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GFP
Kinds of GFP Terms modified by GFP Selected AbstractsFunctional studies of an evolutionarily conserved, cytochrome b5 domain protein reveal a specific role in axonemal organisation and the general phenomenon of post-division axonemal growth in trypanosomesCYTOSKELETON, Issue 1 2009Helen Farr Abstract Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Several recent proteomic studies of cilia and flagella have been published, including a proteome of the flagellum of the protozoan parasite Trypanosoma brucei. Comparing proteomes reveals many novel proteins that appear to be widely conserved in evolution. Amongst these, we found a previously uncharacterised protein which localised to the axoneme in T. brucei, and therefore named it Trypanosome Axonemal protein (TAX)-2. Ablation of the protein using RNA interference in the procyclic form of the parasite has no effect on growth but causes a reduction in motility. Using transmission electron microscopy, various structural defects were seen in some axonemes, most frequently with microtubule doublets missing from the 9+2 arrangement. RNAi knockdown of TAX-2 expression in the bloodstream form of the parasite caused defects in growth and cytokinesis, a further example of the effects caused by loss of flagellar function in bloodstream form T. brucei. In procyclic cells we used a new set of vectors to ablate protein expression in cells expressing a GFP:TAX-2 fusion protein, which enabled us to easily quantify protein reduction and visualise axonemes made before and after RNAi induction. This establishes a useful generic technique but also revealed a specific observation that the new flagellum on the daughter trypanosome continues growth after cytokinesis. Our results provide evidence for TAX-2 function within the axoneme, where we suggest that it is involved in processes linking the outer doublet microtubules and the central pair. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Live-cell analysis of mitotic spindle formation in taxol-treated cellsCYTOSKELETON, Issue 8 2008Jessica E. Hornick Abstract Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-, tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible,but before NEB is complete,results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D cultureCYTOSKELETON, Issue 6 2007N. Lakshman Abstract The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Shaggy/GSK-3, kinase localizes to the centrosome and to specialized cytoskeletal structures in DrosophilaCYTOSKELETON, Issue 6 2006Yves Bobinnec Abstract The assembly of a functional bipolar mitotic spindle requires an exquisite regulation of microtubule behavior in time and space. To characterize new elements of this machinery we carried out a GFP based "protein trap" screen and selected fusion proteins which localized to the spindle apparatus. By this method we identified Shaggy, the Drosophila homologue of glycogen synthase kinase-3, (GSK-3,), as a component of centrosomes. GSK-3, acting in the Wingless signaling pathway is involved in a vast range of developmental processes, from pattern formation to cell-fate specification, and is a key factor for cell proliferation in most animals. We exploited our Shaggy::GFP Drosophila line to analyze the subcellular localizations of GSK-3,/Shaggy and shed light on its multiple roles during embryogenesis. We found that Shaggy becomes enriched transiently in a variety of specialized cytoskeletal structures of the embryo, including centrosomes throughout mitosis, suggesting that this kinase is involved in the regulation of many aspects of the cytoskeleton function. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,CYTOSKELETON, Issue 4 2004Mike Lorenz Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. © 2004 Wiley-Liss, Inc. [source] Role of the two type II myosins, Myo2 and Myp2, in cytokinetic actomyosin ring formation and function in fission yeastCYTOSKELETON, Issue 3 2003Daniel P. Mulvihill Abstract The formation and contraction of a cytokinetic actomyosin ring (CAR) is essential for the execution of cytokinesis in fission yeast. Unlike most organisms in which its composition has been investigated, the fission yeast CAR contains two type II myosins encoded by the genes myo2+ and myp2+. myo2+ is an essential gene whilst myp2+ is dispensable under normal growth conditions. Myo2 is hence the major contractile protein of the CAR whilst Myp2 plays a more subtle and, as yet, incompletely documented role. Using a fission yeast strain in which the chromosomal copy of the myo2+ gene is fused to the gene encoding green fluorescent protein (GFP), we analysed CAR formation and function in the presence and absence of Myp2. No change in the rate of CAR contraction was observed when Myp2 was absent although the CAR persisted longer in the contracted state and was occasionally observed to split into two discrete rings. This was also observed in myp2, cells following actin depolymerisation with latrunculin. CAR contraction in the absence of Myp2 was completely abolished in the presence of elevated levels of chloride ions. Thus, Myp2 appears to contribute to the stability of the CAR, in particular at a late stage of CAR contraction, and to be a component of the signalling pathway that regulates cytokinesis in response to elevated levels of chloride. To determine whether the presence of two type II myosins was a feature of cytokinesis in other fungi that divide by septation, we searched the genomes of two filamentous fungi, Aspergillus fumigatus and Neurospora crassa, for myosin genes. As in fission yeast, both A. fumigatus and N. crassa contained myosins of classes I, II, and V. Unlike fission yeast, both contained a single type II myosin gene that, on the basis of its tail structure, was more reminiscent of Myp2 than Myo2. The significance of these observations to our understanding of septum to formation and cleavage is discussed. Cell Motil. Cytoskeleton 54:208,216, 2003. © 2003 Wiley-Liss, Inc. [source] Hemidesmosome protein dynamics in live epithelial cellsCYTOSKELETON, Issue 2 2003Daisuke Tsuruta Abstract Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin ,4 subunit (GFP-h,4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-h,4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw,like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw,like clusters of both GFP-h,4 and GFP-BP180 are stable over periods of >60 min, other GFP-h,4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-h,4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-h,4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin ,4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions. Cell Motil. Cytoskeleton 54:122,134, 2003. © 2003 Wiley-Liss, Inc. [source] Simultaneous quantification of cell motility and protein-membrane-association using active contoursCYTOSKELETON, Issue 4 2002Dirk Dormann Abstract We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns. Cell Motil. Cytoskeleton 52:221,230, 2002. © 2002 Wiley-Liss, Inc. [source] Dermal fibroblasts contribute to multiple tissues in the accessory limb modelDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2010Ayako Hirata The accessory limb model has become an alternative model for performing investigations of limb regeneration in an amputated limb. In the accessory limb model, a complete patterned limb can be induced as a result of an interaction between the wound epithelium, a nerve and dermal fibroblasts in the skin. Studies should therefore focus on examining these tissues. To date, however, a study of cellular contributions in the accessory limb model has not been reported. By using green fluorescent protein (GFP) transgenic axolotl tissues, we can trace cell fate at the tissue level. Therefore, in the present study, we transgrafted GFP skin onto the limb of a non-GFP host and induced an accessory limb to investigate cellular contributions. Previous studies of cell contribution to amputation-induced blastemas have demonstrated that dermal cells are the progenitors of many of the early blastema cells, and that these cells contribute to regeneration of the connective tissues, including cartilage. In the present study, we have determined that this same population of progenitor cells responds to signaling from the nerve and wound epithelium in the absence of limb amputation to form an ectopic blastema and regenerate the connective tissues of an ectopic limb. Blastema cells from dermal fibroblasts, however, did not differentiate into either muscle or neural cells, and we conclude that dermal fibroblasts are dedifferentiated along its developmental lineage. [source] Ploidy mosaicism in well-developed nuclear transplants produced by transfer of adult somatic cell nuclei to nonenucleated eggs of medaka (Oryzias latipes)DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2007Elena Kaftanovskaya Chromosomal abnormalities such as ploidy mosaicism have constituted a major obstacle to the successful nuclear transfer of adult somatic cell nuclei in lower vertebrates to date. Euploid mosaicism has been reported previously in well-developed amphibian transplants. Here, we investigated ploidy mosaicisms in well-developed transplants of adult somatic cell nuclei in medaka fish (Oryzias latipes). Donor nuclei from primary cultured cells from the adult caudal fin of a transgenic strain carrying the green fluorescent protein gene (GFP) were transferred to recipient nonenucleated eggs of a wild-type strain to produce 662 transplants. While some of the transplants developed beyond the body formation stage and several hatched, all exhibited varying degrees of abnormal morphology, limited growth and subsequent death. Twenty-one transplants, 19 embryos and two larvae, were selected for chromosomal analysis; all were well-developed 6-day-old or later embryonic stages exhibiting slight morphological abnormalities and the same pattern of GFP expression as that of the donor strain. In addition, all exhibited various levels of euploid mosaicism with haploid-diploid, haploid-triploid or haploid-diploid-triploid chromosome sets. No visible chromosomal abnormalities were observed. Thus, euploid mosaicism similar to that observed in amphibians was confirmed in well-developed nuclear transplants of fish. [source] Real-time observation of Wnt ,-catenin signaling in the chick embryoDEVELOPMENTAL DYNAMICS, Issue 1 2010Anne C. Rios Abstract A critical mediator of cell,cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Reporter constructs containing multimerized TCF DNA binding sites have been used to detect Wnt ,-catenin dependent activity during animal development. In this report, we have constructed and compared several TCF green fluorescent protein (GFP) reporter constructs. They contained 3, 8, or 12 TCF binding sites upstream of a minimal promoter driving native or destabilized enhanced GFP (EGFP). We have used the electroporation of somites in the chick embryo as a paradigm to test them in vivo. We have verified that they all respond to Wnt signaling in vivo. We have then assessed their efficiency at reflecting the activity of the Wnt pathway. Using destabilized EGFP reporter constructs, we show that somite cells dynamically regulate Wnt/,-catenin,dependent signaling, a finding that was confirmed by performing time-lapse video confocal observation of electroporated embryos. Developmental Dynamics 239:346,353, 2010. © 2009 Wiley-Liss, Inc. [source] Phenotypic analysis of deflated/Ints7 function in Drosophila developmentDEVELOPMENTAL DYNAMICS, Issue 5 2009Rachael J. Rutkowski Abstract The Drosophila gene deflated (CG18176; renamed after the pupal lethal abdominal phenotype of mutant individuals) is a member of a conserved gene family found in all multicellular organisms. The human orthologue of deflated (Ints7) encodes a subunit of the Integrator complex that associates with RNA polymerase II and has been implicated in snRNA processing. Since loss-of-function analyses of deflated have not yet been reported, we undertook to investigate deflated expression patterns and mutant phenotypes. deflated mRNA was detected at low levels in proliferating cells in postblastoderm embryos and GFP tagged protein is predominately nuclear. Generation and analysis of four mutant alleles revealed deflated is essential for normal development, as mutant individuals displayed pleiotropic defects affecting many stages of development, consistent with perturbation of cell signalling or cell proliferation. Our data demonstrate multiple roles in development for an Ints7 homologue and to demonstrate its requirement for normal cell signalling and proliferation. Developmental Dynamics 238:1131,1139, 2009. © 2009 Wiley-Liss, Inc. [source] Enhancer detection in the ascidian Ciona intestinalis with transposase-expressing lines of MinosDEVELOPMENTAL DYNAMICS, Issue 1 2008Yasunori Sasakura Abstract Germline transgenesis with a Tc1/mariner superfamily Minos transposon was achieved in the ascidian Ciona intestinalis. Transgenic lines that express transposases in germ cells are very useful for remobilizing transposon copies. In the present study, we created transposase-expressing lines of Minos in Ciona. A Ciona gene encoding protamine (Ci - prm) is expressed in the testes and sperm. Transgenic lines expressing Minos transposase in the testes and sperm were created with a cis -element of Ci - prm, and used for enhancer detection. Double-transgenic animals between transposase lines and a transgenic line with an enhancer detection vector passed on several independent enhancer detection events to subsequent progeny. This technique allowed us to isolate transgenic lines that express GFP in restricted tissues. This system provides an easy and efficient method for large-scale enhancer detection in Ciona intestinalis. Developmental Dynamics 237:39,50, 2008. © 2007 Wiley-Liss, Inc. [source] Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryosDEVELOPMENTAL DYNAMICS, Issue 2 2006Robert W. Zeller Abstract The green fluorescent protein (GFP) is used extensively to monitor gene expression and protein localization in living cells, particularly in developing embryos from a variety of species. Several GFP mutations have been characterized that improve protein expression and alter the emission spectra to produce proteins that emit green, blue, cyan, and yellow wavelengths. DsRed and its variants encode proteins that emit in the orange to red wavelengths. Many of these commercially available fluorescent proteins have been "codon optimized" for maximal levels of expression in mammalian cells. We have generated several fluorescent protein color variants that have been codon optimized for maximal expression in the ascidian Ciona intestinalis. By analyzing quantitative time-lapse recordings of transgenic embryos, we demonstrate that, in general, our Ciona optimized variants are detected and expressed at higher levels than commercially available fluorescent proteins. We show that three of these proteins, expressed simultaneously in different spatial domains within the same transgenic embryo are easily detectable using optimized fluorescent filter sets for epifluorescent microscopy. Coupled with recently developed quantitative imaging techniques, our GFP variants should provide useful reagents for monitoring the simultaneous expression of multiple genes in transgenic ascidian embryos. Developmental Dynamics 235:456,467, 2006. © 2005 Wiley-Liss, Inc. [source] Conditional expression of a myocardium-specific transgene in zebrafish transgenic linesDEVELOPMENTAL DYNAMICS, Issue 4 2005Chiu-Ju Huang Abstract To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage. Developmental Dynamics 233:1294,1303, 2005. © 2005 Wiley-Liss, Inc. [source] Quantitative evaluation of morpholino-mediated protein knock down of GFP, MSX1, and PAX7 during tail regeneration in Ambystoma mexicanumDEVELOPMENTAL DYNAMICS, Issue 3 2005Esther Schnapp No abstract is available for this article. [source] Pod1 is required in stromal cells for glomerulogenesisDEVELOPMENTAL DYNAMICS, Issue 3 2003Shiying Cui Abstract Pod1 (capsulin/epicardin/Tcf21) is a basic-helix-loop-helix transcription factor that is highly expressed in the mesenchyme of developing organs that include the kidney, lung, gut, and heart. Null Pod1 mice are born but die shortly after birth due to a lack of alveoli in the lungs and cardiac defects. In addition, the kidneys are hypoplastic and demonstrate disrupted branching morphogenesis of the ureteric bud epithelium, a marked reduction in the number of nephrons, a delay in glomerulogenesis, and blood vessel abnormalities. To further dissect the cellular function of Pod1 during kidney development, chimeric mice were generated through aggregations of null Pod1 embryonic stem cells and murine embryos ubiquitously expressing enhanced green fluorescent protein (GFP). Histologic, immunohistochemical, and in situ hybridization analysis of the resulting chimeric offspring demonstrated both cell autonomous and non,cell autonomous roles for Pod1 in the differentiation of specific renal cell lineages that include peritubular interstitial cells and pericytes. Most strikingly, the glomerulogenesis defect was rescued by the presence of wild-type stromal cells, suggesting a non,cell autonomous role for Pod1 in this cell population. © 2003 Wiley-Liss, Inc. [source] Misregulation of gene expression in the redox-sensitive NF-,b-dependent limb outgrowth pathway by thalidomideDEVELOPMENTAL DYNAMICS, Issue 2 2002Jason M. Hansen Abstract Thalidomide is known to induce oxidative stress, but mechanisms have not been described through which oxidative stress could contribute to thalidomide-induced terata. Oxidative stress modulates intracellular glutathione (GSH) and redox status and can perturb redox-sensitive processes, such as transcription factor activation and/or binding. Nuclear factor-kappa B (NF-,B), a redox-sensitive transcription factor involved in limb outgrowth, may be modulated by thalidomide-induced redox shifts. Thalidomide-resistant Sprague-Dawley rat embryos (gestation day [GD] 13) treated with thalidomide in utero showed no changes in GSH distribution in the limb but thalidomide-sensitive New Zealand White rabbit embryos (GD 12) showed selective GSH depletion in the limb bud progress zone (PZ). NF-,B and regulatory genes that initiate and maintain limb outgrowth and development, such as Twist and Fgf-10, are selectively expressed in the PZ. Green fluorescent protein (GFP) reporter vectors containing NF-,B binding promoter sites were transfected into both rat and rabbit limb bud cells (LBCs). Treatment with thalidomide caused a preferential decrease in GFP expression in rabbit LBCs but not in rat LBCs. N-acetylcysteine and ,-N-t-phenylbutyl nitrone (PBN), a free radical trapping agent, rescued GFP expression in thalidomide-treated cultures compared with cultures that received thalidomide only. In situ hybridization showed a preferential decrease in Twist, Fgf-8, and Fgf-10 expression after thalidomide treatment (400 mg/kg per day) in rabbit embryos. Expression in rat embryos was not affected. Intravenous cotreatment with PBN and thalidomide (gavage) in rabbits restored normal patterns and localization of Twist, Fgf-8, and Fgf-10 expression. These findings show that NF-,B binding is diminished due to selective thalidomide-induced redox changes in the rabbit, resulting in the significant attenuation of expression of genes necessary for limb outgrowth. © 2002 Wiley-Liss, Inc. [source] The migratory behavior of immature enteric neuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2009M.M. Hao Abstract While they are migrating caudally along the developing gut, around 10%,20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP+ cells) migrated, with an average speed of around 15 ,m/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009. [source] FACS-array gene expression analysis during early development of mouse telencephalic interneuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2008Eric D. Marsh Abstract Cortical interneuron dysfunction has been implicated in multiple human disorders including forms of epilepsy, mental retardation, and autism. Although significant advances have been made, understanding the biologic basis of these disorders will require a level of anatomic, molecular, and genetic detail of interneuron development that currently does not exist. To further delineate the pathways modulating interneuron development we performed fluorescent activated cell sorting (FACs) on genetically engineered mouse embryos that selectively express green fluorescent protein (GFP) in developing interneurons followed by whole genome microarray expression profiling on the isolated cells. Bioinformatics analysis revealed expression of both predicted and unexpected genes in developing cortical interneurons. Two unanticipated pathways discovered to be up regulated prior to interneurons differentiating in the cortex were ion channels/neurotransmitters and synaptic/vesicular related genes. A significant association of neurological disease related genes to the population of developing interneurons was found. These results have defined new and potentially important data on gene expression changes during the development of cortical interneurons. In addition, these data can be mined to uncover numerous novel genes involved in the generation of interneurons and may suggest genes/pathways potentially involved in a number of human neurological disorders. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesisDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2007Jos W.G. van Rosmalen Abstract The cellular prion protein (PrPC) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrPC functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrPC fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrPC, the newly synthesized GFP-PrPCK81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrPC, the mature GFP-PrPCK81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrPC at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrPC,GPI and GFP-PrPCocta mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrPC for its correct biosynthesis. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptidesDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2006Ryan T. Birse Abstract Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Liposome-mediated transfection of mature taste cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2005Ana Marie Landin Abstract The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used ,-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common "promiscuous" promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005 [source] Cationic and anionic lipid-based nanoparticles in CEC for protein separationELECTROPHORESIS, Issue 11 2010Christian Nilsson Abstract The development of new separation techniques is an important task in protein science. Herein, we describe how anionic and cationic lipid-based liquid crystalline nanoparticles can be used for protein separation. The potential of the suggested separation methods is demonstrated on green fluorescent protein (GFP) samples for future use on more complex samples. Three different CEC-LIF approaches for protein separation are described. (i) GFP and GFP N212Y, which are equally charged, were separated with high resolution by using anionic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (ii) High efficiency (800,000 plates/m) and peak capacity were demonstrated separating GFP samples from Escherichia coli with cationic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (iii) Three single amino-acid-substituted GFP variants were separated with high resolution using an approach based on a physical attached double-layer coating of cationic and anionic nanoparticles combined with anionic lipid nanoparticles suspended in the electrolyte. The soft and porous lipid-based nanoparticles were synthesized by a one-step procedure based on the self-assembly of lipids, and were biocompatible with a large surface-to-volume ratio. The methodology is still under development and the optimization of the nanoparticle chemistry and separation conditions can further improve the separation system. In contrast to conventional LC, a new interaction phase is introduced for every analysis, which minimizes carry-over and time-consuming column regeneration. [source] Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGEELECTROPHORESIS, Issue 17 2009Few Ne Chew Abstract Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%. [source] Affinity monolith preconcentrators for polymer microchip capillary electrophoresisELECTROPHORESIS, Issue 16 2008Weichun Yang Abstract Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate- co -ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25,000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples. [source] Applications of PAT-Process Analytical Technology in Recombinant Protein Processes with Escherichia coliENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2008C. Kaiser Abstract Monitoring of bioprocesses and thus observation and identification of such processes is one of the main aims of bioprocess engineering. It is of vital importance in bioprocess development to improve the overall productivity by avoiding unintentional limitations to ensure not only optimal process conditions but also the observation of established production processes. Furthermore, reproducibility needs to be improved and final product quality and quantity be guaranteed. Therefore, an advanced monitoring and control system has been developed, which is based on different in-line, on-line and at-line measurements for substrates and products. Observation of cell viability applying in-line radio frequency impedance measurement and on-line determination of intracellular recombinant target protein using the reporter protein T-Sapphire GFP based on in-line fluorescence measurement show the ability for the detection of critical process states. In this way, the possibility for the on-line recognition of optimal harvest times arises and disturbances in the scheduled process route can be perceived. [source] The exopolysaccharide of Rhizobium sp.ENVIRONMENTAL MICROBIOLOGY, Issue 8 2008Brassica napus roots but contributes to root colonization, YAS34 is not necessary for biofilm formation on Arabidopsis thaliana Summary Microbial exopolysaccharides (EPSs) play key roles in plant,microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots (Arabidopsis thaliana and Brassica napus). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene (gta). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root,soil interface, root colonization, but not in biofilm formation. [source] A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxesENVIRONMENTAL MICROBIOLOGY, Issue 4 2006Robin Tecon Summary The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor ,Burkholderia sp. strain RP037 , produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed. [source] Toxicity of manufactured zinc oxide nanoparticles in the nematode Caenorhabditis elegansENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2009Hongbo Ma Abstract Information describing the possible impacts of manufactured nanoparticles on human health and ecological receptors is limited. The objective of the present study was to evaluate the potential toxicological effects of manufactured zinc oxide nanoparticles (ZnO-NPs; 1.5 nm) compared to aqueous zinc chloride (ZnCl2) in the free-living nematode Caenorhabditis elegans. Toxicity of both types of Zn was investigated using the ecologically relevant endpoints of lethality, behavior, reproduction, and transgene expression in a mtl-2::GFP (gene encoding green fluorescence protein fused onto the metallothionein-2 gene promoter) transgenic strain of C. elegans. Zinc oxide nanoparticles showed no significant difference from ZnCl2 regarding either lethality or reproduction in C. elegans, as indicated by their median lethal concentrations (LC50s; p = 0.29, n = 3) and median effective concentrations (EC50s; Z = 0.835, p = 0.797). Also, no significant difference was found in EC50s for behavioral change between ZnO-NPs (635 mg Zn/L; 95% confidence interval [CI], 477,844 mg Zn/L) and ZnCl2 (546 mg Zn/L; 95% CI, 447,666 mg Zn/L) (Z = 0.907, p = 0.834). Zinc oxide nanoparticles induced transgene expression in the mtl-2::GFP transgenic C. elegans in a manner similar to that of ZnCl2, suggesting that intracellular biotransformation of the nanoparticles might have occurred or the nanoparticles have dissolved to Zn2+ to enact toxicity. These findings demonstrate that manufactured ZnO-NPs have toxicity to the nematode C. elegans similar to that of aqueous ZnCl2. [source] | ||||||||||||||||||||||||||||||||||||||||