains compose the adhesive part of the ADAM protein. A small loop within the disintegrin domain, known as the disintegrin loop, can bind to several receptors of the integrin family (11). The best example of this is the interaction of ADAM2 and -3 on the mature mouse sperm with the ␣ 6 ␤...

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Section: Methodsmentioning

confidence: 99%

ADAM13 Disintegrin and Cysteine-rich Domains Bind to the Second Heparin-binding Domain of Fibronectin

Gaultier¹,

Cousin²,

Darribère³

et al. 2002

Journal of Biological Chemistry

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“…We therefore examined the temporal expression of both X-ATM mRNA and protein between fertilization and the swimming tadpole stage in Xenopus. Figure 3a shows an RNAse protection assay for the XTC cell line (Pudney et al, 1973) and the indicated stages of development. X-ATM mRNA was detected in unfertilized eggs and expressed at relatively constant levels throughout cleavage, gastrulation, neurulation and the tadpole stage, although we consistently detected higher X-ATM mRNA levels in unfertilized eggs.…”

Section: Expression Of X-atm During Early Developmentmentioning

confidence: 99%

Isolation and characterization of Xenopus ATM (X-ATM): expression, localization, and complex formation during oogenesis and early development

1999

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ATM, the gene product mutated in Ataxia Telangiectasia (A-T) encodes a 350-kDa protein involved in the regulation of several cellular responses to DNA breaks. We used a degenerate PCR-based strategy to isolate a partial clone of X-ATM, the Xenopus homologue of human ATM. Sequence analysis and con®rmed that the clone was most closely related to human ATM. Xenopus ATM protein (X-ATM) is 85% identical to human ATM within the kinase domain and 71% identical over the carboxyl-terminal half of the protein. Polyclonal antibodies raised against recombinant X-ATM are highly speci®c for the ATM protein and recognize a single polypeptide of 370-kDa in oocytes, embryos, egg extracts and a Xenopus cell line. We found that X-ATM was expressed maternally in eggs and as early as stage II pre-vitellogenic oocytes, and the protein and mRNA were present at relatively constant levels throughout development. Subcellular fractionation showed that the protein was nuclear in both the female and male germlines. The level of X-ATM protein did not change throughout the meiotic divisions or the synchronous mitotic cycles of cleavage stage embryos. In addition, we did not observe any change in the level or mobility of X-ATM protein following g-irradiation of embryos. Finally, we also demonstrated that X-ATM was present in a high molecular weight complex of approximately 500 kDa containing the X-ATM protein and other, as yet unidenti®ed component(s).

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“…To confirm the presence of a functional promoter in this region of DNA, the 2.1-kb DNA fragment (À2199: À55) was subcloned into a luciferase reporter system. The engineered construct was used to transiently transfect two Xenopus cell lines, A6 and XTC: The A6 cell line derives from adult Xenopus kidney, and XTC is a fibroblast cell line obtained from X. laevis tadpoles (Rafferty and Sherwin, 1969;Pudney et al, 1973). Both cell lines express endogenous Xenopus HpaL, as determined by RT-PCR (Fig.…”

Section: Expression Of Heparanase In X Laevis Is Driven By Two Promomentioning

confidence: 99%

Two promoters with distinct activities in different tissues drive the expression of heparanase in Xenopus

Bertolesi

Michaiel

et al. 2011

Developmental Dynamics

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In Xenopus laevis embryos, heparanase, the enzyme that degrades heparan sulfate, is synthesized as a preproheparanase (XHpaL) and processed to become enzymatically active (XHpa active). A short nonenzymatic heparanase splice variant (XHpaS) is also expressed. Using immunohistochemistry, Western blot, and heparanase promoter analysis, we studied the dynamic developmental expression of the three heparanases. Our results indicate that (1) all three isoforms are maternally expressed; (2) XHpaS is a developmental variant; (3) in the early embryo, heparanase is localized to both the plasma membrane and the nucleus; (4) several tissues express heparanase, but expression in the developing nervous system is most evident; (5) two promoters with distinct activities in different tissues drive heparanase expression; (6) Oct binding transcription factors may modulate heparanase promoter activity in the early embryo. These data argue that heparanase is expressed widely during development, but localization and levels are finely regulated.

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Establishment of a cell line (XTC-2) from the South African clawed toad,Xenopus laevis

Cited by 120 publications

References 7 publications

ADAM13 Disintegrin and Cysteine-rich Domains Bind to the Second Heparin-binding Domain of Fibronectin

ADAM13 Disintegrin and Cysteine-rich Domains Bind to the Second Heparin-binding Domain of Fibronectin

Isolation and characterization of Xenopus ATM (X-ATM): expression, localization, and complex formation during oogenesis and early development

Two promoters with distinct activities in different tissues drive the expression of heparanase in Xenopus

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