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Sample records for gfp expressing salamander

  1. [Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

    Science.gov (United States)

    Han, Xiang-ping; Shi, Gui-lan; Wang, Cheng-feng; Li, Jie; Zhang, Jian-wei; Zhang, Yu; Zhang, Shu-ren; Liu, Bin-lei

    2012-12-01

    To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

  2. Prolongation of GFP-expressed skin graft after intrathymic injection of GFP positive splenocytes in adult rat

    Science.gov (United States)

    Hakamata, Yoji; Igarashi, Yuka; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    GFP is a fluorescent product of the jellyfish Aequorea victoria and has been used for a variety of biological experiments as a reporter molecule. While GFP possesses advantages for the non-invasive imaging of viable cells, GFP-positive cells are still considered potential xeno-antigens. It is difficult to observe the precise fate of transplanted cells/organs in recipients without immunological control. The aim of this study was to determine whether intrathymic injection of GFP to recipients and the depletion of peripheral lymphocytes could lead to donor-specific unresponsiveness to GFP-expressed cell. LEW rats were administered intraperitoneally with 0.2 ml of anti-rat lymphocyte serum (ALS) 1 day prior to intrathymic injection of donor splenocytes or adeno-GFP vector. Donor cells and vector were non-invasively inoculated into the thymus under high frequency ultrasound imaging using an echo-guide. All animals subsequently received a 7 days GFP-expressed skin graft from the same genetic background GFP LEW transgenic rat. Skin graft survival was greater in rats injected with donor splenocytes (23.6+/-9.1) compared with adeno-GFP (13.0+/-3.7) or untreated control rats (9.5+/-1.0). Intrathymic injection of donor antigen into adult rats can induce donor-specific unresponsiveness. Donor cells can be observed for a long-term in recipients with normal immunity using this strategy.

  3. Expression Analysis of CB2-GFP BAC Transgenic Mice.

    Science.gov (United States)

    Schmöle, Anne-Caroline; Lundt, Ramona; Gennequin, Benjamin; Schrage, Hanna; Beins, Eva; Krämer, Alexandra; Zimmer, Till; Limmer, Andreas; Zimmer, Andreas; Otte, David-Marian

    2015-01-01

    The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

  4. Chemical clearing and dehydration of GFP expressing mouse brains.

    Science.gov (United States)

    Becker, Klaus; Jährling, Nina; Saghafi, Saiedeh; Weiler, Reto; Dodt, Hans-Ulrich

    2012-01-01

    Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.

  5. Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Zhou

    2017-07-01

    Full Text Available The Chinese giant salamander iridovirus (CGSIV, belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV, expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL and confirmed by Western blot and indirect immunofluorescence (IIF assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9 cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

  6. Molecular characterization and expression analysis of cathepsin C in Chinese giant salamander (Andrias davidianus after Aeromonas hydrophila infection

    Directory of Open Access Journals (Sweden)

    Zisheng Wang

    2018-03-01

    Full Text Available Background: Cathepsin C (CTSC (dipeptidyl peptidase I, DPPI, is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus, an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5′-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3′-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander. Keywords: cDNA, CTSC, Dipeptidyl peptidase I, Gene expression, Hydrophila, Immune, Peptide, Sequence, Tissue

  7. Expression Analysis of CB2-GFP BAC Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Anne-Caroline Schmöle

    Full Text Available The endocannabinoid system (ECS is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2. As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

  8. Chemical clearing and dehydration of GFP expressing mouse brains.

    Directory of Open Access Journals (Sweden)

    Klaus Becker

    Full Text Available Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4 can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9 is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.

  9. Variable expression of GFP in different populations of peripheral cholinergic neurons of ChATBAC-eGFP transgenic mice.

    Science.gov (United States)

    Brown, T Christopher; Bond, Cherie E; Hoover, Donald B

    2018-03-01

    Immunohistochemistry is used widely to identify cholinergic neurons, but this approach has some limitations. To address these problems, investigators developed transgenic mice that express enhanced green fluorescent protein (GFP) directed by the promoter for choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme. Although, it was reported that these mice express GFP in all cholinergic neurons and non-neuronal cholinergic cells, we could not detect GFP in cardiac cholinergic nerves in preliminary experiments. Our goals for this study were to confirm our initial observation and perform a qualitative screen of other representative autonomic structures for the presences of GFP in cholinergic innervation of effector tissues. We evaluated GFP fluorescence of intact, unfixed tissues and the cellular localization of GFP and vesicular acetylcholine transporter (VAChT), a specific cholinergic marker, in tissue sections and intestinal whole mounts. Our experiments identified two major tissues where cholinergic neurons and/or nerve fibers lacked GFP: 1) most cholinergic neurons of the intrinsic cardiac ganglia and all cholinergic nerve fibers in the heart and 2) most cholinergic nerve fibers innervating airway smooth muscle. Most cholinergic neurons in airway ganglia stained for GFP. Cholinergic systems in the bladder and intestines were fully delineated by GFP staining. GFP labeling of input to ganglia with long preganglionic projections (vagal) was sparse or weak, while that to ganglia with short preganglionic projections (spinal) was strong. Total absence of GFP might be due to splicing out of the GFP gene. Lack of GFP in nerve projections from GFP-positive cell bodies might reflect a transport deficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    International Nuclear Information System (INIS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yamamoto, Kenji; Yasuhara, Masato; Kondo, Akihiko

    2008-01-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  11. The expression of GFP under the control of fibroin promotor in ...

    Indian Academy of Sciences (India)

    ... mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector (pGFP-N2/Fib) was constructed by use of replacing the CMV promoter with the fibroin promoter. The results of visual screening under a fluorescent inverted microscope and Western blot analysis indicated that the GFP gene was expressed in ...

  12. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

    Science.gov (United States)

    Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

    2017-07-03

    We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Polarized expression of the GFP-tagged rat V(1a) vasopressin receptor.

    Science.gov (United States)

    Campos, D M; Reyes, C E; Sarmiento, J; Navarro, J; González, C B

    2001-11-30

    We investigated the targeting of the V(1a) receptor fused with the green fluorescence protein (V(1a)R-GFP) in polarized MDCK cells. Cells expressing V(1a)R-GFP displayed binding to vasopressin (AVP) and AVP-induced calcium responses, similar to cells expressing the wild-type V1a receptor. Interestingly, as with the wild-type V(1a)R, V(1a)R-GFP is preferentially distributed in the basolateral side of MDCK cells as monitored by confocal microscopy. Furthermore, AVP induced internalization of GFP-tagged receptors. Therefore, the GFP-tagged V(1a) receptor retains all the sorting signals of the wild-type receptor and offers an excellent system to elucidate the mechanisms of cell trafficking of V(1a) receptors.

  14. Detection of gfp expression from gfp-labelled bacteria spot ...

    African Journals Online (AJOL)

    SERVER

    Green fluorescent protein (GFP) as a marker gene has facilitated biological research ... behaviour of B501gfp1 in sugarcane plant tissues over .... Bacteria population changes over time on the stem tissue (parenchyma tissues and intercellular.

  15. Ghrelin receptor expression and colocalization with anterior pituitary hormones using a GHSR-GFP mouse line.

    Science.gov (United States)

    Reichenbach, Alex; Steyn, Frederik J; Sleeman, Mark W; Andrews, Zane B

    2012-11-01

    Ghrelin is the endogenous ligand for the GH secretagogue receptor (GHSR) and robustly stimulates GH release from the anterior pituitary gland. Ghrelin also regulates the secretion of anterior pituitary hormones including TSH, LH, prolactin (PRL), and ACTH. However, the relative contribution of a direct action at the GHSR in the anterior pituitary gland vs. an indirect action at the GHSR in the hypothalamus remains undefined. We used a novel GHSR-enhanced green fluorescent protein (eGFP) reporter mouse to quantify GHSR coexpression with GH, TSH, LH, PRL, and ACTH anterior pituitary cells in males vs. females and in chow-fed or calorie-restricted (CR) mice. GHSR-eGFP-expressing cells were only observed in anterior pituitary. The number of GHSR-eGFP-expressing cells was higher in male compared with females, and CR did not affect the GHSR-eGFP cell number. Double staining revealed 77% of somatotrophs expressed GHSR-eGFP in both males and females. Nineteen percent and 12.6% of corticotrophs, 21% and 9% of lactotrophs, 18% and 19% of gonadotrophs, and 3% and 9% of males and females, respectively, expressed GHSR-eGFP. CR increased the number of TSH cells, but suppressed the number of lactotrophs and gonadotrophs, expressing GHSR-eGFP compared with controls. These studies support a robust stimulatory action of ghrelin via the GHSR on GH secretion and identify a previously unknown sexual dimorphism in the GHSR expression in the anterior pituitary. CR affects GHSR-eGFP expression on lactotrophs, gonadotrophs, and thyrotrophs, which may mediate reproductive function and energy metabolism during periods of negative energy balance. The low to moderate expression of GHSR-eGFP suggests that ghrelin plays a minor direct role on remaining anterior pituitary cells.

  16. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

    Directory of Open Access Journals (Sweden)

    Arthur M Talman

    2010-02-01

    Full Text Available The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

  17. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

    Science.gov (United States)

    Talman, Arthur M; Blagborough, Andrew M; Sinden, Robert E

    2010-02-10

    The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

  18. A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Ishag, H Z A; Liu, M J; Yang, R S; Xiong, Q Y; Feng, Z X; Shao, G Q

    2016-04-28

    Mycoplasma hyopneumoniae (M. hyopneumoniae) causes porcine enzootic pneumonia (PEP) that significantly affects the pig industry worldwide. Despite the availability of the whole genome sequence, studies on the pathogenesis of this organism have been limited due to the lack of a genetic manipulation system. Therefore, the aim of the current study was to generate a general GFP reporter vector based on a replicating plasmid. Here, we describe the feasibility of GFP reporter expression in M. hyopneumoniae (strain 168L) controlled by the p97 gene promoter of this mycoplasma. An expression plasmid (pMD18-TOgfp) containing the p97 gene promoter, and origin of replication (oriC) of M. hyopneumoniae, tetracycline resistant marker (tetM), and GFP was constructed and used to transform competent M. hyopneumoniae cells. We observed green fluorescence in M. hyopneumoniae transformants under fluorescence microscopy, which indicates that there was expression of the GFP reporter that was driven by the p97 gene promoter. Additionally, an electroporation method for M. hyopneumoniae with an efficiency of approximately 1 x 10(-6) transformants/μg plasmid DNA was optimized and is described herein. In conclusion, our data demonstrate the susceptibility of M. hyopneumoniae to genetic manipulation whereby foreign genes are expressed. This work may encourage the development of genetic tools to manipulate the genome of M. hyopneumoniae for functional genomic analyses.

  19. Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

    Science.gov (United States)

    Weld, R; Heinemann, J; Eady, C

    2001-03-01

    The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

  20. Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

    DEFF Research Database (Denmark)

    Huber, Reinhard C.; Remuge, Liliana; Carlisle, Ailsa

    2012-01-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology—animal welfare—has not been approached through systematic assessment...... and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals...... months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs...

  1. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Science.gov (United States)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  2. Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150+ long-term repopulating murine hematopoietic stem cells.

    Science.gov (United States)

    Tornack, Julia; Kawano, Yohei; Garbi, Natalio; Hämmerling, Günter J; Melchers, Fritz; Tsuneto, Motokazu

    2017-09-01

    The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long-term repopulating HSCs (LT-HSC) are routinely enriched as Lin - Sca1 + c-Kit + CD34 - Flt3 - CD150 + CD48 - cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L-eGFP reporter mice, we show that endogenous Flt3L-eGFP-reporter RNA expression correlates with eGFP-protein expression. This Flt3L-eGFP-reporter expression distinguishes two LT-HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L-eGFP-reporter low cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporter high cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporter low cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporter high HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporter low cells develop to Flt3L-eGFP-reporter high cells in vitro, although Flt3L-eGFP-reporter high cells remain Flt3L-eGFP-reporter high . CD150 + Flt3L-eGFP-reporter low cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporter high cells do express EPCR but not CD41. Thus, FACS-enrichment of Flt3/ Flt3L-eGFP-reporter negative, Lin - CD150 + CD48 - EPCR + CD41 + HSCs allows a further 5-fold enrichment of functional LT-HSCs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A Plasmodium falciparum Strain Expressing GFP throughout the Parasite's Life-Cycle

    OpenAIRE

    Talman, Arthur M.; Blagborough, Andrew M.; Sinden, Robert E.

    2010-01-01

    The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete spo...

  4. Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle.

    Science.gov (United States)

    Ono, Takeshi; Tadakuma, Takushi; Rodriguez, Ana

    2007-03-01

    Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.

  5. AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells.

    Science.gov (United States)

    Fassina, L; Magenes, G; Inzaghi, A; Palumbo, S; Allavena, G; Miracco, C; Pirtoli, L; Biggiogera, M; Comincini, S

    2012-10-11

    An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

  6. The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

    Science.gov (United States)

    Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

    2011-02-01

    In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

  7. Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

    Science.gov (United States)

    Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

  8. Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

    Science.gov (United States)

    2010-01-01

    Background The Mexican axolotl (Ambystoma mexicanum) is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic) form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum) that typically undergo a metamorphosis. Results Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph) and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs) were identified as unique to the axolotl (n = 76) and tiger salamander (n = 292) than were identified as shared (n = 108). All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. Conclusions Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome-wide reduction in mRNA abundance

  9. Microarray analysis of a salamander hopeful monster reveals transcriptional signatures of paedomorphic brain development

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    Putta Srikrishna

    2010-06-01

    Full Text Available Abstract Background The Mexican axolotl (Ambystoma mexicanum is considered a hopeful monster because it exhibits an adaptive and derived mode of development - paedomorphosis - that has evolved rapidly and independently among tiger salamanders. Unlike related tiger salamanders that undergo metamorphosis, axolotls retain larval morphological traits into adulthood and thus present an adult body plan that differs dramatically from the ancestral (metamorphic form. The basis of paedomorphic development was investigated by comparing temporal patterns of gene transcription between axolotl and tiger salamander larvae (Ambystoma tigrinum tigrinum that typically undergo a metamorphosis. Results Transcript abundances from whole brain and pituitary were estimated via microarray analysis on four different days post hatching (42, 56, 70, 84 dph and regression modeling was used to independently identify genes that were differentially expressed as a function of time in both species. Collectively, more differentially expressed genes (DEGs were identified as unique to the axolotl (n = 76 and tiger salamander (n = 292 than were identified as shared (n = 108. All but two of the shared DEGs exhibited the same temporal pattern of expression and the unique genes tended to show greater changes later in the larval period when tiger salamander larvae were undergoing anatomical metamorphosis. A second, complementary analysis that directly compared the expression of 1320 genes between the species identified 409 genes that differed as a function of species or the interaction between time and species. Of these 409 DEGs, 84% exhibited higher abundances in tiger salamander larvae at all sampling times. Conclusions Many of the unique tiger salamander transcriptional responses are probably associated with metamorphic biological processes. However, the axolotl also showed unique patterns of transcription early in development. In particular, the axolotl showed a genome

  10. Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

    Science.gov (United States)

    Balic, Anamaria; Mina, Mina

    2011-01-01

    Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

  11. Invasive Asian Earthworms Negatively Impact Keystone Terrestrial Salamanders.

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    Julie L Ziemba

    Full Text Available Asian pheretimoid earthworms (e.g. Amynthas and Metaphire spp. are invading North American forests and consuming the vital detrital layer that forest floor biota [including the keystone species Plethodon cinereus (Eastern Red-backed Salamander], rely on for protection, food, and habitat. Plethodon cinereus population declines have been associated with leaf litter loss following the invasion of several exotic earthworm species, but there have been few studies on the specific interactions between pheretimoid earthworms and P. cinereus. Since some species of large and active pheretimoids spatially overlap with salamanders beneath natural cover objects and in detritus, they may distinctively compound the negative consequences of earthworm-mediated resource degradation by physically disturbing important salamander activities (foraging, mating, and egg brooding. We predicted that earthworms would exclude salamanders from high quality microhabitat, reduce foraging efficiency, and negatively affect salamander fitness. In laboratory trials, salamanders used lower quality microhabitat and consumed fewer flies in the presence of earthworms. In a natural field experiment, conducted on salamander populations from "non-invaded" and "pheretimoid invaded" sites in Ohio, salamanders and earthworms shared cover objects ~60% less than expected. Earthworm abundance was negatively associated with juvenile and male salamander abundance, but had no relationship with female salamander abundance. There was no effect of pheretimoid invasion on salamander body condition. Juvenile and non-resident male salamanders do not hold stable territories centered beneath cover objects such as rocks or logs, which results in reduced access to prey, greater risk of desiccation, and dispersal pressure. Habitat degradation and physical exclusion of salamanders from cover objects may hinder juvenile and male salamander performance, ultimately reducing recruitment and salamander abundance

  12. Invasive Asian Earthworms Negatively Impact Keystone Terrestrial Salamanders.

    Science.gov (United States)

    Ziemba, Julie L; Hickerson, Cari-Ann M; Anthony, Carl D

    2016-01-01

    Asian pheretimoid earthworms (e.g. Amynthas and Metaphire spp.) are invading North American forests and consuming the vital detrital layer that forest floor biota [including the keystone species Plethodon cinereus (Eastern Red-backed Salamander)], rely on for protection, food, and habitat. Plethodon cinereus population declines have been associated with leaf litter loss following the invasion of several exotic earthworm species, but there have been few studies on the specific interactions between pheretimoid earthworms and P. cinereus. Since some species of large and active pheretimoids spatially overlap with salamanders beneath natural cover objects and in detritus, they may distinctively compound the negative consequences of earthworm-mediated resource degradation by physically disturbing important salamander activities (foraging, mating, and egg brooding). We predicted that earthworms would exclude salamanders from high quality microhabitat, reduce foraging efficiency, and negatively affect salamander fitness. In laboratory trials, salamanders used lower quality microhabitat and consumed fewer flies in the presence of earthworms. In a natural field experiment, conducted on salamander populations from "non-invaded" and "pheretimoid invaded" sites in Ohio, salamanders and earthworms shared cover objects ~60% less than expected. Earthworm abundance was negatively associated with juvenile and male salamander abundance, but had no relationship with female salamander abundance. There was no effect of pheretimoid invasion on salamander body condition. Juvenile and non-resident male salamanders do not hold stable territories centered beneath cover objects such as rocks or logs, which results in reduced access to prey, greater risk of desiccation, and dispersal pressure. Habitat degradation and physical exclusion of salamanders from cover objects may hinder juvenile and male salamander performance, ultimately reducing recruitment and salamander abundance following Asian

  13. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    International Nuclear Information System (INIS)

    Semaan, Sheila J; Nickells, Robert W

    2010-01-01

    Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116 BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. HCT116 BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the

  14. Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

    International Nuclear Information System (INIS)

    Wang Hongmin; Monteiro, Mervyn J.

    2007-01-01

    Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases

  15. Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

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    François Vromman

    Full Text Available Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

  16. Protective Immunity Induced by DNA Vaccination against Ranavirus Infection in Chinese Giant Salamander Andrias davidianus

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    Zhong-Yuan Chen

    2018-01-01

    Full Text Available Andrias davidianus ranavirus (ADRV is an emerging viral pathogen that causes severe systemic hemorrhagic disease in Chinese giant salamanders. There is an urgent need for developing an effective vaccine against this fatal disease. In this study, DNA vaccines containing the ADRV 2L gene (pcDNA-2L and the 58L gene (pcDNA-58L were respectively constructed, and their immune protective effects were evaluated in Chinese giant salamanders. In vitro and in vivo expression of the vaccine plasmids were confirmed in transfected cells and muscle tissues of vaccinated Chinese giant salamanders by using immunoblot analysis or RT-PCR. Following ADRV challenge, the Chinese giant salamanders vaccinated with pcDNA-2L showed a relative percent survival (RPS of 66.7%, which was significant higher than that in Chinese giant salamanders immunized with pcDNA-58L (RPS of 3.3%. Moreover, the specific antibody against ADRV was detected in Chinese giant salamanders vaccinated with pcDNA-2L at 14 and 21 days post-vaccination by indirect enzyme-linked immunosorbent assay (ELISA. Transcriptional analysis revealed that the expression levels of immune-related genes including type I interferon (IFN, myxovirus resistance (Mx, major histocompatibility complex class IA (MHC IA, and immunoglobulin M (IgM were strongly up-regulated after vaccination with pcDNA-2L. Furthermore, vaccination with pcDNA-2L significantly suppressed the virus replication, which was seen by a low viral load in the spleen of Chinese giant salamander survivals after ADRV challenge. These results indicated that pcDNA-2L could induce a significant innate immune response and an adaptive immune response involving both humoral and cell-mediated immunity that conferred effective protection against ADRV infection, and might be a potential vaccine candidate for controlling ADRV disease in Chinese giant salamanders.

  17. Efficient transformation and expression of gfp gene in Valsa mali var. mali.

    Science.gov (United States)

    Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

    2015-01-01

    Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

  18. Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

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    Francis M. F. Nunes

    2013-01-01

    Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  19. Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

    Science.gov (United States)

    Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

    2013-01-04

    RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  20. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    Directory of Open Access Journals (Sweden)

    Semaan Sheila J

    2010-10-01

    Full Text Available Abstract Background Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Methods Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. Results HCT116BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Conclusion Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of

  1. A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

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    Carlos R. Arias-Barreiro

    2010-06-01

    Full Text Available A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2. Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm were determined as 1.0 x 10−7 (arsenite, 1.0 x 10−4 (naphthalene, 1.0 x 10−4 (Cu2+, 3.8 x 10−4 (H2O2, 1.0 x 10−3 (Cd2+, 1.0 x 10−3 (Zn2+, 1.0 x 10−2 (menadione, 1.0 (triphenyltin, 1.56 (zinc pyrithione, 3.1 (selenite and 6.3 (Pb2+, respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.

  2. Parallel habitat acclimatization is realized by the expression of different genes in two closely related salamander species (genus Salamandra).

    Science.gov (United States)

    Goedbloed, D J; Czypionka, T; Altmüller, J; Rodriguez, A; Küpfer, E; Segev, O; Blaustein, L; Templeton, A R; Nolte, A W; Steinfartz, S

    2017-12-01

    The utilization of similar habitats by different species provides an ideal opportunity to identify genes underlying adaptation and acclimatization. Here, we analysed the gene expression of two closely related salamander species: Salamandra salamandra in Central Europe and Salamandra infraimmaculata in the Near East. These species inhabit similar habitat types: 'temporary ponds' and 'permanent streams' during larval development. We developed two species-specific gene expression microarrays, each targeting over 12 000 transcripts, including an overlapping subset of 8331 orthologues. Gene expression was examined for systematic differences between temporary ponds and permanent streams in larvae from both salamander species to establish gene sets and functions associated with these two habitat types. Only 20 orthologues were associated with a habitat in both species, but these orthologues did not show parallel expression patterns across species more than expected by chance. Functional annotation of a set of 106 genes with the highest effect size for a habitat suggested four putative gene function categories associated with a habitat in both species: cell proliferation, neural development, oxygen responses and muscle capacity. Among these high effect size genes was a single orthologue (14-3-3 protein zeta/YWHAZ) that was downregulated in temporary ponds in both species. The emergence of four gene function categories combined with a lack of parallel expression of orthologues (except 14-3-3 protein zeta) suggests that parallel habitat adaptation or acclimatization by larvae from S. salamandra and S. infraimmaculata to temporary ponds and permanent streams is mainly realized by different genes with a converging functionality.

  3. Identification of Cells at Early and Late Stages of Polarization During Odontoblast Differentiation Using pOBCol3.6GFP and pOBCol2.3GFP Transgenic Mice

    Science.gov (United States)

    Balic, Anamaria; Aguila, H. Leonardo; Mina, Mina

    2010-01-01

    Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stage of differentiation along a lineage. In the present study we used primary cell cultures derived from the dental pulp from pOBCol3.6GFP and pOBCol2.3GFP transgenic mice as a model to develop markers for early stages of odontoblast differentiation from progenitor cells. We analyzed the temporal and spatial expression of 2.3-GFP and 3.6-GFP during in vitro mineralization. Using FACS to separate cells based on GFP expression, we obtained relatively homogenous sub-populations of cells and analyzed their dentinogenic potentials and their progression into odontoblasts. Our observations showed that these transgenes were activated before the onset of matrix deposition and in cells at different stages of polarization. The 3.6-GFP transgene was activated in cells in early stages of polarization whereas the 2.3-GFP transgene was activated at a later stage of polarization just before or at the time of formation of secretory odontoblast. PMID:20728593

  4. Activity of cardiorespiratory networks revealed by transsynaptic virus expressing GFP.

    Science.gov (United States)

    Irnaten, M; Neff, R A; Wang, J; Loewy, A D; Mettenleiter, T C; Mendelowitz, D

    2001-01-01

    A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alpha herpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

  5. Conservation assessment for the Siskiyou Mountains salamander and Scott Bar salamander in northern California.

    Energy Technology Data Exchange (ETDEWEB)

    Vinikour, W. S.; LaGory, K. E.; Adduci, J. J.; Environmental Science Division

    2006-10-20

    The purpose of this conservation assessment is to summarize existing knowledge regarding the biology and ecology of the Siskiyou Mountains salamander and Scott Bar salamander, identify threats to the two species, and identify conservation considerations to aid federal management for persistence of the species. The conservation assessment will serve as the basis for a conservation strategy for the species.

  6. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Rachel M. Woodfint

    2017-01-01

    Full Text Available Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2 expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2, GATA binding protein 4 (GATA4, hepatocyte nuclear factor 4 α (HNF4A, and transcription factor 4 (TCF4 that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  7. The Impact of GFP Reporter Gene Transduction and Expression on Metabolomics of Placental Mesenchymal Stem Cells Determined by UHPLC-Q/TOF-MS

    Directory of Open Access Journals (Sweden)

    Jinfeng Yang

    2017-01-01

    Full Text Available Introduction. Green fluorescent protein (GFP is widely used as a reporter gene in regenerative medicine research to label and track stem cells. Here, we examined whether expressing GFP gene may impact the metabolism of human placental mesenchymal stem cells (hPMSCs. Methods. The GFP gene was transduced into hPMSCs using lentiviral-based infection to establish GFP+hPMSCs. A sensitive 13C/12C-dansyl labeling LC-MS method targeting the amine/phenol submetabolome was used for in-depth cell metabolome profiling. Results. A total of 1151 peak pairs or metabolites were detected from 12 LC-MS runs. Principal component analysis and partial least squares discriminant analysis showed poor separation, and the volcano plots demonstrated that most of the metabolites were not significantly changed when hPMSCs were tagged with GFP. Overall, 739 metabolites were positively or putatively identified. Only 11 metabolites showed significant changes. Metabolic pathway analyses indicated that three of the identified metabolites were involved in nine pathways. However, these metabolites are unlikely to have a large impact on the metabolic pathways due to their nonessential roles and limited hits in pathway analysis. Conclusion. This study indicated that the expression of ectopic GFP reporter gene did not significantly alter the metabolomics pathways covered by the amine/phenol submetabolome.

  8. Isolation, Culture, and Motility Measurements of Epidermal Melanocytes from GFP-Expressing Reporter Mice.

    Science.gov (United States)

    Dagnino, Lina; Crawford, Melissa

    2018-03-27

    In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26 mT/mG reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity. The cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as capacity for migration. Melanocytes are slow moving cells, and we also provide a method to measure motility using individual cell tracking and data analysis.

  9. The effect of waist twisting on walking speed of an amphibious salamander like robot

    Science.gov (United States)

    Yin, Xin-Yan; Jia, Li-Chao; Wang, Chen; Xie, Guang-Ming

    2016-06-01

    Amphibious salamanders often swing their waist to coordinate quadruped walking in order to improve their crawling speed. A robot with a swing waist joint, like an amphibious salamander, is used to mimic this locomotion. A control method is designed to allow the robot to maintain the rotational speed of its legs continuous and avoid impact between its legs and the ground. An analytical expression is established between the amplitude of the waist joint and the step length. Further, an optimization amplitude is obtained corresponding to the maximum stride. The simulation results based on automatic dynamic analysis of mechanical systems (ADAMS) and physical experiments verify the rationality and validity of this expression.

  10. Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase

    Science.gov (United States)

    Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

    2016-01-01

    Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins. PMID:26258682

  11. Reproductive biology of Ambystoma salamanders in the southeastern United States

    Science.gov (United States)

    Glorioso, Brad M.; Waddle, J. Hardin; Hefner, J. M.

    2015-01-01

    Reproductive aspects of Ambystoma salamanders were investigated at sites in Louisiana (2010–12) and Mississippi (2013). Three species occurred at the Louisiana site, Spotted Salamander (A. maculatum), Marbled Salamander (A. opacum), and Mole Salamander (A. talpoideum), whereas only Spotted Salamanders were studied at the Mississippi site. A total of 162 and 71 egg masses of Spotted Salamanders were examined at the Louisiana and Mississippi sites, respectively. Significantly more Spotted Salamander eggs per egg mass were observed at the Mississippi site (x̄ = 78.2) than the Louisiana site (x̄ = 53.8; P < 0.001). The mean snout–vent length of female Spotted Salamanders at the Mississippi site (82.9 mm) was significantly larger than the Louisiana site (76.1 mm; P < 0.001). Opaque Spotted Salamander egg masses were not found at the Mississippi site, but accounted for 11% of examined egg masses at the Louisiana site. The mean number of eggs per egg mass at the Louisiana site did not differ between opaque (47.3) and clear (54.6) egg masses (P = 0.21). A total of 47 egg masses of the Mole Salamander were examined, with a mean number of 6.7 embryos per mass. Twenty-three individual nests of the Marbled Salamander were found either under or in decaying logs in the dry pond basins. There was no difference between the mean numbers of eggs per mass of attended nests (93.0) versus those that were discovered unattended (86.6; P = 0.67). Females tended to place their nests at intermediate heights within the pond basin.

  12. Evolution of gigantism in amphiumid salamanders.

    Directory of Open Access Journals (Sweden)

    Ronald M Bonett

    2009-05-01

    Full Text Available The Amphiumidae contains three species of elongate, permanently aquatic salamanders with four diminutive limbs that append one, two, or three toes. Two of the species, Amphiuma means and A. tridactylum, are among the largest salamanders in the world, reaching lengths of more than one meter, whereas the third species (A. pholeter, extinct amphiumids, and closely related salamander families are relatively small. Amphiuma means and A. tridactylum are widespread species and live in a wide range of lowland aquatic habitats on the Coastal Plain of the southeastern United States, whereas A. pholeter is restricted to very specialized organic muck habitats and is syntopic with A. means. Here we present analyses of sequences of mitochondrial and nuclear loci from across the distribution of the three taxa to assess lineage diversity, relationships, and relative timing of divergence in amphiumid salamanders. In addition we analyze the evolution of gigantism in the clade. Our analyses indicate three lineages that have diverged since the late Miocene, that correspond to the three currently recognized species, but the two gigantic species are not each other's closest relatives. Given that the most closely related salamander families and fossil amphiumids from the Upper Cretaceous and Paleocene are relatively small, our results suggest at least two extreme changes in body size within the Amphuimidae. Gigantic body size either evolved once as the ancestral condition of modern amphiumas, with a subsequent strong size reduction in A. pholeter, or gigantism independently evolved twice in the modern species, A. means and A. tridactylum. These patterns are concordant with differences in habitat breadth and range size among lineages, and have implications for reproductive isolation and diversification of amphiumid salamanders.

  13. Terrestrial salamander abundance on reclaimed mountaintop removal mines

    Science.gov (United States)

    Wood, Petra Bohall; Williams, Jennifer M.

    2013-01-01

    Mountaintop removal mining, a large-scale disturbance affecting vegetation, soil structure, and topography, converts landscapes from mature forests to extensive grassland and shrubland habitats. We sampled salamanders using drift-fence arrays and coverboard transects on and near mountaintop removal mines in southern West Virginia, USA, during 2000–2002. We compared terrestrial salamander relative abundance and species richness of un-mined, intact forest with habitats on reclaimed mountaintop removal mines (reclaimed grassland, reclaimed shrubland, and fragmented forest). Salamanders within forests increased in relative abundance with increasing distance from reclaimed mine edge. Reclaimed grassland and shrubland habitats had lower relative abundance and species richness than forests. Characteristics of reclaimed habitats that likely contributed to lower salamander abundance included poor soils (dry, compacted, little organic matter, high rock content), reduced vertical structure of vegetation and little tree cover, and low litter and woody debris cover. Past research has shown that salamander populations reduced by clearcutting may rebound in 15–24 years. Time since disturbance was 7–28 years in reclaimed habitats on our study areas and salamander populations had not reached levels found in adjacent mature forests.

  14. Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

    Science.gov (United States)

    Iqbal, Asif J; McNeill, Eileen; Kapellos, Theodore S; Regan-Komito, Daniel; Norman, Sophie; Burd, Sarah; Smart, Nicola; Machemer, Daniel E W; Stylianou, Elena; McShane, Helen; Channon, Keith M; Chawla, Ajay; Greaves, David R

    2014-10-09

    The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. © 2014 by The American Society of Hematology.

  15. Salamander occupancy in headwater stream networks

    Science.gov (United States)

    Grant, E.H.C.; Green, L.E.; Lowe, W.H.

    2009-01-01

    1. Stream ecosystems exhibit a highly consistent dendritic geometry in which linear habitat units intersect to create a hierarchical network of connected branches. 2. Ecological and life history traits of species living in streams, such as the potential for overland movement, may interact with this architecture to shape patterns of occupancy and response to disturbance. Specifically, large-scale habitat alteration that fragments stream networks and reduces connectivity may reduce the probability a stream is occupied by sensitive species, such as stream salamanders. 3. We collected habitat occupancy data on four species of stream salamanders in first-order (i.e. headwater) streams in undeveloped and urbanised regions of the eastern U.S.A. We then used an information-theoretic approach to test alternative models of salamander occupancy based on a priori predictions of the effects of network configuration, region and salamander life history. 4. Across all four species, we found that streams connected to other first-order streams had higher occupancy than those flowing directly into larger streams and rivers. For three of the four species, occupancy was lower in the urbanised region than in the undeveloped region. 5. These results demonstrate that the spatial configuration of stream networks within protected areas affects the occurrences of stream salamander species. We strongly encourage preservation of network connections between first-order streams in conservation planning and management decisions that may affect stream species.

  16. Effects of red-backed salamanders on ecosystem functions.

    Directory of Open Access Journals (Sweden)

    Daniel J Hocking

    Full Text Available Ecosystems provide a vast array of services for human societies, but understanding how various organisms contribute to the functions that maintain these services remains an important ecological challenge. Predators can affect ecosystem functions through a combination of top-down trophic cascades and bottom-up effects on nutrient dynamics. As the most abundant vertebrate predator in many eastern US forests, woodland salamanders (Plethodon spp. likely affect ecosystems functions. We examined the effects of red-backed salamanders (Plethodon cinereus on a variety of forest ecosystem functions using a combined approach of large-scale salamander removals (314-m(2 plots and small-scale enclosures (2 m(2 where we explicitly manipulated salamander density (0, 0.5, 1, 2, 4 m(-2. In these experiments, we measured the rates of litter and wood decomposition, potential nitrogen mineralization and nitrification rates, acorn germination, and foliar insect damage on red oak seedlings. Across both experimental venues, we found no significant effect of red-backed salamanders on any of the ecosystem functions. We also found no effect of salamanders on intraguild predator abundance (carabid beetles, centipedes, spiders. Our study adds to the already conflicting evidence on effects of red-backed salamander and other amphibians on terrestrial ecosystem functions. It appears likely that the impact of terrestrial amphibians on ecosystem functions is context dependent. Future research would benefit from explicitly examining terrestrial amphibian effects on ecosystem functions under a variety of environmental conditions and in different forest types.

  17. Effects of red-backed salamanders on ecosystem functions.

    Science.gov (United States)

    Hocking, Daniel J; Babbitt, Kimberly J

    2014-01-01

    Ecosystems provide a vast array of services for human societies, but understanding how various organisms contribute to the functions that maintain these services remains an important ecological challenge. Predators can affect ecosystem functions through a combination of top-down trophic cascades and bottom-up effects on nutrient dynamics. As the most abundant vertebrate predator in many eastern US forests, woodland salamanders (Plethodon spp.) likely affect ecosystems functions. We examined the effects of red-backed salamanders (Plethodon cinereus) on a variety of forest ecosystem functions using a combined approach of large-scale salamander removals (314-m(2) plots) and small-scale enclosures (2 m(2)) where we explicitly manipulated salamander density (0, 0.5, 1, 2, 4 m(-2)). In these experiments, we measured the rates of litter and wood decomposition, potential nitrogen mineralization and nitrification rates, acorn germination, and foliar insect damage on red oak seedlings. Across both experimental venues, we found no significant effect of red-backed salamanders on any of the ecosystem functions. We also found no effect of salamanders on intraguild predator abundance (carabid beetles, centipedes, spiders). Our study adds to the already conflicting evidence on effects of red-backed salamander and other amphibians on terrestrial ecosystem functions. It appears likely that the impact of terrestrial amphibians on ecosystem functions is context dependent. Future research would benefit from explicitly examining terrestrial amphibian effects on ecosystem functions under a variety of environmental conditions and in different forest types.

  18. Isolation of progenitor cells from GFP-transgenic pigs and transplantation to the retina of allorecipients

    DEFF Research Database (Denmark)

    Klassen, Henry; Warfvinge, Karin; Schwartz, Philip H

    2008-01-01

    to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower...... in conjunction with photoreceptor markers and glial fibrillary acid protein (GFAP), thus suggesting downregulation of GFP during differentiation. Following transplantation, GFP expression allowed histological visualization of integrated cells and extension of fine processes to adjacent plexiform layers. GFP...

  19. Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2000-01-01

    Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

  20. Long bone histology of the stem salamander Kokartus honorarius (Amphibia: Caudata) from the Middle Jurassic of Kyrgyzstan.

    Science.gov (United States)

    Skutschas, Pavel; Stein, Koen

    2015-04-01

    Kokartus honorarius from the Middle Jurassic (Bathonian) of Kyrgyzstan is one of the oldest salamanders in the fossil record, characterized by a mixture of plesiomorphic morphological features and characters shared with crown-group salamanders. Here we present a detailed histological analysis of its long bones. The analysis of a growth series demonstrates a significant histological maturation during ontogeny, expressed by the progressive appearance of longitudinally oriented primary vascular canals, primary osteons, growth marks, remodelling features in primary bone tissues, as well as progressive resorption of the calcified cartilage, formation of endochondral bone and development of cartilaginous to bony trabeculae in the epiphyses. Apart from the presence of secondary osteons, the long bone histology of Kokartus is very similar to that of miniaturized temnospondyls, other Jurassic stem salamanders, miniaturized seymouriamorphs and modern crown-group salamanders. We propose that the presence of secondary osteons in Kokartus honorarius is a plesiomorphic feature, and the loss of secondary osteons in the long bones of crown-group salamanders as well as in those of miniaturized temnospondyls is the result of miniaturization processes. Hitherto, all stem salamander long bong histology (Kokartus, Marmorerpeton and 'salamander A') has been generally described as having paedomorphic features (i.e. the presence of Katschenko's Line and a layer of calcified cartilage), these taxa were thus most likely neotenic forms. The absence of clear lines of arrested growth and annuli in long bones of Kokartus honorarius suggests that the animals lived in an environment with stable local conditions. © 2015 Anatomical Society.

  1. Bromeliad Selection by Two Salamander Species in a Harsh Environment

    Science.gov (United States)

    Ruano-Fajardo, Gustavo; Rovito, Sean M.; Ladle, Richard J.

    2014-01-01

    Bromeliad phytotelmata are frequently used by several Neotropical amphibian taxa, possibly due to their high humidity, microclimatic stability, and role as a refuge from predators. Indeed, the ability of phytotelmata to buffer against adverse environmental conditions may be instrumental in allowing some amphibian species to survive during periods of environmental change or to colonize sub-optimal habitats. Association between bromeliad traits and salamanders has not been studied at a fine scale, despite the intimate association of many salamander species with bromeliads. Here, we identify microhabitat characteristics of epiphytic bromeliads used by two species of the Bolitoglossa morio group (B. morio and B. pacaya) in forest disturbed by volcanic activity in Guatemala. Specifically, we measured multiple variables for bromeliads (height and position in tree, phytotelma water temperature and pH, canopy cover, phytotelma size, leaf size, and tree diameter at breast height), as well as salamander size. We employed a DNA barcoding approach to identify salamanders. We found that B. morio and B. pacaya occurred in microsympatry in bromeliads and that phytotelmata size and temperature of bromeliad microhabitat were the most important factors associated with the presence of salamanders. Moreover, phytotelmata with higher pH contained larger salamanders, suggesting that larger salamanders or aggregated individuals might modify pH. These results show that bromeliad selection is nonrandom with respect to microhabitat characteristics, and provide insight into the relationship between salamanders and this unique arboreal environment. PMID:24892414

  2. Bromeliad selection by two salamander species in a harsh environment.

    Directory of Open Access Journals (Sweden)

    Gustavo Ruano-Fajardo

    Full Text Available Bromeliad phytotelmata are frequently used by several Neotropical amphibian taxa, possibly due to their high humidity, microclimatic stability, and role as a refuge from predators. Indeed, the ability of phytotelmata to buffer against adverse environmental conditions may be instrumental in allowing some amphibian species to survive during periods of environmental change or to colonize sub-optimal habitats. Association between bromeliad traits and salamanders has not been studied at a fine scale, despite the intimate association of many salamander species with bromeliads. Here, we identify microhabitat characteristics of epiphytic bromeliads used by two species of the Bolitoglossa morio group (B. morio and B. pacaya in forest disturbed by volcanic activity in Guatemala. Specifically, we measured multiple variables for bromeliads (height and position in tree, phytotelma water temperature and pH, canopy cover, phytotelma size, leaf size, and tree diameter at breast height, as well as salamander size. We employed a DNA barcoding approach to identify salamanders. We found that B. morio and B. pacaya occurred in microsympatry in bromeliads and that phytotelmata size and temperature of bromeliad microhabitat were the most important factors associated with the presence of salamanders. Moreover, phytotelmata with higher pH contained larger salamanders, suggesting that larger salamanders or aggregated individuals might modify pH. These results show that bromeliad selection is nonrandom with respect to microhabitat characteristics, and provide insight into the relationship between salamanders and this unique arboreal environment.

  3. Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

    Science.gov (United States)

    Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

    2017-08-01

    Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.

  4. eGFP expression under the Uchl1 promoter labels corticospinal motor neurons and a subpopulation of degeneration resistant spinal motor neurons in ALS mouse models

    Science.gov (United States)

    Yasvoina, Marina V.

    Current understanding of basic cellular and molecular mechanisms for motor neuron vulnerability during motor neuron disease initiation and progression is incomplete. The complex cytoarchitecture and cellular heterogeneity of the cortex and spinal cord greatly impedes our ability to visualize, isolate, and study specific neuron populations in both healthy and diseased states. We generated a novel reporter line, the Uchl1-eGFP mouse, in which cortical and spinal components of motor neuron circuitry are genetically labeled with eGFP under the Uchl1 promoter. A series of cellular and anatomical analyses combined with retrograde labeling, molecular marker expression, and electrophysiology were employed to determine identity of eGFP expressing cells in the motor cortex and the spinal cord of novel Uchl1-eGFP reporter mice. We conclude that eGFP is expressed in corticospinal motor neurons (CSMN) in the motor cortex and a subset of S-type alpha and gamma spinal motor neurons (SMN) in the spinal cord. hSOD1G93A and Alsin-/- mice, mouse models for amyotrophic lateral sclerosis (ALS), were bred to Uchl1-eGFP reporter mouse line to investigate the pathophysiology and underlying mechanisms of CSMN degeneration in vivo. Evidence suggests early and progressive degeneration of CSMN and SMN in the hSOD1G93A transgenic mice. We show an early increase of autophagosome formation in the apical dendrites of vulnerable CSMN in hSOD1G93A-UeGFP mice, which is localized to the apical dendrites. In addition, labeling S-type alpha and gamma SMN in the hSOD1G93A-UeGFP mice provide a unique opportunity to study basis of their resistance to degeneration. Mice lacking alsin show moderate clinical phenotype and mild CSMN axon degeneration in the spinal cord, which suggests vulnerability of CSMN. Therefore, we investigated the CSMN cellular and axon defects in aged Alsin-/- mice bred to Uchl1-eGFP reporter mouse line. We show that while CSMN are preserved and lack signs of degeneration, CSMN axons

  5. Impact of valley fills on streamside salamanders in southern West Virginia

    Science.gov (United States)

    Wood, Petra Bohall; Williams, Jennifer M.

    2013-01-01

    Valley fills associated with mountaintop-removal mining bury stream headwaters and affect water quality and ecological function of reaches below fills. We quantified relative abundance of streamside salamanders in southern West Virginia during 2002 in three streams below valley fills (VFS) and in three reference streams (RS). We surveyed 36 10- × 2-m stream transects, once in summer and fall, paired by order and structure. Of 2,343 salamanders captured, 66.7% were from RS. Total salamanders (adults plus larvae) were more abundant in RS than VFS for first-order and second-order reaches. Adult salamanders had greater abundance in first-order reaches of RS than VFS. Larval salamanders were more abundant in second-order reaches of RS than VFS. No stream width or mesohabitat variables differed between VFS and RS. Only two cover variables differed. Silt cover, greater in VFS than RS first-order reaches, is a likely contributor to reduced abundance of salamanders in VFS. Second-order RS had more boulder cover than second-order VFS, which may have contributed to the higher total and larval salamander abundance in RS. Water chemistry assessments of our VFS and RS reported elevated levels of metal and ion concentrations in VFS, which can depress macroinvertebrate populations and likely affect salamander abundance. Valley fills appear to have significant negative effects on stream salamander abundance due to alterations in habitat structure, water quality and chemistry, and macroinvertebrate communities in streams below fills.

  6. Environmental and Anthropogenic Factors Influencing Salamanders in Riparian Forests: A Review

    Directory of Open Access Journals (Sweden)

    Hannah L. Clipp

    2014-11-01

    Full Text Available Salamanders and riparian forests are intimately interconnected. Salamanders are integral to ecosystem functions, contributing to vertebrate biomass and complex food webs in riparian forests. In turn, these forests are critical ecosystems that perform many environmental services, facilitate high biodiversity and species richness, and provide habitat to salamander populations. Due to the global decline of amphibians, it is important to understand, as thoroughly and holistically as possible, the roles of environmental parameters and the impact of human activities on salamander abundance and diversity in riparian forests. To determine the population responses of salamanders to a variety of environmental factors and anthropogenic activities, we conducted a review of published literature that compared salamander abundance and diversity, and then summarized and synthesized the data into general patterns. We identify stream quality, leaf litter and woody debris, riparian buffer width, and soil characteristics as major environmental factors influencing salamander populations in riparian forests, describe and explain salamander responses to those factors, and discuss the effects of anthropogenic activities such as timber harvest, prescribed fires, urbanization, road construction, and habitat fragmentation. This review can assist land and natural resource managers in anticipating the consequences of human activities and preparing strategic conservation plans.

  7. Ontogenetic evidence for the Paleozoic ancestry of salamanders.

    Science.gov (United States)

    Schoch, Rainer R; Carroll, Robert L

    2003-01-01

    The phylogenetic positions of frogs, salamanders, and caecilians have been difficult to establish. Data matrices based primarily on Paleozoic taxa support a monophyletic origin of all Lissamphibia but have resulted in widely divergent hypotheses of the nature of their common ancestor. Analysis that concentrates on the character states of the stem taxa of the extant orders, in contrast, suggests a polyphyletic origin from divergent Paleozoic clades. Comparison of patterns of larval development in Paleozoic and modern amphibians provides a means to test previous phylogenies based primarily on adult characteristics. This proves to be highly informative in the case of the origin of salamanders. Putative ancestors of salamanders are recognized from the Permo-Carboniferous boundary of Germany on the basis of ontogenetic changes observed in fossil remains of larval growth series. The entire developmental sequence from hatching to metamorphosis is revealed in an assemblage of over 600 specimens from a single locality, all belonging to the genus Apateon. Apateon forms the most speciose genus of the neotenic temnospondyl family Branchiosauridae. The sequence of ossification of individual bones and the changing configuration of the skull closely parallel those observed in the development of primitive living salamanders. These fossils provide a model of how derived features of the salamander skull may have evolved in the context of feeding specializations that appeared in early larval stages of members of the Branchiosauridae. Larvae of Apateon share many unique derived characters with salamanders of the families Hynobiidae, Salamandridae, and Ambystomatidae, which have not been recognized in any other group of Paleozoic amphibians.

  8. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    Science.gov (United States)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  9. Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium

    NARCIS (Netherlands)

    Franz, Eelco; Visser, Anna A; Van Diepeningen, Anne D; Klerks, Michel M; Termorshuizen, Aad J; van Bruggen, Ariena H C

    The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca

  10. Tiger salamanders' (Ambystoma tigrinum) response learning and usage of visual cues.

    Science.gov (United States)

    Kundey, Shannon M A; Millar, Roberto; McPherson, Justin; Gonzalez, Maya; Fitz, Aleyna; Allen, Chadbourne

    2016-05-01

    We explored tiger salamanders' (Ambystoma tigrinum) learning to execute a response within a maze as proximal visual cue conditions varied. In Experiment 1, salamanders learned to turn consistently in a T-maze for reinforcement before the maze was rotated. All learned the initial task and executed the trained turn during test, suggesting that they learned to demonstrate the reinforced response during training and continued to perform it during test. In a second experiment utilizing a similar procedure, two visual cues were placed consistently at the maze junction. Salamanders were reinforced for turning towards one cue. Cue placement was reversed during test. All learned the initial task, but executed the trained turn rather than turning towards the visual cue during test, evidencing response learning. In Experiment 3, we investigated whether a compound visual cue could control salamanders' behaviour when it was the only cue predictive of reinforcement in a cross-maze by varying start position and cue placement. All learned to turn in the direction indicated by the compound visual cue, indicating that visual cues can come to control their behaviour. Following training, testing revealed that salamanders attended to stimuli foreground over background features. Overall, these results suggest that salamanders learn to execute responses over learning to use visual cues but can use visual cues if required. Our success with this paradigm offers the potential in future studies to explore salamanders' cognition further, as well as to shed light on how features of the tiger salamanders' life history (e.g. hibernation and metamorphosis) impact cognition.

  11. Excited state proton transfer in strongly enhanced GFP (sGFP2).

    Science.gov (United States)

    van Oort, Bart; ter Veer, Mirelle J T; Groot, Marie Louise; van Stokkum, Ivo H M

    2012-07-07

    Proton transfer is an elementary process in biology. Green fluorescent protein (GFP) has served as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. We have used pump-dump-probe spectroscopy to study how proton transfer through the 'proton-wire' around the chromophore is affected by a combination of mutations in a modern GFP variety (sGFP2). The results indicate that in H(2)O, after absorption of a photon, a proton is transferred (A* → I*) in 5 ps, and back-transferred from a ground state intermediate (I → A) in 0.3 ns, similar to time constants found with GFPuv, although sGFP2 shows less heterogeneous proton transfer. This suggests that the mutations left the proton-transfer largely unchanged, indicating the robustness of the proton-wire. We used pump-dump-probe spectroscopy in combination with target analysis to probe suitability of the sGFP2 fluorophore for super-resolution microscopy.

  12. Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum

    Directory of Open Access Journals (Sweden)

    Vahid NASIRI

    2016-10-01

    Full Text Available Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development. 

  13. A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis.

    Science.gov (United States)

    Song, Kai; Xue, Yiqun; Wang, Xiaohua; Wan, Yinglang; Deng, Xin; Lin, Jinxing

    2017-06-01

    Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFP A206K driven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFP A206K line was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFP A206K can be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Effects of mercury on behavior and performance of northern two-lined salamanders (Eurycea bislineata)

    Energy Technology Data Exchange (ETDEWEB)

    Burke, John N; Bergeron, Christine M; Todd, Brian D [Wildlife Ecotoxicology and Physiological Ecology Program, Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, 100 Cheatham Hall, Blacksburg, VA 24061 (United States); Hopkins, William A., E-mail: hopkinsw@vt.ed [Wildlife Ecotoxicology and Physiological Ecology Program, Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, 100 Cheatham Hall, Blacksburg, VA 24061 (United States)

    2010-12-15

    Mercury (Hg) causes a range of deleterious effects in wildlife, but little is known about its effects on amphibians. Our objective was to determine whether Hg affects performance and behavior in two-lined salamanders (Eurycea bislineata). We collected salamanders from Hg-contaminated and reference sites and assessed speed, responsiveness, and prey capture ability. Mercury concentrations were >17x higher in salamanders from the contaminated sites and were among the highest documented in amphibians. In the first, but not in the second, locomotion trial, we found a significant effect of Hg on speed and responsiveness. In the prey capture experiment, reference salamanders ate approximately twice as many prey items as the contaminated salamanders. Together, our results suggest that sublethal Hg concentrations may negatively affect salamanders by reducing their ability to successfully execute tasks critical to survival. Future work is warranted to determine whether Hg has other sublethal effects on salamanders and whether other amphibians are similarly affected. - Mercury contamination may alter behavior and performance in the northern two-lined salamander (Eurycea bislineata).

  15. Effects of mercury on behavior and performance of northern two-lined salamanders (Eurycea bislineata)

    International Nuclear Information System (INIS)

    Burke, John N.; Bergeron, Christine M.; Todd, Brian D.; Hopkins, William A.

    2010-01-01

    Mercury (Hg) causes a range of deleterious effects in wildlife, but little is known about its effects on amphibians. Our objective was to determine whether Hg affects performance and behavior in two-lined salamanders (Eurycea bislineata). We collected salamanders from Hg-contaminated and reference sites and assessed speed, responsiveness, and prey capture ability. Mercury concentrations were >17x higher in salamanders from the contaminated sites and were among the highest documented in amphibians. In the first, but not in the second, locomotion trial, we found a significant effect of Hg on speed and responsiveness. In the prey capture experiment, reference salamanders ate approximately twice as many prey items as the contaminated salamanders. Together, our results suggest that sublethal Hg concentrations may negatively affect salamanders by reducing their ability to successfully execute tasks critical to survival. Future work is warranted to determine whether Hg has other sublethal effects on salamanders and whether other amphibians are similarly affected. - Mercury contamination may alter behavior and performance in the northern two-lined salamander (Eurycea bislineata).

  16. Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events

    Science.gov (United States)

    Docherty, D.E.; Meteyer, C.U.; Wang, Jingyuan; Mao, J.; Case, S.T.; Chinchar, V.G.

    2003-01-01

    In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to

  17. Better than fish on land? Hearing across metamorphosis in salamanders

    DEFF Research Database (Denmark)

    Christensen, Christian Bech; Lauridsen, Henrik; Christensen-Dalsgaard, Jakob

    2015-01-01

    hearing in early tetrapods. Here, we combine imaging techniques with neurophysiological measurements to resolve how the change from aquatic larvae to terrestrial adult affects the ear morphology and sensory capabilities of salamanders. We show that air-induced pressure detection enhances underwater...... hearing sensitivity of salamanders at frequencies above 120 Hz, and that both terrestrial adults and fully aquatic juvenile salamanders can detect airborne sound. Collectively, these findings suggest that early atympanic tetrapods may have been pre-equipped to aerial hearing and are able to hear airborne...

  18. Evolution of coprophagy and nutrient absorption in a Cave Salamander

    Directory of Open Access Journals (Sweden)

    Daphne Soares

    2017-11-01

    Full Text Available The transition from carnivory to omnivory is poorly understood. The ability to feed at more than one trophic level theoretically increases an animal’s fitness in a novel environment. Because of the absence of light and photosynthesis, most subterranean ecosystems are characterized by very few trophic levels, such that food scarcity is a challenge in many subterranean habitats. One strategy against starvation is to expand diet breadth. Grotto Salamanders (Eurycea spelaea (Stejneger, 1892 are known to ingest bat guano deliberately, challenging the general understanding that salamanders are strictly carnivorous. Here we tested the hypothesis that grotto salamanders have broadened their diet related to cave adaptation and found that, although coprophagous behavior is present, salamanders are unable to acquire sufficient nutrition from bat guano alone. Our results suggest that the coprophagic behavior has emerged prior to physiological or gut biome adaptations.

  19. Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum.

    Science.gov (United States)

    Page, Robert B; Monaghan, James R; Samuels, Amy K; Smith, Jeramiah J; Beachy, Christopher K; Voss, S Randal

    2007-02-01

    Ambystomatid salamanders offer several advantages for endocrine disruption research, including genomic and bioinformatics resources, an accessible laboratory model (Ambystoma mexicanum), and natural lineages that are broadly distributed among North American habitats. We used microarray analysis to measure the relative abundance of transcripts isolated from A. mexicanum epidermis (skin) after exogenous application of thyroid hormone (TH). Only one gene had a >2-fold change in transcript abundance after 2 days of TH treatment. However, hundreds of genes showed significantly different transcript levels at days 12 and 28 in comparison to day 0. A list of 123 TH-responsive genes was identified using statistical, BLAST, and fold level criteria. Cluster analysis identified two groups of genes with similar transcription patterns: up-regulated versus down-regulated. Most notably, several keratins exhibited dramatic (1000 fold) increases or decreases in transcript abundance. Keratin gene expression changes coincided with morphological remodeling of epithelial tissues. This suggests that keratin loci can be developed as sensitive biomarkers to assay temporal disruptions of larval-to-adult gene expression programs. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander, thus setting the stage for future investigations of TH disruption in the Mexican axolotl and other salamanders of the genus Ambystoma.

  20. Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization

    DEFF Research Database (Denmark)

    Xu, Wen-Ming; Zhang, Si-Zhong; Qiu, Wei-Min

    2004-01-01

    To use green fluorescent protein as a marker to study the localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification, and then cloned into pGEM-Teasy vector. After the double enzyme...... cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1. Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP). Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed...

  1. Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis

    Directory of Open Access Journals (Sweden)

    Bailey Kirra

    2010-11-01

    Full Text Available Abstract Background Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green and mCherry (red. Sporulation in the Gram positive bacterium Bacillus subtilis has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns. Findings While observing the recruitment of the transcription machinery into the forespore of sporulating Bacillus subtilis, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores. Conclusions Great care should be taken

  2. Stream water temperature limits occupancy of salamanders in mid-Atlantic protected areas

    Science.gov (United States)

    Grant, Evan H. Campbell; Wiewel, Amber N. M.; Rice, Karen C.

    2014-01-01

    Stream ecosystems are particularly sensitive to urbanization, and tolerance of water-quality parameters is likely important to population persistence of stream salamanders. Forecasted climate and landscape changes may lead to significant changes in stream flow, chemical composition, and temperatures in coming decades. Protected areas where landscape alterations are minimized will therefore become increasingly important for salamander populations. We surveyed 29 streams at three national parks in the highly urbanized greater metropolitan area of Washington, DC. We investigated relationships among water-quality variables and occupancy of three species of stream salamanders (Desmognathus fuscus, Eurycea bislineata, and Pseudotriton ruber). With the use of a set of site-occupancy models, and accounting for imperfect detection, we found that stream-water temperature limits salamander occupancy. There was substantial uncertainty about the effects of the other water-quality variables, although both specific conductance (SC) and pH were included in competitive models. Our estimates of occupancy suggest that temperature, SC, and pH have some importance in structuring stream salamander distribution.

  3. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

    Science.gov (United States)

    Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

    2012-12-01

    Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Pancreatic differentiation of Pdx1-GFP reporter mouse induced pluripotent stem cells.

    Science.gov (United States)

    Porciuncula, Angelo; Kumar, Anujith; Rodriguez, Saray; Atari, Maher; Araña, Miriam; Martin, Franz; Soria, Bernat; Prosper, Felipe; Verfaillie, Catherine; Barajas, Miguel

    2016-12-01

    Efficient induction of defined lineages in pluripotent stem cells constitutes the determinant step for the generation of therapeutically relevant replacement cells to potentially treat a wide range of diseases, including diabetes. Pancreatic differentiation has remained an important challenge in large part because of the need to differentiate uncommitted pluripotent stem cells into highly specialized hormone-secreting cells, which has been shown to require a developmentally informed step-by-step induction procedure. Here, in the framework of using induced pluripotent stem cells (iPSCs) to generate pancreatic cells for pancreatic diseases, we have generated and characterized iPSCs from Pdx1-GFP transgenic mice. The use of a GFP reporter knocked into the endogenous Pdx1 promoter allowed us to monitor pancreatic induction based on the expression of Pdx1, a pancreatic master transcription factor, and to isolate a pure Pdx1-GFP + population for downstream applications. Differentiated cultures timely expressed markers specific to each stage and end-stage progenies acquired a rather immature beta-cell phenotype, characterized by polyhormonal expression even among cells highly expressing the Pdx1-GFP reporter. Our findings highlight the utility of employing a fluorescent protein reporter under the control of a master developmental gene in order to devise novel differentiation protocols for relevant cell types for degenerative diseases such as pancreatic beta cells for diabetes. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  5. Woodland salamander responses to a shelterwood harvest-prescribed burn silvicultural treatment within Appalachian mixed-oak forests

    Science.gov (United States)

    Ford, W. Mark; Mahoney, Kathleen R.; Russell, Kevin R.; Rodrigue, Jane L.; Riddle, Jason D.; Schuler, Thomas M.; Adams, Mary Beth

    2015-01-01

    Forest management practices that mimic natural canopy disturbances, including prescribed fire and timber harvests, may reduce competition and facilitate establishment of favorable vegetative species within various ecosystems. Fire suppression in the central Appalachian region for almost a century has contributed to a transition from oak-dominated to more mesophytic, fire-intolerant forest communities. Prescribed fire coupled with timber removal is currently implemented to aid in oak regeneration and establishment but responses of woodland salamanders to this complex silvicultural system is poorly documented. The purpose of our research was to determine how woodland salamanders respond to shelterwood harvests following successive burns in a central Appalachian mixed-oak forest. Woodland salamanders were surveyed using coverboard arrays in May, July, and August–September 2011 and 2012. Surveys were conducted within fenced shelterwood-burn (prescribed fires, shelterwood harvest, and fencing to prevent white-tailed deer [Odocoileus virginianus] herbivory), shelterwood-burn (prescribed fires and shelterwood harvest), and control plots. Relative abundance was modeled in relation to habitat variables measured within treatments for mountain dusky salamanders (Desmognathus ochrophaeus), slimy salamanders (Plethodon glutinosus), and eastern red-backed salamanders (Plethodon cinereus). Mountain dusky salamander relative abundance was positively associated with canopy cover and there were significantly more individuals within controls than either shelterwood-burn or fenced shelterwood-burn treatments. Conversely, habitat variables associated with slimy salamanders and eastern red-backed salamanders did not differ among treatments. Salamander age-class structure within controls did not differ from shelterwood-burn or fenced shelterwood-burn treatments for any species. Overall, the woodland salamander assemblage remained relatively intact throughout the shelterwoodburn

  6. Effects of host species and environment on the skin microbiome of Plethodontid salamanders

    Science.gov (United States)

    Muletz-Wolz, Carly R.; Yarwood, Stephanie A.; Grant, Evan H. Campbell; Fleischer, Robert C.; Lips, Karen R.

    2018-01-01

    The amphibian skin microbiome is recognized for its role in defence against pathogens, including the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). Yet, we have little understanding of evolutionary and ecological processes that structure these communities, especially for salamanders and closely related species. We investigated patterns in the distribution of bacterial communities on Plethodon salamander skin across host species and environments.Quantifying salamander skin microbiome structure contributes to our understanding of how host-associated bacteria are distributed across the landscape, among host species, and their putative relationship with disease.We characterized skin microbiome structure (alpha-diversity, beta-diversity and bacterial operational taxonomic unit [OTU] abundances) using 16S rRNA gene sequencing for co-occurring Plethodon salamander species (35 Plethodon cinereus, 17 Plethodon glutinosus, 10 Plethodon cylindraceus) at three localities to differentiate the effects of host species from environmental factors on the microbiome. We sampled the microbiome of P. cinereus along an elevational gradient (n = 50, 700–1,000 m a.s.l.) at one locality to determine whether elevation predicts microbiome structure. Finally, we quantified prevalence and abundance of putatively anti-Bd bacteria to determine if Bd-inhibitory bacteria are dominant microbiome members.Co-occurring salamanders had similar microbiome structure, but among sites salamanders had dissimilar microbiome structure for beta-diversity and abundance of 28 bacterial OTUs. We found that alpha-diversity increased with elevation, beta-diversity and the abundance of 17 bacterial OTUs changed with elevation (16 OTUs decreasing, 1 OTU increasing). We detected 11 putatively anti-Bd bacterial OTUs that were present on 90% of salamanders and made up an average relative abundance of 83% (SD ± 8.5) per salamander. All salamanders tested negative for Bd.We conclude that

  7. Excited state proton transfer in strongly enhanced GFP (sGFP2)

    NARCIS (Netherlands)

    van Oort, B.F.; ter Veer, M.J.T.; Groot, M.L.; van Stokkum, I.H.M.

    2012-01-01

    Proton transfer is an elementary process in biology. Green fluorescent protein (GFP) has served as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. We have used pump-dump-probe spectroscopy to study

  8. Environmental influences on egg and clutch sizes in lentic- and lotic-breeding salamanders

    Directory of Open Access Journals (Sweden)

    Jon M. Davenport

    2010-12-01

    Full Text Available Recent research indicates that social and environmental factors influence egg and clutch sizes in amphibians. However, most of this work is based on the reproductively diverse order Anura (frogs and toads, whereas less research has been conducted on Caudata (salamanders and Gymnophiona (caecilians. Researchers have suggested that a relationship exists between social and environmental factors and egg and clutch sizes in salamanders, but studies controlling for phylogenetic context are lacking. We could not identify a sufficient number of comparisons for social influences on egg and clutch sizes; therefore, we focused on environmental influences for this study. Data on egg size, clutch size, environmental factors, and phylogenies for salamanders were assembled from the scientific literature. We used independent, pair-wise comparisons to investigate the association of larval salamander habitat and egg size and the association of larval salamander habitat with clutch sizes within a phylogenetic framework. There is a significant association between larval habitat and egg size; specifically, stream-breeding species produce larger eggs. There is no significant association between larval habitat and clutchsize. Our study confirms earlier reports that salamander egg size is associated with larval environments, but is the first to use phylogenetically independent contrasts to account for the lack of phylogenetic independence of the traits measured (egg size and clutch size associated with many of the diverse lineages. Our study shows that environmental selection pressure can be quite strong on one aspect of salamander reproduction—egg size.

  9. The hTH-GFP reporter rat model for the study of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Lorraine Iacovitti

    Full Text Available Parkinson disease (PD is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH-expressing dopaminergic (DA neurons of the substantia nigra pars compacta (SNpc. These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus. As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson's disease research.

  10. Seasonality and microhabitat selection in a forest-dwelling salamander

    Science.gov (United States)

    Basile, Marco; Romano, Antonio; Costa, Andrea; Posillico, Mario; Scinti Roger, Daniele; Crisci, Aldo; Raimondi, Ranieri; Altea, Tiziana; Garfì, Vittorio; Santopuoli, Giovanni; Marchetti, Marco; Salvidio, Sebastiano; De Cinti, Bruno; Matteucci, Giorgio

    2017-10-01

    Many small terrestrial vertebrates exhibit limited spatial movement and are considerably exposed to changes in local environmental variables. Among such vertebrates, amphibians at present experience a dramatic decline due to their limited resilience to environmental change. Since the local survival and abundance of amphibians is intrinsically related to the availability of shelters, conservation plans need to take microhabitat requirements into account. In order to gain insight into the terrestrial ecology of the spectacled salamander Salamandrina perspicillata and to identify appropriate forest management strategies, we investigated the salamander's seasonal variability in habitat use of trees as shelters in relation to tree features (size, buttresses, basal holes) and environmental variables in a beech forest in Italy. We used the occupancy approach to assess tree suitability on a non-conventional spatial scale. Our approach provides fine-grained parameters of microhabitat suitability and elucidates many aspects of the salamander's terrestrial ecology . Occupancy changed with the annual life cycle and was higher in autumn than in spring, when females were found closer to the stream in the study area. Salamanders showed a seasonal pattern regarding the trees they occupied and a clear preference for trees with a larger diameter and more burrows. With respect to forest management, we suggest maintaining a suitable number of trees with a trunk diameter exceeding 30 cm. A practice of selective logging along the banks of streams could help maintain an adequate quantity of the appropriate microhabitat. Furthermore, in areas with a presence of salamanders, a good forest management plan requires leaving an adequate buffer zone around streams, which should be wider in autumn than in spring.

  11. 77 FR 36287 - Proposed Low-Effect Habitat Conservation Plan for the California Tiger Salamander, Calaveras...

    Science.gov (United States)

    2012-06-18

    ...-FXES11120800000F2-123-F2] Proposed Low-Effect Habitat Conservation Plan for the California Tiger Salamander... animal, the threatened Central California Distinct Population Segment of the California tiger salamander (tiger salamander). The applicant would implement a conservation program to minimize and mitigate the...

  12. The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

    Science.gov (United States)

    Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

    2008-12-01

    Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

  13. A nondestructive technique to monitor the relative abundance of terrestrial salamanders

    Science.gov (United States)

    Richard M. DeGraaf; Mariko. Yamasaki

    1992-01-01

    Salamanders are abundant vertebrates in many forest ecosystems, and their annual biomass production can be important in forest food webs (Pough et al. 1987). Population densities of eastern redback salamanders (Plethodon cinereus) can exceed 2 individuals/m2 in deciduous forests of the United States (Heatwole 1962, Jaeger 1980...

  14. Effects of microhabitat and land use on stream salamander abundance in the southwest Virginia coalfields

    Science.gov (United States)

    Sweeten, Sara E.; Ford, W. Mark

    2015-01-01

    Large-scale land uses such as residential wastewater discharge and coal mining practices, particularly surface coal extraction and associated valley fills, are of particular ecological concern in central Appalachia. Identification and quantification of both alterations across scales are a necessary first-step to mitigate negative consequences to biota. In central Appalachian headwater streams absent of fish, salamanders are the dominant, most abundant vertebrate predator providing a significant intermediate trophic role. Stream salamander species are considered to be sensitive to aquatic stressors and environmental alterations, and past research has shown linkages among microhabitat parameters, large-scale land use such as urbanization and logging with salamander abundances. However, little is known about these linkages in the coalfields of central Appalachia. In the summer of 2013, we visited 70 sites (sampled three times each) in the southwest Virginia coalfields to survey salamanders and quantify stream and riparian microhabitat parameters. Using an information-theoretic framework we compared the effects of microhabitat and large-scale land use on salamander abundances. Our findings indicate that dusky salamander (Desmognathus spp.) abundances are more correlated to microhabitat parameters such as canopy cover than to subwatershed land uses. Brook salamander (Eurycea spp.) abundances show strong negative associations to the suspended sediments and stream substrate embeddedness. Neither Desmognathus spp. nor Eurycea spp. abundances were influenced by water conductivity. These suggest protection or restoration of riparian habitats and erosion control is an important conservation component for maintaining stream salamanders in the mined landscapes of central Appalachia.

  15. Cannibalistic-morph Tiger Salamanders in unexpected ecological contexts

    Science.gov (United States)

    McLean, Kyle I.; Stockwell, Craig A.; Mushet, David M.

    2016-01-01

    Barred tiger salamanders [Ambystoma mavortium (Baird, 1850)] exhibit two trophic morphologies; a typical and a cannibalistic morph. Cannibalistic morphs, distinguished by enlarged vomerine teeth, wide heads, slender bodies, and cannibalistic tendencies, are often found where conspecifics occur at high density. During 2012 and 2013, 162 North Dakota wetlands and lakes were sampled for salamanders. Fifty-one contained A. mavortium populations; four of these contained cannibalistic morph individuals. Two populations with cannibalistic morphs occurred at sites with high abundances of conspecifics. However, the other two populations occurred at sites with unexpectedly low conspecific but high fathead minnow [Pimephales promelas (Rafinesque, 1820)] abundances. Further, no typical morphs were observed in either of these later two populations, contrasting with earlier research suggesting cannibalistic morphs only occur at low frequencies in salamander populations. Another anomaly of all four populations was the occurrence of cannibalistic morphs in permanent water sites, suggesting their presence was due to factors other than faster growth allowing them to occupy ephemeral habitats. Therefore, our findings suggest environmental factors inducing the cannibalistic morphism may be more complex than previously thought.

  16. Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.

    Science.gov (United States)

    Öster, Carl; Svensson Bonde, Johan; Bülow, Leif; Dicko, Cedric

    2014-04-01

    Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-silk fusion protein is predominantly α-helical, and that pH can trigger a α- to β-transition resulting in aggregation. Structural analysis by small angle X-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution. Copyright © 2013 Wiley Periodicals, Inc.

  17. Superparamagnetic iron oxide nanoparticle-labeled cells as an effective vehicle for tracking the GFP gene marker using magnetic resonance imaging

    Science.gov (United States)

    Zhang, Z; Mascheri, N; Dharmakumar, R; Fan, Z; Paunesku, T; Woloschak, G; Li, D

    2010-01-01

    Background Detection of a gene using magnetic resonance imaging (MRI) is hindered by the magnetic resonance (MR) targeting gene technique. Therefore it may be advantageous to image gene-expressing cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles by MRI. Methods The GFP-R3230Ac (GFP) cell line was incubated for 24 h using SPIO nanoparticles at a concentration of 20 μg Fe/mL. Cell samples were prepared for iron content analysis and cell function evaluation. The labeled cells were imaged using fluorescent microscopy and MRI. Results SPIO was used to label GFP cells effectively, with no effects on cell function and GFP expression. Iron-loaded GFP cells were successfully imaged with both fluorescent microscopy and T2*-weighted MRI. Prussian blue staining showed intracellular iron accumulation in the cells. All cells were labeled (100% labeling efficiency). The average iron content per cell was 4.75±0.11 pg Fe/cell (P<0.05 versus control). Discussion This study demonstrates that the GFP expression of cells is not altered by the SPIO labeling process. SPIO-labeled GFP cells can be visualized by MRI; therefore, GFP, a gene marker, was tracked indirectly with the SPIO-loaded cells using MRI. The technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker and enhancing the understanding of cell- and gene-based therapeutic strategies. PMID:18956269

  18. Mechanics of lung ventilation in a post-metamorphic salamander, Ambystoma Tigrinum.

    Science.gov (United States)

    Simons, R S; Bennett, W O; Brainerd, E L

    2000-03-01

    The mechanics of lung ventilation in frogs and aquatic salamanders has been well characterized, whereas lung ventilation in terrestrial-phase (post-metamorphic) salamanders has received little attention. We used electromyography (EMG), X-ray videography, standard videography and buccal and body cavity pressure measurements to characterize the ventilation mechanics of adult (post-metamorphic) tiger salamanders (Ambystoma tigrinum). Three results emerged: (i) under terrestrial conditions or when floating at the surface of the water, adult A. tigrinum breathed through their nares using a two-stroke buccal pump; (ii) in addition to this narial two-stroke pump, adult tiger salamanders also gulped air in through their mouths using a modified two-stroke buccal pump when in an aquatic environment; and (iii) exhalation in adult tiger salamanders is active during aquatic gulping breaths, whereas exhalation appears to be passive during terrestrial breathing at rest. Active exhalation in aquatic breaths is indicated by an increase in body cavity pressure during exhalation and associated EMG activity in the lateral hypaxial musculature, particularly the M. transversus abdominis. In terrestrial breathing, no EMG activity in the lateral hypaxial muscles is generally present, and body cavity pressure decreases during exhalation. In aquatic breaths, tidal volume is larger than in terrestrial breaths, and breathing frequency is much lower (approximately 1 breath 10 min(-)(1 )versus 4-6 breaths min(-)(1)). The use of hypaxial muscles to power active exhalation in the aquatic environment may result from the need for more complete exhalation and larger tidal volumes when breathing infrequently. This hypothesis is supported by previous findings that terrestrial frogs ventilate their lungs with small tidal volumes and exhale passively, whereas aquatic frogs and salamanders use large tidal volumes and and exhale actively.

  19. Genic regions of a large salamander genome contain long introns and novel genes

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 × 109 bp were isolated and sequenced to characterize the structure of genic regions. Results Annotation of genes within BACs showed that axolotl introns are on average 10× longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86% of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5× larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases! Conclusion This study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders.

  20. Detection of an enigmatic plethodontid Salamander using Environmental DNA

    Science.gov (United States)

    Pierson, Todd W.; Mckee, Anna; Spear, Stephen F.; Maerz, John C.; Camp, Carlos D.; Glenn, Travis C.

    2016-01-01

    The isolation and identification of environmental DNA (eDNA) offers a non-invasive and efficient method for the detection of rare and secretive aquatic wildlife, and it is being widely integrated into inventory and monitoring efforts. The Patch-Nosed Salamander (Urspelerpes brucei) is a tiny, recently discovered species of plethodontid salamander known only from headwater streams in a small region of Georgia and South Carolina. Here, we present results of a quantitative PCR-based eDNA assay capable of detecting Urspelerpes in more than 75% of 33 samples from five confirmed streams. We deployed the method at 31 additional streams and located three previously undocumented populations of Urspelerpes. We compare the results of our eDNA assay with our attempt to use aquatic leaf litterbags for the rapid detection of Urspelerpes and demonstrate the relative efficacy of the eDNA assay. We suggest that eDNA offers great potential for use in detecting other aquatic and semi-aquatic plethodontid salamanders.

  1. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  2. Slow Lives in the Fast Landscape: Conservation and Management of Plethodontid Salamanders in Production Forests of the United States

    Directory of Open Access Journals (Sweden)

    Jessica A. Homyack

    2014-11-01

    Full Text Available Intensively-managed forest (IMF ecosystems support environmental processes, retain biodiversity and reduce pressure to extract wood products from other forests, but may affect species, such as plethodontid salamanders, that are associated with closed canopies and possess limited vagility. We describe: (1 critical aspects of IMF ecosystems; (2 effectiveness of plethodontid salamanders as barometers of forest change; (3 two case studies of relationships between salamanders and coarse woody debris (CWD; and (4 research needs for effective management of salamanders in IMF ecosystems. Although plethodontid salamanders are sensitive to microclimate changes, their role as ecological indicators rarely have been evaluated quantitatively. Our case studies of CWD and salamanders in western and eastern forests demonstrated effects of species, region and spatial scale on the existence and strength of relationships between plethodontid species and a “critical” microhabitat variable. Oregon slender salamanders (Batrachoseps wrighti were more strongly associated with abundance of CWD in managed second growth forests than ensatina salamanders (Ensatina eschscholtzii. Similarly, CWD was not an important predictor of abundance of Appalachian salamanders in managed hardwood forest. Gaining knowledge of salamanders in IMF ecosystems is critical to reconciling ecological and economic objectives of intensive forest management, but faces challenges in design and implementation.

  3. Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability.

    Science.gov (United States)

    Muletz-Wolz, Carly R; DiRenzo, Graziella V; Yarwood, Stephanie A; Campbell Grant, Evan H; Fleischer, Robert C; Lips, Karen R

    2017-05-01

    Diverse bacteria inhabit amphibian skin; some of those bacteria inhibit growth of the fungal pathogen Batrachochytrium dendrobatidis Yet there has been no systematic survey of anti- B. dendrobatidis bacteria across localities, species, and elevations. This is important given geographic and taxonomic variations in amphibian susceptibility to B. dendrobatidis Our collection sites were at locations within the Appalachian Mountains where previous sampling had indicated low B. dendrobatidis prevalence. We determined the numbers and identities of anti- B. dendrobatidis bacteria on 61 Plethodon salamanders (37 P. cinereus , 15 P. glutinosus , 9 P. cylindraceus ) via culturing methods and 16S rRNA gene sequencing. We sampled co-occurring species at three localities and sampled P. cinereus along an elevational gradient (700 to 1,000 meters above sea level [masl]) at one locality. We identified 50 anti- B. dendrobatidis bacterial operational taxonomic units (OTUs) and found that the degree of B. dendrobatidis inhibition was not correlated with relatedness. Five anti- B. dendrobatidis bacterial strains occurred on multiple amphibian species at multiple localities, but none were shared among all species and localities. The prevalence of anti- B. dendrobatidis bacteria was higher at Shenandoah National Park (NP), VA, with 96% (25/26) of salamanders hosting at least one anti- B. dendrobatidis bacterial species compared to 50% (7/14) at Catoctin Mountain Park (MP), MD, and 38% (8/21) at Mt. Rogers National Recreation Area (NRA), VA. At the individual level, salamanders at Shenandoah NP had more anti- B. dendrobatidis bacteria per individual (μ = 3.3) than those at Catoctin MP (μ = 0.8) and at Mt. Rogers NRA (μ = 0.4). All salamanders tested negative for B. dendrobatidis Anti- B. dendrobatidis bacterial species are diverse in central Appalachian Plethodon salamanders, and their distribution varied geographically. The antifungal bacterial species that we identified may play a

  4. Design tradeoffs in long-term research for stream salamanders

    Science.gov (United States)

    Brand, Adrianne B,; Grant, Evan H. Campbell

    2017-01-01

    Long-term research programs can benefit from early and periodic evaluation of their ability to meet stated objectives. In particular, consideration of the spatial allocation of effort is key. We sampled 4 species of stream salamanders intensively for 2 years (2010–2011) in the Chesapeake and Ohio Canal National Historical Park, Maryland, USA to evaluate alternative distributions of sampling locations within stream networks, and then evaluated via simulation the ability of multiple survey designs to detect declines in occupancy and to estimate dynamic parameters (colonization, extinction) over 5 years for 2 species. We expected that fine-scale microhabitat variables (e.g., cobble, detritus) would be the strongest determinants of occupancy for each of the 4 species; however, we found greater support for all species for models including variables describing position within the stream network, stream size, or stream microhabitat. A monitoring design focused on headwater sections had greater power to detect changes in occupancy and the dynamic parameters in each of 3 scenarios for the dusky salamander (Desmognathus fuscus) and red salamander (Pseudotriton ruber). Results for transect length were more variable, but across all species and scenarios, 25-m transects are most suitable as a balance between maximizing detection probability and describing colonization and extinction. These results inform sampling design and provide a general framework for setting appropriate goals, effort, and duration in the initial planning stages of research programs on stream salamanders in the eastern United States.

  5. Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation

    Science.gov (United States)

    Walker, Donald M.; Leys, Jacob E.; Dunham, Kelly E.; Oliver, Joshua C.; Schiller, Emily E.; Stephenson, Kelsey S.; Kimrey, John T.; Wooten, Jessica; Rogers, Mark W.

    2017-01-01

    Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations.

  6. Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

    Directory of Open Access Journals (Sweden)

    Thomas Wurster

    Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

  7. YGFP: a spectral variant of GFP

    DEFF Research Database (Denmark)

    Hansen, Flemming G.; Atlung, Tove

    2011-01-01

    We describe YGFP, a slow bleaching green fluorescent protein (GFP) with unique spectral properties. YGFP is derived from an Escherichia coli codon-optimized synthetic gfp, mutant 2 derivative. In addition to the GFP-mut 2 changes, it also carries S202F and T203I substitutions. YGFP can be used...

  8. Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

    International Nuclear Information System (INIS)

    Fujimura, Juri; Ogawa, Rei; Mizuno, Hiroshi; Fukunaga, Yoshitaka; Suzuki, Hidenori

    2005-01-01

    Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders

  9. Predicted Changes in Climatic Niche and Climate Refugia of Conservation Priority Salamander Species in the Northeastern United States

    Directory of Open Access Journals (Sweden)

    William B. Sutton

    2014-12-01

    Full Text Available Global climate change represents one of the most extensive and pervasive threats to wildlife populations. Amphibians, specifically salamanders, are particularly susceptible to the effects of changing climates due to their restrictive physiological requirements and low vagility; however, little is known about which landscapes and species are vulnerable to climate change. Our study objectives included, (1 evaluating species-specific predictions (based on 2050 climate projections and vulnerabilities to climate change and (2 using collective species responses to identify areas of climate refugia for conservation priority salamanders in the northeastern United States. All evaluated salamander species were projected to lose a portion of their climatic niche. Averaged projected losses ranged from 3%–100% for individual species, with the Cow Knob Salamander (Plethodon punctatus, Cheat Mountain Salamander (Plethodon nettingi, Shenandoah Mountain Salamander (Plethodon virginia, Mabee’s Salamander (Ambystoma mabeei, and Streamside Salamander (Ambystoma barbouri predicted to lose at least 97% of their landscape-scale climatic niche. The Western Allegheny Plateau was predicted to lose the greatest salamander climate refugia richness (i.e., number of species with a climatically-suitable niche in a landscape patch, whereas the Central Appalachians provided refugia for the greatest number of species during current and projected climate scenarios. Our results can be used to identify species and landscapes that are likely to be further affected by climate change and potentially resilient habitats that will provide consistent climatic conditions in the face of environmental change.

  10. A new approach for surveying the Alpine Salamander (Salamandra atra in Austria

    Directory of Open Access Journals (Sweden)

    Ursula Reinthaler-Lottermoser

    2010-12-01

    Full Text Available The Alpine Salamander is a small pitch black amphibian which is endemic to the European Alps and the Dinarides. It is strictly protected according to the European FFH guidelines. Despite its central role in the alpine ecosystem our actual published record in Austria is small. In order to resolve this shortcoming our project explores its distribution in Austria. It uses a participatory and community based approach to gather data. Everybody can enter and look at Alpine Salamander observations on our website www.alpensalamander.eu. This approach also allows us to establish an “oral history” of Salamander observations in the past 50 years by conducting interviews in the local community. Since July 2009 the website and salamander report database are online. From the actual data (more than 5600 records we already obtained an overview about the present distribution and data quality. The data are an excellent basis for detailed scientific studies on these remarkable amphibians. With this new and highly interactive approach science and education are combined to initiate protection measures with the public.

  11. Data from proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaofang Geng

    2015-06-01

    Full Text Available The Chinese giant salamander (Andrias davidianus, renowned as a living fossil, is the largest and longest-lived amphibian species in the world. Its skin is rich in collagens, and has developed mucous gland which could secrete a large amount of mucus under the scraping and electric stimulation. The molting is the degraded skin stratum corneum. To establish the functional skin proteome of Chinese giant salamander, two-dimensional gel electrophoresis (2DE and mass spectrometry (MS were applied to detect the composition and relative abundance of the proteins in the skin, mucus and molting. The determination of the general proteome in the skin can potentially serve as a foundation for future studies characterizing the skin proteomes from diseased salamander to provide molecular and mechanistic insights into various disease states and potential therapeutic interventions. Data presented here are also related to the research article “Proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus” in the Journal of Proteomics [1].

  12. Generation and characterization of neurogenin1-GFP transgenic medaka with potential for rapid developmental neurotoxicity screening

    International Nuclear Information System (INIS)

    Fan Chunyang; Simmons, Steven O.; Law, Sheran H.W.; Jensen, Karl; Cowden, John; Hinton, David; Padilla, Stephanie; Ramabhadran, Ram

    2011-01-01

    Fish models such as zebrafish and medaka are increasingly used as alternatives to rodents in developmental and toxicological studies. These developmental and toxicological studies can be facilitated by the use of transgenic reporters that permit the real-time, noninvasive observation of the fish. Here we report the construction and characterization of transgenic medaka lines expressing green fluorescent protein (GFP) under the control of the zebrafish neurogenin 1 (ngn1) gene promoter. Neurogenin (ngn1) is a helix-loop-helix transcription factor expressed in proliferating neuronal progenitor cells early in neuronal differentiation and plays a crucial role in directing neurogenesis. GFP expression was detected from 24 h post-fertilization until hatching, in a spatial pattern consistent with the previously reported zebrafish ngn1 expression. Temporal expression of the transgene parallels the expression profile of the endogenous medaka ngn1 transcript. Further, we demonstrate that embryos from the transgenic line permit the non-destructive, real-time screening of ngn1 promoter-directed GFP expression in a 96-well format, enabling higher throughput studies of developmental neurotoxicants. This strain has been deposited with and maintained by the National BioResource Project and is available on request ( (http://www.shigen.nig.ac.jp/medaka/strainDetailAction.do?quickSearch=true and strainId=5660)).

  13. Effects of edge contrast on redback salamander distribution in even-aged northern hardwoods

    Science.gov (United States)

    Richard M. DeGraaf; Mariko. Yamasaki

    2002-01-01

    Terrestrial salamanders are sensitive to forest disturbance associated with even-aged management. We studied the distribution of redback salamanders (Plethodon cinereus) for 4 yr at edges between even-aged northern hardwood stands along three replicate transects in each of three edge contrast types: regeneration/mature, sapling/mature, and...

  14. Acid precipitation and reproductive success of Ambystoma salamanders

    Energy Technology Data Exchange (ETDEWEB)

    Pough, R H; Wilson, R E

    1976-01-01

    The two species of mole salamander that occur in the Ithaca, New York, region (Ambystoma maculatum and A. jeffersonianum) breed in temporary ponds that are formed by accumulation of melted snow and spring rains. Water in many of these pools during the breeding season is acid; pH values as low as 3.5 have been measured. In laboratory experiments A. maculatum tolerated pHs from 6 to 10 and had greatest hatching success at pH 7 to 9. Ambystoma Jeffersonianum tolerated pH 4 to 8 and was most successful at pH 5 to 6. Mortality rose abruptly beyond the tolerance limits. The pH optimum shifted upward with increasing temperature for A. jeffersonianum and downward for A. maculatum. Judging from our laboratory studies, the acidity measured in breeding ponds should cause mortality in A. maculatum and permit normal development in A. jeffersonianum. In a four-year study of a large acidic vernal pond, 938 adult A. maculatum produced 486 metamorphosed juveniles (0.52 juvenile/adult), while 686 adult A. jeffersonianum produced 2157 juveniles (3.14 juveniles/adult). Because the effects of acid precipitation on the salamanders' breeding ponds are cumulative from year to year, profound changes in the salamander populations can be anticipated.

  15. Acid precipitation and reproductive success of Ambystoma salamanders

    Energy Technology Data Exchange (ETDEWEB)

    Pough, F H; Wilson, R E

    1977-03-01

    The two species of mole salamander that occur in the Ithaca, New York, region (Ambystoma maculatum and A. jeffersonianum) breed in temporary ponds that are formed by accumulation of melted snow and spring rains. Water in many of these pools during the breeding season is acid; pH values as low as 3.5 have been measured. In laboratory experiments A. maculatum tolerated pHs from 6 to 10 and had greatest hatching success at pH 7 to 9. Ambystoma jeffersonianum tolerated pH 4 to 8 and was most successful at pH 5 to 6. Mortality rose abruptly beyond the tolerance limits. The pH optimum shifted upward with increasing temperature for A. jeffersonianum and downward for A. maculatum. Judging from our laboratory studies, the acidity measured in breeding ponds should cause mortality in A. maculatum and permit normal development in A. jeffersonianum. In a 4 yr study of a large, acidic vernal pond, 938 adult A. maculatum produced 486 metamorphosed juveniles (0.52 juvenile/adult), while 686 adult A. jeffersonianum produced 2157 juveniles (3.14 juveniles/adult). Because the effects of acid precipitation on the salamanders' breeding ponds are cumulative from year to year, profound changes in the salamander populations can be anticipated.

  16. Cytogenetics of the Brazilian Bolitoglossa paraensis (Unterstein, 1930 salamanders (Caudata, Plethodontidae

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    Jéssica Barata da Silva

    2014-09-01

    Full Text Available Plethodontid salamanders of genus Bolitoglossa constitute the largest and most diverse group of salamanders, including around 20% of living caudate species. Recent studies have indicated the occurrence of five recognized species in the Brazilian Amazon Rainforest. We present here the first cytogenetic data of a Brazilian salamander, which may prove to be a useful by contribution to the cytotaxonomy of the genus. Specimens were collected near the "type" locality (Utinga, Belém, PA, Brazil. Chromosomal preparations from duodenal epithelial cells and testes were subjected to Giemsa staining, C-banding and DAPI/CMA3 fluorochrome staining. All specimens showed a karyotype with 13 bi-armed chromosome pairs (2n = 26. Nucleolar Organizer Regions, evidenced by CMA3, were located distally on the long arm of pair 7 (7q. DAPI+ heterochromatin was predominantly centromeric, with some small pericentromeric bands. Although the C-banding patterns of other Bolitoglossa species are so far unknown, cytogenetic studies conducted in other Plethodontid salamanders have demonstrated that pericentromeric heterochromatin is a useful cytological marker for identifying interspecific homeologies. Species diversification is usually accompanied by chromosomal changes. Therefore, the cytogenetic characterization of Bolitoglossa populations from the middle and western Brazilian Amazon Basin could identify differences which may lead to the identification of new species.

  17. Anatomy, function, and evolution of jaw and hyobranchial muscles in cryptobranchoid salamander larvae.

    Science.gov (United States)

    Kleinteich, Thomas; Herzen, Julia; Beckmann, Felix; Matsui, Masafumi; Haas, Alexander

    2014-02-01

    Larval salamanders (Lissamphibia: Caudata) are known to be effective suction feeders in their aquatic environments, although they will eventually transform into terrestrial tongue feeding adults during metamorphosis. Early tetrapods may have had a similar biphasic life cycle and this makes larval salamanders a particularly interesting model to study the anatomy, function, development, and evolution of the feeding apparatus in terrestrial vertebrates. Here, we provide a description of the muscles that are involved in the feeding strike in salamander larvae of the Hynobiidae and compare them to larvae of the paedomorphic Cryptobranchidae. We provide a functional and evolutionary interpretation for the observed muscle characters. The cranial muscles in larvae from species of the Hynobiidae and Cryptobranchidae are generally very similar. Most notable are the differences in the presence of the m. hyomandibularis, a muscle that connects the hyobranchial apparatus with the lower jaw. We found this muscle only in Onychodactylus japonicus (Hynobiidae) but not in other hynobiid or cryptobranchid salamanders. Interestingly, the m. hyomandibularis in O. japonicus originates from the ceratobranchial I and not the ceratohyal, and thus exhibits what was previously assumed to be the derived condition. Finally, we applied a biomechanical model to simulate suction feeding in larval salamanders. We provide evidence that a flattened shape of the hyobranchial apparatus in its resting position is beneficial for a fast and successful suction feeding strike. Copyright © 2013 Wiley Periodicals, Inc.

  18. Decadal changes in phenology of peak abundance patterns of woodland pond salamanders in northern Wisconsin

    Science.gov (United States)

    Donner, Deahn M.; Ribic, Christine; Beck, Albert J.; Higgins, Dale; Eklund, Dan; Reinecke, Susan

    2015-01-01

    Woodland ponds are important landscape features that help sustain populations of amphibians that require this aquatic habitat for successful reproduction. Species abundance patterns often reflect site-specific differences in hydrology, physical characteristics, and surrounding vegetation. Large-scale processes such as changing land cover and environmental conditions are other potential drivers influencing amphibian populations in the Upper Midwest, but little information exists on the combined effects of these factors. We used Blue-spotted (Ambystoma laterale Hallowell) and Spotted Salamander (A. maculatum Shaw) monitoring data collected at the same woodland ponds thirteen years apart to determine if changing environmental conditions and vegetation cover in surrounding landscapes influenced salamander movement phenology and abundance. Four woodland ponds in northern Wisconsin were sampled for salamanders in April 1992-1994 and 2005-2007. While Blue-spotted Salamanders were more abundant than Spotted Salamanders in all ponds, there was no change in the numbers of either species over the years. However, peak numbers of Blue-spotted Salamanders occurred 11.7 days earlier (range: 9-14 days) in the 2000s compared to the 1990s; Spotted Salamanders occurred 9.5 days earlier (range: 3 - 13 days). Air and water temperatures (April 13- 24) increased, on average, 4.8°C and 3.7°C, respectively, between the decades regardless of pond. There were no discernible changes in canopy openness in surrounding forests between decades that would have warmed the water sooner (i.e., more light penetration). Our finding that salamander breeding phenology can vary by roughly 10 days in Wisconsin contributes to growing evidence that amphibian populations have responded to changing climate conditions by shifting life-cycle events. Managers can use this information to adjust monitoring programs and forest management activities in the surrounding landscape to avoid vulnerable amphibian

  19. Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP zebrafish embryos.

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    François Brion

    Full Text Available The tg(cyp19a1b-GFP transgenic zebrafish expresses GFP (green fluorescent protein under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i it is only expressed in radial glial progenitors in the brain of fish and (ii it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture, including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective.

  20. Proton transfer events in GFP

    NARCIS (Netherlands)

    Di Donato, M.; van Wilderen, L.J.G.W.; van Stokkum, I.H.M.; Cohen Stuart, T.A.; Kennis, J.T.M.; Hellingwerf, K.J.; van Grondelle, R.; Groot, M.L.

    2011-01-01

    Proton transfer is one of the most important elementary processes in biology. Green fluorescent protein (GFP) serves as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. Illumination initiates proton

  1. Asparaginase II-GFP fusion as a tool for studying the secretion of the enzyme under nitrogen starvation Fusão asparaginase II-GFP como ferramenta para estudo da via secretora de enzima sobre depleção por nitrogênio

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    Adriana Sotero-Martins

    2003-12-01

    Full Text Available Production of asparaginase II of Saccharomyces cerevisiae is regulated by nitrogen and can be used as a model system for studying other secreted proteins in yeast. Green fluorescent protein (GFP from Aequorea victoria was fused to the carboxy-terminus of the enzyme by genomic integration to the locus ASP3 of S. cerevisiae. We determined asparaginase II activity, mRNA ASP3, mRNA ASP3-GFP and GFP fluorescence. Nitrogen starvation in cells carrying the chimera ASP3-GFP caused an increase in fluorescence and in the expression of ASP3. We have shown that cells producing the chimera Asp3-GFPp displayed the same response to nitrogen starvation as control cells. We demonstrated that Asp3-GFPp can be used for studying asparaginase II secretion under nitrogen starvation in vivo.A produção de asparaginase II de Saccharomyces cerevisiae é regulada por nitrogênio e pode ser utilizada como um sistema modelo para estudar outras proteínas secretadas, em leveduras. A proteína "green fluorescent protein" (GFP de Aequorea victoria foi fusionada à porção carboxi-terminal de Asp3p por integração genômica da sequência de GFP ao locus ASP3. Determinaram-se os níveis de atividade de asparaginase II, mRNA ASP3, mRNA ASP3-GFP e de fluorescência para GFP. A depleção para nitrogênio, em células portadoras do gene quimérico ASP3-GFP, fez aumentar a fluorescência, assim como a expressão de ASP3. Demonstramos que Asp3-GFPp pode ser utilizada para estudar a secreção de asparaginase II em células submetidas à privação de nitrogênio in vivo.

  2. Survey of Pathogenic Chytrid Fungi (Batrachochytrium dendrobatidis and B. salamandrivorans) in Salamanders from Three Mountain Ranges in Europe and the Americas.

    Science.gov (United States)

    Parrott, Joshua Curtis; Shepack, Alexander; Burkart, David; LaBumbard, Brandon; Scimè, Patrick; Baruch, Ethan; Catenazzi, Alessandro

    2017-06-01

    Batrachochytrium salamandrivorans (Bsal) is a virulent fungal pathogen that infects salamanders. It is implicated in the recent collapse of several populations of fire salamanders in Europe. This pathogen seems much like that of its sister species, Batrachochytrium dendrobatidis (Bd), the agent responsible for anuran extinctions and extirpations worldwide, and is considered to be an emerging global threat to salamander communities. Bsal thrives at temperatures found in many mountainous regions rich in salamander species; because of this, we have screened specimens of salamanders representing 17 species inhabiting mountain ranges in three continents: The Smoky Mountains, the Swiss Alps, and the Peruvian Andes. We screened 509 salamanders, with 192 representing New World salamanders that were never tested for Bsal previously. Bsal was not detected, and Bd was mostly present at low prevalence except for one site in the Andes.

  3. Multiple drivers, scales, and interactions influence southern Appalachian stream salamander occupancy

    Science.gov (United States)

    Cecala, Kristen K.; Maerz, John C.; Halstead, Brian J.; Frisch, John R.; Gragson, Ted L.; Hepinstall-Cymerman, Jeffrey; Leigh, David S.; Jackson, C. Rhett; Peterson, James T.; Pringle, Catherine M.

    2018-01-01

    Understanding how factors that vary in spatial scale relate to population abundance is vital to forecasting species responses to environmental change. Stream and river ecosystems are inherently hierarchical, potentially resulting in organismal responses to fine‐scale changes in patch characteristics that are conditional on the watershed context. Here, we address how populations of two salamander species are affected by interactions among hierarchical processes operating at different scales within a rapidly changing landscape of the southern Appalachian Mountains. We modeled reach‐level occupancy of larval and adult black‐bellied salamanders (Desmognathus quadramaculatus) and larval Blue Ridge two‐lined salamanders (Eurycea wilderae) as a function of 17 different terrestrial and aquatic predictor variables that varied in spatial extent. We found that salamander occurrence varied widely among streams within fully forested catchments, but also exhibited species‐specific responses to changes in local conditions. While D. quadramaculatus declined predictably in relation to losses in forest cover, larval occupancy exhibited the strongest negative response to forest loss as well as decreases in elevation. Conversely, occupancy of E. wilderae was unassociated with watershed conditions, only responding negatively to higher proportions of fast‐flowing stream habitat types. Evaluation of hierarchical relationships demonstrated that most fine‐scale variables were closely correlated with broad watershed‐scale variables, suggesting that local reach‐scale factors have relatively smaller effects within the context of the larger landscape. Our results imply that effective management of southern Appalachian stream salamanders must first focus on the larger scale condition of watersheds before management of local‐scale conditions should proceed. Our findings confirm the results of some studies while refuting the results of others, which may indicate that

  4. Regional Differences in Striatal Neuronal Ensemble Excitability Following Cocaine and Extinction Memory Retrieval in Fos-GFP Mice.

    Science.gov (United States)

    Ziminski, Joseph J; Sieburg, Meike C; Margetts-Smith, Gabriella; Crombag, Hans S; Koya, Eisuke

    2018-03-01

    Learned associations between drugs of abuse and the drug administration environment have an important role in addiction. In rodents, exposure to a drug-associated environment elicits conditioned psychomotor activation, which may be weakened following extinction (EXT) learning. Although widespread drug-induced changes in neuronal excitability have been observed, little is known about specific changes within neuronal ensembles activated during the recall of drug-environment associations. Using a cocaine-conditioned locomotion (CL) procedure, the present study assessed the excitability of neuronal ensembles in the nucleus accumbens core and shell (NAc core and NAc shell ), and dorsal striatum (DS) following cocaine conditioning and EXT in Fos-GFP mice that express green fluorescent protein (GFP) in activated neurons (GFP+). During conditioning, mice received repeated cocaine injections (20 mg/kg) paired with a locomotor activity chamber (Paired) or home cage (Unpaired). Seven to 13 days later, both groups were re-exposed to the activity chamber under drug-free conditions and Paired, but not Unpaired, mice exhibited CL. In a separate group of mice, CL was extinguished by repeatedly exposing mice to the activity chamber under drug-free conditions. Following the expression and EXT of CL, GFP+ neurons in the NAc core (but not NAc shell and DS) displayed greater firing capacity compared to surrounding GFP- neurons. This difference in excitability was due to a generalized decrease in GFP- excitability following CL and a selective increase in GFP+ excitability following its EXT. These results suggest a role for both widespread and ensemble-specific changes in neuronal excitability following recall of drug-environment associations.

  5. The membrane skeleton in Paramecium: Molecular characterization of a novel epiplasmin family and preliminary GFP expression results.

    Science.gov (United States)

    Pomel, Sébastien; Diogon, Marie; Bouchard, Philippe; Pradel, Lydie; Ravet, Viviane; Coffe, Gérard; Viguès, Bernard

    2006-02-01

    Previous attempts to identify the membrane skeleton of Paramecium cells have revealed a protein pattern that is both complex and specific. The most prominent structural elements, epiplasmic scales, are centered around ciliary units and are closely apposed to the cytoplasmic side of the inner alveolar membrane. We sought to characterize epiplasmic scale proteins (epiplasmins) at the molecular level. PCR approaches enabled the cloning and sequencing of two closely related genes by amplifications of sequences from a macronuclear genomic library. Using these two genes (EPI-1 and EPI-2), we have contributed to the annotation of the Paramecium tetraurelia macronuclear genome and identified 39 additional (paralogous) sequences. Two orthologous sequences were found in the Tetrahymena thermophila genome. Structural analysis of the 43 sequences indicates that the hallmark of this new multigenic family is a 79 aa domain flanked by two Q-, P- and V-rich stretches of sequence that are much more variable in amino-acid composition. Such features clearly distinguish members of the multigenic family from epiplasmic proteins previously sequenced in other ciliates. The expression of Green Fluorescent Protein (GFP)-tagged epiplasmin showed significant labeling of epiplasmic scales as well as oral structures. We expect that the GFP construct described herein will prove to be a useful tool for comparative subcellular localization of different putative epiplasmins in Paramecium.

  6. Exceptional soft tissues preservation in a mummified frog-eating Eocene salamander

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    Jérémy Tissier

    2017-10-01

    Full Text Available Fossils are almost always represented by hard tissues but we present here the exceptional case of a three-dimensionally preserved specimen that was ‘mummified’ (likely between 40 and 34 million years ago in a terrestrial karstic environment. This fossil is the incomplete body of a salamander, Phosphotriton sigei, whose skeleton and external morphology are well preserved, as revealed by phase-contrast synchrotron X-ray microtomography. In addition, internal structures composed of soft tissues preserved in three dimensions are now identified: a lung, the spinal cord, a lumbosacral plexus, the digestive tract, muscles and urogenital organs that may be cloacal glands. These are among the oldest known cases of three-dimensional preservation of these organs in vertebrates and shed light on the ecology of this salamander. Indeed, the digestive tract contains remains of a frog, which represents the only known case of an extinct salamander that fed on a frog, an extremely rare type of predation in extant salamanders. These new data improve our scarce knowledge on soft tissue anatomy of early urodeles and should prove useful for future biologists and palaeontologists working on urodele evolutionary biology. We also suggest that the presence of bat guano and carcasses represented a close source of phosphorus, favouring preservation of soft tissues. Bone microanatomy indicates that P. sigei was likely amphibious or terrestrial, and was probably not neotenic.

  7. Exceptional soft tissues preservation in a mummified frog-eating Eocene salamander.

    Science.gov (United States)

    Tissier, Jérémy; Rage, Jean-Claude; Laurin, Michel

    2017-01-01

    Fossils are almost always represented by hard tissues but we present here the exceptional case of a three-dimensionally preserved specimen that was 'mummified' (likely between 40 and 34 million years ago) in a terrestrial karstic environment. This fossil is the incomplete body of a salamander, Phosphotriton sigei , whose skeleton and external morphology are well preserved, as revealed by phase-contrast synchrotron X-ray microtomography. In addition, internal structures composed of soft tissues preserved in three dimensions are now identified: a lung, the spinal cord, a lumbosacral plexus, the digestive tract, muscles and urogenital organs that may be cloacal glands. These are among the oldest known cases of three-dimensional preservation of these organs in vertebrates and shed light on the ecology of this salamander. Indeed, the digestive tract contains remains of a frog, which represents the only known case of an extinct salamander that fed on a frog, an extremely rare type of predation in extant salamanders. These new data improve our scarce knowledge on soft tissue anatomy of early urodeles and should prove useful for future biologists and palaeontologists working on urodele evolutionary biology. We also suggest that the presence of bat guano and carcasses represented a close source of phosphorus, favouring preservation of soft tissues. Bone microanatomy indicates that P. sigei was likely amphibious or terrestrial, and was probably not neotenic.

  8. Digits lost or gained? Evidence for pedal evolution in the dwarf salamander complex (Eurycea, Plethodontidae.

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    Trip Lamb

    Full Text Available Change in digit number, particularly digit loss, has occurred repeatedly over the evolutionary history of tetrapods. Although digit loss has been documented among distantly related species of salamanders, it is relatively uncommon in this amphibian order. For example, reduction from five to four toes appears to have evolved just three times in the morphologically and ecologically diverse family Plethodontidae. Here we report a molecular phylogenetic analysis for one of these four-toed lineages--the Eurycea quadridigitata complex (dwarf salamanders--emphasizing relationships to other species in the genus. A multilocus phylogeny reveals that dwarf salamanders are paraphyletic with respect to a complex of five-toed, paedomorphic Eurycea from the Edwards Plateau in Texas. We use this phylogeny to examine evolution of digit number within the dwarf-Edwards Plateau clade, testing contrasting hypotheses of digit loss (parallelism among dwarf salamanders versus digit gain (re-evolution in the Edwards Plateau complex. Bayes factors analysis provides statistical support for a five-toed common ancestor at the dwarf-Edwards node, favoring, slightly, the parallelism hypothesis for digit loss. More importantly, our phylogenetic results pinpoint a rare event in the pedal evolution of plethodontid salamanders.

  9. Effects of Buffering Key Habitat for Terrestrial Salamanders: Implications for the Management of the Federally Threatened Red Hills Salamander (Phaeognathus hubrichti and Other Imperiled Plethodontids

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    Joseph J. Apodaca

    2015-03-01

    Full Text Available Forestry practices are placing ever increasing emphasis on sustainability and the maintenance of ecological processes, biodiversity, and endangered species or populations. Balancing timber harvest and the management of imperiled species presents a particularly difficult challenge during this shift, as we often know very little about these species’ natural history and how and why silviculture practices affect their populations. Accordingly, investigation of and improvement on current management practices for threatened species is imperative. We investigated the effectiveness of habitat buffers as a management technique for the imperiled Red Hills salamander (Phaeognathus hubrichti by combining genetic, transect, and body-condition data. We found that populations where habitat buffers have been employed have higher genetic diversity and higher population densities, and individuals have better overall body condition. These results indicate that buffering the habitat of imperiled species can be an effective management tool for terrestrial salamanders. Additionally, they provide further evidence that leaving the habitat of imperiled salamanders unbuffered can have both immediate and long-term negative impacts on populations.

  10. Toxicological responses of red-backed salamanders (Plethodon cinereus) to subchronic soil exposures of 2,4-dinitrotoluene

    International Nuclear Information System (INIS)

    Johnson, Mark S.; Suski, Jamie; Bazar, Matthew A.

    2007-01-01

    Dinitrotoluenes are used as propellants and in explosives by the military and as such have been found at relatively high concentrations in the soil. To determine whether concentrations of 2,4-dinitrotoluene (2,4-DNT) in soil are toxic to amphibians, 100 red-backed salamanders (Plethodon cinereus) were exposed to either 1500, 800, 200, 75 or 0 mg 2,4-DNT/kg soil for 28 days and evaluated for indicators of toxicity. Concentrations of 2,4-DNT were less than targets and varied with time. Most salamanders exposed to concentrations exceeding 1050 mg/kg died or were moribund within the first week. Salamanders exposed to soil concentrations exceeding 345 mg/kg lost >6% of their body mass though no mortality occurred. Overt effects included a reduction in feed consumption and an increase in bucco-pharyngeal oscillations in salamanders. These results suggest that only high soil concentrations of 2,4-DNT have the potential to cause overtly toxic effects in terrestrial salamanders. - Exposures of 2,4-dinitrotoluene in soil exceeding 345 mg/kg causes toxicity to P. cinereus

  11. Climate-mediated competition in a high-elevation salamander community

    Science.gov (United States)

    Dallalio, Eric A.; Brand, Adrianne B,; Grant, Evan H. Campbell

    2017-01-01

    The distribution of the federally endangered Shenandoah Salamander (Plethodon shenandoah) is presumed to be limited by competition with the Red-backed Salamander (Plethodon cinereus). In particular, the current distribution of P. shenandoah is understood to be restricted to warmer and drier habitats because of interspecific interactions. These habitats may be particularly sensitive to climate change, though the influence of competition may also be affected by temperature and relative humidity. We investigated the response of P. shenandoah to competition with P. cinereus under four climate scenarios in 3-dimensional mesocosms. The results suggest that, although climate change may alleviate competitive pressure from P. cinereus, warmer temperatures may also significantly influence the persistence of the species across its known range.

  12. A novel binary T-vector with the GFP reporter gene for promoter characterization.

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    Shu-Ye Jiang

    Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

  13. Rosa26-GFP direct repeat (RaDR-GFP mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

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    Michelle R Sukup-Jackson

    2014-06-01

    Full Text Available Homologous recombination (HR is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

  14. Role of habitat complexity in predator-prey dynamics between an introduced fish and larval Long-toed Salamanders (Ambystoma macrodactylum)

    Science.gov (United States)

    Kenison, Erin K; Litt, Andrea R.; Pilliod, David S.; McMahon, Tom E

    2016-01-01

    Predation by nonnative fishes has reduced abundance and increased extinction risk for amphibian populations worldwide. Although rare, fish and palatable amphibians have been observed to coexist where aquatic vegetation and structural complexity provide suitable refugia. We examined whether larval long-toed salamanders (Ambystoma macrodactylum Baird, 1849) increased use of vegetation cover in lakes with trout and whether adding vegetation structure could reduce predation risk and nonconsumptive effects (NCEs), such as reductions in body size and delayed metamorphosis. We compared use of vegetation cover by larval salamanders in lakes with and without trout and conducted a field experiment to investigate the influence of added vegetation structure on salamander body morphology and life history. The probability of catching salamanders in traps in lakes with trout was positively correlated with the proportion of submerged vegetation and surface cover. Growth rates of salamanders in enclosures with trout cues decreased as much as 85% and the probability of metamorphosis decreased by 56%. We did not find evidence that adding vegetation reduced NCEs in experimental enclosures, but salamanders in lakes with trout utilized more highly-vegetated areas which suggests that adding vegetation structure at the scale of the whole lake may facilitate coexistence between salamanders and introduced trout.

  15. Habitat relationships of eastern red-backed salamanders (Plethodon cinereus) in Appalachian agroforestry and grazing systems

    Science.gov (United States)

    Breanna L. Riedel; Kevin R. Russell; W. Mark Ford; Katherine P. O' Neill; Harry W. Godwin

    2008-01-01

    Woodland salamander responses to either traditional grazing or silvopasture systems are virtually unknown. An information-theoretic modelling approach was used to evaluate responses of red-backed salamanders (Plethodon cinereus) to silvopasture and meadow conversions in southern West Virginia. Searches of area-constrained plots and artificial...

  16. Evaluating multi-level models to test occupancy state responses of Plethodontid salamanders

    Science.gov (United States)

    Kroll, Andrew J.; Garcia, Tiffany S.; Jones, Jay E.; Dugger, Catherine; Murden, Blake; Johnson, Josh; Peerman, Summer; Brintz, Ben; Rochelle, Michael

    2015-01-01

    Plethodontid salamanders are diverse and widely distributed taxa and play critical roles in ecosystem processes. Due to salamander use of structurally complex habitats, and because only a portion of a population is available for sampling, evaluation of sampling designs and estimators is critical to provide strong inference about Plethodontid ecology and responses to conservation and management activities. We conducted a simulation study to evaluate the effectiveness of multi-scale and hierarchical single-scale occupancy models in the context of a Before-After Control-Impact (BACI) experimental design with multiple levels of sampling. Also, we fit the hierarchical single-scale model to empirical data collected for Oregon slender and Ensatina salamanders across two years on 66 forest stands in the Cascade Range, Oregon, USA. All models were fit within a Bayesian framework. Estimator precision in both models improved with increasing numbers of primary and secondary sampling units, underscoring the potential gains accrued when adding secondary sampling units. Both models showed evidence of estimator bias at low detection probabilities and low sample sizes; this problem was particularly acute for the multi-scale model. Our results suggested that sufficient sample sizes at both the primary and secondary sampling levels could ameliorate this issue. Empirical data indicated Oregon slender salamander occupancy was associated strongly with the amount of coarse woody debris (posterior mean = 0.74; SD = 0.24); Ensatina occupancy was not associated with amount of coarse woody debris (posterior mean = -0.01; SD = 0.29). Our simulation results indicate that either model is suitable for use in an experimental study of Plethodontid salamanders provided that sample sizes are sufficiently large. However, hierarchical single-scale and multi-scale models describe different processes and estimate different parameters. As a result, we recommend careful consideration of study questions

  17. Evaluating Multi-Level Models to Test Occupancy State Responses of Plethodontid Salamanders.

    Directory of Open Access Journals (Sweden)

    Andrew J Kroll

    Full Text Available Plethodontid salamanders are diverse and widely distributed taxa and play critical roles in ecosystem processes. Due to salamander use of structurally complex habitats, and because only a portion of a population is available for sampling, evaluation of sampling designs and estimators is critical to provide strong inference about Plethodontid ecology and responses to conservation and management activities. We conducted a simulation study to evaluate the effectiveness of multi-scale and hierarchical single-scale occupancy models in the context of a Before-After Control-Impact (BACI experimental design with multiple levels of sampling. Also, we fit the hierarchical single-scale model to empirical data collected for Oregon slender and Ensatina salamanders across two years on 66 forest stands in the Cascade Range, Oregon, USA. All models were fit within a Bayesian framework. Estimator precision in both models improved with increasing numbers of primary and secondary sampling units, underscoring the potential gains accrued when adding secondary sampling units. Both models showed evidence of estimator bias at low detection probabilities and low sample sizes; this problem was particularly acute for the multi-scale model. Our results suggested that sufficient sample sizes at both the primary and secondary sampling levels could ameliorate this issue. Empirical data indicated Oregon slender salamander occupancy was associated strongly with the amount of coarse woody debris (posterior mean = 0.74; SD = 0.24; Ensatina occupancy was not associated with amount of coarse woody debris (posterior mean = -0.01; SD = 0.29. Our simulation results indicate that either model is suitable for use in an experimental study of Plethodontid salamanders provided that sample sizes are sufficiently large. However, hierarchical single-scale and multi-scale models describe different processes and estimate different parameters. As a result, we recommend careful consideration of

  18. Using a GIS model to assess terrestrial salamander response to alternative forest management plans

    Science.gov (United States)

    Eric J. Gustafson; Nathan L. Murphy; Thomas R. Crow

    2001-01-01

    A GIS model predicting the spatial distribution of terrestrial salamander abundance based on topography and forest age was developed using parameters derived from the literature. The model was tested by sampling salamander abundance across the full range of site conditions used in the model. A regression of the predictions of our GIS model against these sample data...

  19. Apparent survival of the salamander Salamandra salamandra is low because of high migratory activity

    Directory of Open Access Journals (Sweden)

    Schaub Michael

    2007-09-01

    Full Text Available Abstract Background Understanding the demographic processes underlying population dynamics is a central theme in ecology. Populations decline if losses from the population (i.e., mortality and emigration exceed gains (i.e., recruitment and immigration. Amphibians are thought to exhibit little movement even though local populations often fluctuate dramatically and are likely to go exinct if there is no rescue effect through immigration from nearby populations. Terrestrial salamanders are generally portrayed as amphibians with low migratory activity. Our study uses demographic analysis as a key to unravel whether emigration or mortality is the main cause of "losses" from the population. In particular, we use the analysis to challenge the common belief that terrestrial salamanders show low migratory activity. Results The mark-recapture analysis of adult salamanders showed that monthly survival was high (> 90% without a seasonal pattern. These estimates, however, translate into rather low rates of local annual survival of only ~40% and suggest that emigration was important. The estimated probability of emigration was 49%. Conclusion Our analysis shows that terrestrial salamanders exhibit more migratory activity than commonly thought. This may be due either because the spatial extent of salamander populations is underestimated or because there is a substantial exchange of individuals between populations. Our current results are in line with several other studies that suggest high migratory activity in amphibians. In particular, many amphibian populations may be characterized by high proportions of transients and/or floaters.

  20. Morphological variation in two genetically distinct groups of the golden-striped salamander, Chioglossa lusitanica (Amphibia: Urodela)

    NARCIS (Netherlands)

    Alexandrino, J.; Ferrand, N.; Arntzen, J.W.

    2005-01-01

    Morphometric and colour pattern variation in the endemic Iberian salamander Chioglossa lusitanica is concordant with the genetic differentiation of two groups of populations separated by the Mondego river in Portugal. Salamanders from the south have shorter digits than those from the north. Clinal

  1. Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.

    Directory of Open Access Journals (Sweden)

    Dipak K. Sahoo

    2014-01-01

    Full Text Available To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

  2. Informing recovery in a human-transformed landscape: Drought-mediated coexistence alters population trends of an imperiled salamander and invasive predators

    Science.gov (United States)

    Hossack, Blake R.; Honeycutt, Richard; Sigafus, Brent H.; Muths, Erin L.; Crawford, Catherine L.; Jones, Thomas R.; Sorensen, Jeff A.; Rorabaugh, James C.; Chambert, Thierry

    2017-01-01

    Understanding the additive or interactive threats of habitat transformation and invasive species is critical for conservation, especially where climate change is expected to increase the severity or frequency of drought. In the arid southwestern USA, this combination of stressors has caused widespread declines of native aquatic and semi-aquatic species. Achieving resilience to drought and other effects of climate change may depend upon continued management, so understanding the combined effects of stressors is important. We used Bayesian hierarchical models fitted with 10-years of pond-based monitoring surveys for the federally-endangered Sonoran Tiger Salamander (Ambystoma mavortium stebbinsi) and invasive predators (fishes and American Bullfrogs, Lithobates catesbeianus) that threaten native species. We estimated trends in occupancy of salamanders and invasive predators while accounting for hydrological dynamics of ponds, then used a two-species interaction model to directly estimate how invasive predators affected salamander occupancy. We also tested a conceptual model that predicted that drought, by limiting the distribution of invasive predators, could ultimately benefit native species. Even though occupancy of invasive predators was stationary and their presence in a pond reduced the probability of salamander presence by 23%, occupancy of Sonoran Tiger Salamanders increased, annually, by 2.2%. Occupancy of salamanders and invasive predators both declined dramatically following the 5th consecutive year of drought. Salamander occupancy recovered quickly after return to non-drought conditions, while occupancy of invasive predators remained suppressed. Models that incorporated three time-lagged periods (1 to 4 years) of local moisture conditions confirmed that salamanders and invasive predators responded differently to drought, reflecting how life-history strategies shape responses to disturbances. The positive 10-year trend in salamander occupancy and their

  3. Salamander blue-sensitive cones lost during metamorphosis.

    NARCIS (Netherlands)

    Chen, Y.; Znoiko, S.; Grip, W.J. de; Crouch, R.K.; Ma, J.X.

    2008-01-01

    The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and

  4. 76 FR 55413 - Proposed Safe Harbor Agreement for California Red-legged Frog, California Tiger Salamander, Smith...

    Science.gov (United States)

    2011-09-07

    ...] Proposed Safe Harbor Agreement for California Red-legged Frog, California Tiger Salamander, Smith's Blue... endangered Smith's blue butterfly (Euphilotes enoptes smithi) under the Endangered Species Act of 1973, as..., California tiger salamander, Smith's blue butterfly, and Yadon's piperia on the property subject to the...

  5. Skin Microbiomes of California Terrestrial Salamanders Are Influenced by Habitat More Than Host Phylogeny

    Directory of Open Access Journals (Sweden)

    Alicia K. Bird

    2018-03-01

    Full Text Available A multitude of microorganisms live on and within plant and animal hosts, yet the ecology and evolution of these microbial communities remains poorly understood in many taxa. This study examined the extent to which environmental factors and host taxonomic identity explain microbiome variation within two salamander genera, Ensatina and Batrachoseps, in the family Plethodontidae. In particular, we assessed whether microbiome differentiation paralleled host genetic distance at three levels of taxonomy: genus and high and low clade levels within Ensatina eschscholtzii. We predicted that more genetically related host populations would have more similar microbiomes than more distantly related host populations. We found that salamander microbiomes possess bacterial species that are most likely acquired from their surrounding soil environment, but the relative representation of those bacterial species is significantly different on the skin of salamanders compared to soil. We found differences in skin microbiome alpha diversity among Ensatina higher and lower clade groups, as well as differences between Ensatina and Batrachoseps. We also found that relative microbiome composition (beta diversity did vary between Ensatina lower clades, but differences were driven by only a few clades and not correlated to clade genetic distances. We conclude this difference was likely a result of Ensatina lower clades being associated with geographic location and habitat type, as salamander identity at higher taxonomic levels (genus and Ensatina higher clades was a weak predictor of microbiome composition. These results lead us to conclude that environmental factors are likely playing a more significant role in salamander cutaneous microbiome assemblages than host-specific traits.

  6. Behavioral and Physiological Responses of Ozark Zigzag Salamanders to Stimuli from an Invasive Predator: The Armadillo

    Directory of Open Access Journals (Sweden)

    Adam L. Crane

    2012-01-01

    Full Text Available When new predators invade a habitat, either through range extensions or introductions, prey may be at a high risk because they do not recognize the predators as dangerous. The nine-banded armadillo (Dasypus novemcinctus has recently expanded its range in North America. Armadillos forage by searching soil and leaf litter, consuming invertebrates and small vertebrates, including salamanders. We tested whether Ozark zigzag salamanders (Plethodon angusticlavius from a population coexisting with armadillos for about 30 years exhibit antipredator behavior in the presence of armadillo chemical cues and whether they can discriminate between stimuli from armadillos and a nonpredatory sympatric mammal (white-tailed deer, Odocoileus virginianus. Salamanders appeared to recognize substrate cues from armadillos as a threat because they increased escape behaviors and oxygen consumption. When exposed to airborne cues from armadillos, salamanders also exhibited an antipredator response by spending more time in an inconspicuous posture. Additionally, individually consistent behaviors across treatments for some response variables suggest the potential for a behavioral syndrome in this species.

  7. Comparative and developmental patterns of amphibious auditory function in salamanders.

    Science.gov (United States)

    Zeyl, Jeffrey N; Johnston, Carol E

    2016-12-01

    Early amphibious tetrapods may have detected aquatic sound pressure using sound-induced lung vibrations, but their lack of tympanic middle ears would have restricted aerial sensitivity. Sharing these characteristics, salamanders could be models for the carryover of auditory function across an aquatic-terrestrial boundary without tympanic middle ears. We measured amphibious auditory evoked potential audiograms in five phylogenetically and ecologically distinct salamanders (Amphiuma means, Notophthalmus viridescens, Ambystoma talpoideum, Eurycea spp., and Plethodon glutinosus) and tested whether metamorphosis and terrestrial niche were linked to aerial sensitivity. Threshold differences between media varied between species. A. means' relative aerial sensitivity was greatest at 100 Hz and decreased with increasing frequency. In contrast, all other salamanders retained greater sensitivity up to 500 Hz, and in A. talpoideum and Eurycea, relative sensitivity at 500 Hz was higher than at 100 Hz. Aerial thresholds of terrestrial P. glutinosus above 200 Hz were similar to A. talpoideum and Eurycea, but lower than N. viridescens and A. means. Metamorphosis did not affect aerial sensitivity in N. viridescens or A. talpoideum. These results fail to support a hypothesis of terrestrial hearing specialization across ontogeny or phylogeny. We discuss methodological limitations to our amphibious comparisons and factors affecting variation in amphibious performance.

  8. GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

    Science.gov (United States)

    Weber, Eva; Guth, Christina; Weiss, Ingrid M

    2012-01-01

    Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

  9. Transcriptional and phylogenetic analysis of five complete ambystomatid salamander mitochondrial genomes.

    Science.gov (United States)

    Samuels, Amy K; Weisrock, David W; Smith, Jeramiah J; France, Katherine J; Walker, John A; Putta, Srikrishna; Voss, S Randal

    2005-04-11

    We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger

  10. in Chinese giant salamander ( Andrias davidianus , Blanchard, 1871)

    African Journals Online (AJOL)

    A disease in farmed Chinese giant salamander (Andrias davidianus) was a common event, being an economically important threat for Chinese farms. Based on the clinical signs, epizootiology and pathogens belonging to the genus, Ranavirus was suspected as the possible etiology. Although in a cultured Chinese giant ...

  11. Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

    Science.gov (United States)

    Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

    2017-01-31

    The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

  12. Variable infection of stream salamanders in the southern Appalachians by the trematode Metagonimoides oregonensis (family: Heterophyidae)

    Science.gov (United States)

    Jennie A. Wyderko; Ernest F. Benfield; John C. Maerz; Kristen C. Cecala; Lisa K. Belden

    2015-01-01

    Many factors contribute to parasites varying in host specificity and distribution among potential hosts. Metagonimoides oregonensis is a digenetic trematode that uses stream-dwelling plethodontid salamanders as second intermediate hosts in the Eastern US. We completed a field survey to identify which stream salamander species, at a regional level, are most...

  13. Effects of microhabitat and large-scale land use on stream salamander occupancy in the coalfields of Central Appalachia

    Science.gov (United States)

    Sweeten, Sara E.; Ford, W. Mark

    2016-01-01

    Large-scale coal mining practices, particularly surface coal extraction and associated valley fills as well as residential wastewater discharge, are of ecological concern for aquatic systems in central Appalachia. Identifying and quantifying alterations to ecosystems along a gradient of spatial scales is a necessary first-step to aid in mitigation of negative consequences to aquatic biota. In central Appalachian headwater streams, apart from fish, salamanders are the most abundant vertebrate predator that provide a significant intermediate trophic role linking aquatic and terrestrial food webs. Stream salamander species are considered to be sensitive to aquatic stressors and environmental alterations, as past research has shown linkages among microhabitat parameters, large-scale land use such as urbanization and logging, and salamander abundances. However, there is little information examining these relationships between environmental conditions and salamander occupancy in the coalfields of central Appalachia. In the summer of 2013, 70 sites (sampled two to three times each) in the southwest Virginia coalfields were visited to collect salamanders and quantify stream and riparian microhabitat parameters. Using an information-theoretic framework, effects of microhabitat and large-scale land use on stream salamander occupancy were compared. The findings indicate that Desmognathus spp. occupancy rates are more correlated to microhabitat parameters such as canopy cover than to large-scale land uses. However, Eurycea spp. occupancy rates had a strong association with large-scale land uses, particularly recent mining and forest cover within the watershed. These findings suggest that protection of riparian habitats is an important consideration for maintaining aquatic systems in central Appalachia. If this is not possible, restoration riparian areas should follow guidelines using quick-growing tree species that are native to Appalachian riparian areas. These types of trees

  14. hNIS-IRES-eGFP Dual Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2005-04-01

    Full Text Available The human and rodent sodium iodide symporters (NIS have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES-linked human NIS (hNIS-enhanced green fluorescent protein (eGFP hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.

  15. A Salamander Tale: Effective Exhibits and Attitude Change

    Science.gov (United States)

    Rollins, Jeffrey; Watson, Sunnie Lee

    2017-01-01

    Little information exists regarding intention behind the design and development of Extension outreach and educational exhibits. An evaluation of response to the exhibit "A Salamander Tale" indicates that the methods used to develop the exhibit resulted in an effective way to present information to an adult audience. Survey questions were…

  16. Low levels of LTR retrotransposon deletion by ectopic recombination in the gigantic genomes of salamanders.

    Science.gov (United States)

    Frahry, Matthew Blake; Sun, Cheng; Chong, Rebecca A; Mueller, Rachel Lockridge

    2015-02-01

    Across the tree of life, species vary dramatically in nuclear genome size. Mutations that add or remove sequences from genomes-insertions or deletions, or indels-are the ultimate source of this variation. Differences in the tempo and mode of insertion and deletion across taxa have been proposed to contribute to evolutionary diversity in genome size. Among vertebrates, most of the largest genomes are found within the salamanders, an amphibian clade with genome sizes ranging from ~14 to ~120 Gb. Salamander genomes have been shown to experience slower rates of DNA loss through small (i.e., genomes. However, no studies have addressed DNA loss from salamander genomes resulting from larger deletions. Here, we focus on one type of large deletion-ectopic-recombination-mediated removal of LTR retrotransposon sequences. In ectopic recombination, double-strand breaks are repaired using a "wrong" (i.e., ectopic, or non-allelic) template sequence-typically another locus of similar sequence. When breaks occur within the LTR portions of LTR retrotransposons, ectopic-recombination-mediated repair can produce deletions that remove the internal transposon sequence and the equivalent of one of the two LTR sequences. These deletions leave a signature in the genome-a solo LTR sequence. We compared levels of solo LTRs in the genomes of four salamander species with levels present in five vertebrates with smaller genomes. Our results demonstrate that salamanders have low levels of solo LTRs, suggesting that ectopic-recombination-mediated deletion of LTR retrotransposons occurs more slowly than in other vertebrates with smaller genomes.

  17. GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

    Directory of Open Access Journals (Sweden)

    Eva Weber

    Full Text Available Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3 (- as the first ionic interaction partner, but not necessarily for Ca(2+. The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

  18. Synthesis and properties of the para-trimethylammonium analogues of green fluorescence protein (GFP) chromophore: The mimic of protonated GFP chromophore.

    Science.gov (United States)

    Fanjiang, Ming-Wei; Li, Ming-Ju; Sung, Robert; Sung, Kuangsen

    2018-04-01

    At low pH, protons from the external, bulk solution can protonate the phenoxide group of the p-HBDI chromophore in wild-type green fluorescent protein (wtGFP) and its mutants, and likely continue to tentatively protonate the phenol hydroxyl group of the same chromophores. Because the protonated GFP chromophore is a transient, we prepare the stable p-trimethylammonium analogues (2a and 2b) of the GFP chromophore to mimic it and explore their properties. What we found is that the p-trimethylammonium analogues of the GFP chromophore have the highly electrophilic amidine carbon, blue-shifted electronic absorption, smaller molar absorptivity, smaller fluorescent quantum yield, and faster E-Z thermoisomerization rate. The amidine carbon of the p-trimethylammonium analogue (2b) of the GFP chromophore is the only site that is attacked by very weak nucleophile of water, resulting in ring-opening of the imidazolinone moiety. The half-life of its decay rate in D 2 O is around 33 days. Actually, acid-catalyzed hydrolysis of p-HBDI also results in ring-opening of the imidazolinone moiety. The ratio of the acid-catalyzed hydrolysis rate constants [k obs (p-HBDI)/k obs (1b)] between p-HBDI and 1b (p-dimethylammonium analogue of the GFP chromophore) is dramatically increased from 0.30 at pH = 2 to 0.63 at pH = 0. This is the evidence that more and more phenol hydroxyl groups of p-HBDI are tentatively protonated in a low-pH aqueous solution and that accelerates hydrolysis of p-HBDI in the way similar to the quaternary ammonium derivatives 2a and 2b in water. With this view point, 2a and 2b still can partially mimic the cationic p-HBDI with the protonated phenol hydroxyl group. Implication of the experiment is that the amidine carbon of the chromophore in wtGFP and its mutants at very low pH should be highly electrophilic. Whether ring-opening of the imidazolinone moiety of the GFP chromophore would occur or not depends on if water molecules can reach the amidine carbon of

  19. Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo.

    Directory of Open Access Journals (Sweden)

    Claudia Laperchia

    Full Text Available Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype.With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity of dendritic cells (DCs, and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.

  20. Reproductive biology of the Del Norte salamander (Plethodon elongatus).

    Science.gov (United States)

    Clara A. Wheeler; Hartwell H. Welsh Jr.; Lisa M. Ollivier

    2013-01-01

    We examined seasonal reproductive patterns of the Del Norte Salamander, Plethodon elongatus, in mixed conifer and hardwood forests of northwestern California and southwestern Oregon. Seasonal size differences in reproductive structures suggested that maximum spermatogenic activity occurred during the late summer, with spermatozoa transfer to the...

  1. Overview of the status of the Cheat Mountain salamander

    Science.gov (United States)

    Thomas K. Pauley

    2010-01-01

    Plethodon nettingi, the Cheat Mountain salamander, is endemic to the high elevations of the Allegheny Mountains in eastern West Virginia. In 1938, N.B. Green named the species from specimens collected at Barton Knob, Randolph County, in honor of his friend and colleague Graham Netting.

  2. Phylogeography of Sardinian cave salamanders (genus Hydromantes) is mainly determined by geomorphology.

    Science.gov (United States)

    Chiari, Ylenia; van der Meijden, Arie; Mucedda, Mauro; Lourenço, João M; Hochkirch, Axel; Veith, Michael

    2012-01-01

    Detecting the factors that determine the interruption of gene flow between populations is key to understanding how speciation occurs. In this context, caves are an excellent system for studying processes of colonization, differentiation and speciation, since they represent discrete geographical units often with known geological histories. Here, we asked whether discontinuous calcareous areas and cave systems represent major barriers to gene flow within and among the five species of Sardinian cave salamanders (genus Hydromantes) and whether intraspecific genetic structure parallels geographic distance within and among caves. We generated mitochondrial cytochrome b gene sequences from 184 individuals representing 48 populations, and used a Bayesian phylogeographic approach to infer possible areas of cladogenesis for these species and reconstruct historical and current dispersal routes among distinct populations. Our results show deep genetic divergence within and among all Sardinian cave salamander species, which can mostly be attributed to the effects of mountains and discontinuities in major calcareous areas and cave systems acting as barriers to gene flow. While these salamander species can also occur outside caves, our results indicate that there is a very poor dispersal of these species between separate cave systems.

  3. Data set for transcriptome analysis of the Chinese giant salamander (Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xuemei Jiang

    2016-03-01

    Full Text Available The Chinese giant salamander (Andrias davidianus occupies a seat at the phylogenetic and species evolution process, which makes it an invaluable model for genetics; however, the genetic information and gene sequences about the Chinese giant salamander in public databases are scanty. Hence, we aimed to perform transcriptome analysis with the help of high-throughput sequencing. In this data, 61,317,940 raw reads were acquired from Chinese giant salamander mRNA using Illumina paired-end sequencing platform. After de novo assembly, a total of 72,072 unigenes were gained, in which 33,834 (46.95% and 29,479 (40.91% transcripts exhibited homology to sequences in the Nr database and Swiss-Prot database, (E-value <10−5, respectively. In the obtained unigenes, 18,019 (25% transcripts were assigned with at least one Gene Ontology term, of which 1218 (6.8% transcripts were assigned to immune system processes. In addition, a total of 17,572 assembled sequences were assigned into 241 predicted KEGG metabolic pathways. Among these, 2552 (14.5% transcripts were assigned to the immune system relevant pathway and 5 transcripts were identified as potential antimicrobial peptides (AMPs. Keywords: Andrias davidianus, Transcriptome

  4. Infection of spotted salamanders (Ambystoma maculatum) with Ichthyophonus-like organisms in Virginia.

    Science.gov (United States)

    Ware, Joy L; Viverette, Cathy; Kleopfer, John D; Pletcher, Leeanna; Massey, Davis; Wright, Anne

    2008-01-01

    Ichthyophonus-like organisms were found in two free-ranging adult spotted salamanders (Ambystoma maculatum) captured within two different vernal ponds in the Virginia Commonwealth University Rice Center for Environmental Life Sciences in Charles City County, Virginia. Histopathologic examination of necropsied specimens revealed large spores, often enclosed by granulomas. These enclosed spores resembled those caused by the fish pathogen Ichthyophonus hoeferi. One salamander displayed an externally visible large swelling beneath the jaws. The other lacked macroscopic abnormalities, but histologic sections of ventral muscle revealed early-stage Ichthyophonus-like organisms and minimal granulomatous reactions. This is the first report of Ichthyophonus-like infection of Ambystoma maculatum in Virginia.

  5. Evolving trends in biosciences: Multi-purpose proteins - GFP and GFP-like proteins

    Digital Repository Service at National Institute of Oceanography (India)

    Krishna, K.; Ingole, B.S.

    The sea is considered as holding a clue to many known and unknown biologically active compounds. A family of protein named Green Fluorescent Proteins (GFP)-like proteins, initially isolated from marine organisms, started a trend in biotechnological...

  6. Energy profile of nanobody-GFP complex under force

    Science.gov (United States)

    Klamecka, Kamila; Severin, Philip M.; Milles, Lukas F.; Gaub, Hermann E.; Leonhardt, Heinrich

    2015-10-01

    Nanobodies (Nbs)—the smallest known fully functional and naturally occuring antigen-binding fragments—have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb-green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that—despite identical epitopes—the Nb binds stronger (41-56 pN) to enhanced GFP than to wild-type GFP (28-45 pN). Measured forces make the Nb-GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo.

  7. Energy profile of nanobody-GFP complex under force.

    Science.gov (United States)

    Klamecka, Kamila; Severin, Philip M; Milles, Lukas F; Gaub, Hermann E; Leonhardt, Heinrich

    2015-09-10

    Nanobodies (Nbs)-the smallest known fully functional and naturally occuring antigen-binding fragments-have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb-green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that-despite identical epitopes-the Nb binds stronger (41-56 pN) to enhanced GFP than to wild-type GFP (28-45 pN). Measured forces make the Nb-GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo.

  8. Deployment of a Prototype Plant GFP Imager at the Arthur Clarke Mars Greenhouse of the Haughton Mars Project

    Directory of Open Access Journals (Sweden)

    Robert J. Ferl

    2008-04-01

    Full Text Available The use of engineered plants as biosensors has made elegant strides in the past decades, providing keen insights into the health of plants in general and particularly in the nature and cellular location of stress responses. However, most of the analytical procedures involve laboratory examination of the biosensor plants. With the advent of the green fluorescence protein (GFP as a biosensor molecule, it became at least theoretically possible for analyses of gene expression to occur telemetrically, with the gene expression information of the plant delivered to the investigator over large distances simply as properly processed fluorescence images. Spaceflight and other extraterrestrial environments provide unique challenges to plant life, challenges that often require changes at the gene expression level to accommodate adaptation and survival. Having previously deployed transgenic plant biosensors to evaluate responses to orbital spaceflight, we wished to develop the plants and especially the imaging devices required to conduct such experiments robotically, without operator intervention, within extraterrestrial environments. This requires the development of an autonomous and remotely operated plant GFP imaging system and concomitant development of the communications infrastructure to manage dataflow from the imaging device. Here we report the results of deploying a prototype GFP imaging system within the Arthur Clarke Mars Greenhouse (ACMG an autonomously operated greenhouse located within the Haughton Mars Project in the Canadian High Arctic. Results both demonstrate the applicability of the fundamental GFP biosensor technology and highlight the difficulties in collecting and managing telemetric data from challenging deployment environments.

  9. Energy profile of nanobody–GFP complex under force

    International Nuclear Information System (INIS)

    Klamecka, Kamila; Severin, Philip M; Milles, Lukas F; Gaub, Hermann E; Leonhardt, Heinrich

    2015-01-01

    Nanobodies (Nbs)—the smallest known fully functional and naturally occuring antigen-binding fragments—have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb–green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that—despite identical epitopes—the Nb binds stronger (41–56 pN) to enhanced GFP than to wild-type GFP (28–45 pN). Measured forces make the Nb–GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo. (paper)

  10. The phenology of a rare salamander (Salamandra infraimmaculata in a population breeding under unpredictable ambient conditions: a 25 year study

    Directory of Open Access Journals (Sweden)

    Michael R. Warburg

    2007-11-01

    Full Text Available This is a long-term study (1974-1999 on the phenology of the rare, xeric- inhabiting salamander Salamandra infraimmaculata in a small isolated population during the breeding season near the breeding ponds on Mt. Carmel. This is a fringe area of the genus’ south-easternmost Palaearctic distribution. Salamanders were captured during the 25 year long study. The first years up to the 1980s the total number of salamanders increased but during the last years there seems to have been a decline. Although this could be a phase in normal population cyclic oscillations nevertheless when compared with long-term data on a European Salamandra it does not seem so. The interpretation of the species’ status is dependent on numbers of salamanders captured as well as on the duration of the study. These subjects are reviewed and discussed in this paper.

  11. From biomedicine to natural history research: EST resources for ambystomatid salamanders

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2004-08-01

    Full Text Available Abstract Background Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum and Eastern tiger salamander (A. tigrinum tigrinum, species with deep and diverse research histories. Results Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. Conclusions Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

  12. From biomedicine to natural history research: EST resources for ambystomatid salamanders

    Science.gov (United States)

    Putta, Srikrishna; Smith, Jeramiah J; Walker, John A; Rondet, Mathieu; Weisrock, David W; Monaghan, James; Samuels, Amy K; Kump, Kevin; King, David C; Maness, Nicholas J; Habermann, Bianca; Tanaka, Elly; Bryant, Susan V; Gardiner, David M; Parichy, David M; Voss, S Randal

    2004-01-01

    Background Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories. Results Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. Conclusions Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research. PMID:15310388

  13. Abundance of western red-backed salamanders (Plethodon vehiculum) in the Washington Coast Range after headwater stream-buffer manipulation

    Science.gov (United States)

    Randall J. Wilk; Jeffrey D. Ricklefs; Martin G. Raphael

    2014-01-01

    We evaluated the effect of forest riparian alternative tree buffer designs on Western Red-backed Salamanders (Plethodon vehiculum) along headwater stream banks in managed forests of the Washington Coast Range. We used pit trap live removals in early autumn to estimate relative abundances of surface-active salamanders before and after 3 levels of riparian buffer...

  14. A new protein-protein interaction sensor based on tripartite split-GFP association.

    Science.gov (United States)

    Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-10-04

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

  15. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    CERN Document Server

    Awazu, K; Tamiya, E

    2002-01-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 mu m. The maximum transfer efficiency was about 0.5%.

  16. Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

    International Nuclear Information System (INIS)

    Moen, Ingrid; Øyan, Anne M; Stuhr, Linda EB; Jevne, Charlotte; Wang, Jian; Kalland, Karl-Henning; Chekenya, Martha; Akslen, Lars A; Sleire, Linda; Enger, Per Ø; Reed, Rolf K

    2012-01-01

    The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O 2 , à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway

  17. Heterogeneous vesicles in mucous epithelial cells of posterior esophagus of Chinese giant salamander (Andrias davidianus

    Directory of Open Access Journals (Sweden)

    H. Zhang

    2015-08-01

    Full Text Available The Chinese giant salamander belongs to an old lineage of salamanders and endangered species. Many studies of breeding and disease regarding this amphibian had been implemented. However, the studies on the ultrastructure of this amphibian are rare. In this work, we provide a histological and ultrastructural investigation on posterior esophagus of Chinese giant salamander. The sections of amphibian esophagus were stained by hematoxylin & eosin (H&E. Moreover, the esophageal epithelium was observed by transmission electron microscopy (TEM. The results showed that esophageal epithelium was a single layer epithelium, which consisted of mucous cells and columnar cells. The esophageal glands were present in submucosa. The columnar cells were ciliated. According to the diverging ultrastructure of mucous vesicles, three types of mucous cells could be identified in the esophageal mucosa: i electron-lucent vesicles mucous cell (ELV-MC; ii electron-dense vesicles mucous cell (EDV-MC; and iii mixed vesicles mucous cell (MV-MC.

  18. Cave exploitation by an usual epigean species: a review on the current knowledge on fire salamander breeding in cave

    Directory of Open Access Journals (Sweden)

    Raoul Manenti

    2017-11-01

    Full Text Available The fire salamander (Salamandra salamandra is a relatively common epigean amphibian, widely distributed throughout Europe, which usually gives birth to aquatic larvae. Even if epigean streams represent the most common places in which the species breeds, in some countries caves with underground waters are also used. To improve our understanding of the habitat features allowing successful breeding of salamanders in underground sites, we combined an exhaustive review of the available literature, especially the grey one, with direct observations performed from 2008 to 2017 in several natural and artificial caves of Lombardy, Liguria and Tuscany (Italy, Ariège and Provence (France. We provide a synthesis of published and unpublished caves in which the fire salamander breeding has been observed, along with a synthesis of the investigated ecological, behavioural and morphological traits. The use of underground sites is reported in several published papers and appears to be a common phenomenon not limited to single karst areas. The absence of predators, the relative stability of the aquatic habitats and the possibility to exploit new ecological resources are environmental factors that favour the breeding of the fire salamander. Our synthesis suggests that breeding of fire salamanders in caves is not a random event, but a widespread phenomenon that may be linked to specific biogeographical factors. Further insights may be obtained by performing genetic analyses on both cave and epigean populations, and considering larger landscape scales for ecological studies as well. Gene flow between salamanders that breed in caves and in streams probably occurs, but on the other hand, assortative mating might limit it, thus allowing the conservation of local adaptations driving successful cave colonisation.

  19. Stoichiometry of excreta and excretion rates of a stream-dwelling plethodontid salamander

    Data.gov (United States)

    U.S. Environmental Protection Agency — Stoichiometry of excreta and excretion rates of a stream-dwelling plethodontid salamander in Cincinnati, OH, USA. This dataset is associated with the following...

  20. Propulsive forces of mudskipper fins and salamander limbs during terrestrial locomotion: implications for the invasion of land.

    Science.gov (United States)

    Kawano, Sandy M; Blob, Richard W

    2013-08-01

    The invasion of land was a pivotal event in vertebrate evolution that was associated with major appendicular modifications. Although fossils indicate that the evolution of fundamentally limb-like appendages likely occurred in aquatic environments, the functional consequences of using early digited limbs, rather than fins, for terrestrial propulsion have had little empirical investigation. Paleontological and experimental analyses both have led to the proposal of an early origin of "hind limb-driven" locomotion among tetrapods or their ancestors. However, the retention of a pectoral appendage that had already developed terrestrial adaptations has been proposed for some taxa, and few data are available from extant functional models that can provide a foundation for evaluating the relative contributions of pectoral and pelvic appendages to terrestrial support among early stem tetrapods. To examine these aspects of vertebrate locomotor evolution during the invasion of land, we measured three-dimensional ground reaction forces (GRFs) produced by isolated pectoral fins of mudskipper fishes (Periophthalmus barbarus) during terrestrial crutching, and compared these to isolated walking footfalls by the forelimbs and hind limbs of tiger salamanders (Ambystoma tigrinum), a species with subequally-sized limbs that facilitate comparisons to early tetrapods. Pectoral appendages of salamanders and mudskippers exhibited numerous differences in GRFs. Compared with salamander forelimbs, isolated fins of mudskippers bear lower vertical magnitudes of GRFs (as a proportion of body weight), and had GRFs that were oriented more medially. Comparing the salamanders' forelimbs and hind limbs, although the peak net GRF occurs later in stance for the forelimb, both limbs experience nearly identical mediolateral and vertical components of GRF, suggesting comparable contributions to support. Thus, forelimbs could also have played a significant locomotor role among basal tetrapods that had limbs

  1. Recombinant nAG (a Salamander-Derived Protein Decreases the Formation of Hypertrophic Scarring in the Rabbit Ear Model

    Directory of Open Access Journals (Sweden)

    Mohammad M. Al-Qattan

    2014-01-01

    Full Text Available nAG (newt-Anterrior Gradient protein is the key mediator of regrowth of amputated limbs in salamanders. In a previous work in our lab, a new nAG gene (suitable for humans was designed and cloned. The cloned vector was transfected into primary human fibroblasts. The expression of nAG in human primary fibroblasts was found to suppress collagen expression. The current study shows that local injection of recombinant nAG reduces scar hypertrophy in the rabbit ear model. This is associated with lower scar elevation index (SEI, lower levels of collagen I & III, higher levels of MMP1, and a higher degree of scar maturation in experimental wounds compared to controls.

  2. [Construction and identification of eukaryotic plasmid pGC-silencer-U6/Neo/GFP/ABCG2].

    Science.gov (United States)

    Yu, Yanping; Zhang, Song; Kong, Weijia

    2010-09-01

    To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, and to detect the RNAi effect of shRNA. Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The recombinant plasmids were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and Western blot. The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed. The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells, at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3 (P < 0.05), with an inhibition ratio of 75% at the mRNA level and 68% at the protein level. pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.

  3. CRISPR/Cas9 Mediated GFP Knock-in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy.

    Science.gov (United States)

    Wu, Zhiqiang; Zhao, Jinlin; Qiu, Minghan; Mi, Zeyun; Meng, Maobin; Guo, Yu; Wang, Hui; Yuan, Zhiyong

    2018-04-19

    Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignores the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable. Here, we generated a knock-in GFP-LC3 reporter via the CRISPR/Cas9 system in 293FT cells to add GFP to the N-terminal of and in frame with endogenous LC3. We proved that this knock-in GFP-LC3 was expressed at biological level driven by the endogenous transcriptional regulatory elements as the wild type alleles. Compared with the ectopically expressed GFP-LC3, the endogenous knock-in reporter exhibited much higher sensitivity and signal-to-noise ratio of GFP-LC3 puncta upon the induction or inhibition of autophagy at certain step for monitoring autophagy activity. Thus, according to the previous reported concerning and the results presented here, we suggest that this knock-in GFP-LC3 reporter is better for bona fide monitoring cellular autophagy and should be employed for further study of autophagy in vitro and in vivo. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice - including relationship of sex differences

    International Nuclear Information System (INIS)

    Ogawa, Rei; Mizuno, Hiroshi; Watanabe, Atsushi; Migita, Makoto; Hyakusoku, Hiko; Shimada, Takashi

    2004-01-01

    We have previously demonstrated that adipose-derived stromal cells (ASCs) as well as bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. Both types are considered to include mesenchymal stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have also previously reported the plasticity of BSCs and ASCs. In this study, we focused on adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice. Moreover, preadipocytes and mature adipocytes were harvested at the same time, and the cells were cultured to compare them with ASCs. Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs, preadipocytes, and mature adipocytes. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks. Adipogenic differentiation of ASCs was assessed by Oil Red O staining and the expression of the adipocyte specific peroxisome proliferative activated receptor γ2 (PPAR-γ2) gene. These ASCs stained positively, and expression of PPAR-γ2 was detected. Moreover, we also tried to characterize the influence of sex differences on the adipogenic differentiation of ASCs harvested from both male and female mice. This was assessed by the expression levels of the PPAR-γ2 gene using real-time PCR. The results showed that the expression levels of ASCs harvested from female mice were a maximum of 2.89 times greater than those harvested from male mice. This suggests that the adipogenic differentiation of ASCs is closely related to sex differences

  5. Efficacy of riparian buffers in mitigating local populations declines and the effects of even-aged timber harvest on larval salamanders

    Science.gov (United States)

    William E Peterman; Raymond D. Semlitch

    2009-01-01

    Headwater streams are an important and prevalent feature of the eastern North American landscape.These streams provide a wealth of ecosystem services and support tremendous biological diversity, which is predominated by salamanders in the Appalachian region. Salamanders are ubiquitous throughout the region, contributing a significant...

  6. Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy

    NARCIS (Netherlands)

    Vermeer, J.E.M.; van Munster, E.B.; Vischer, N.O.; Gadella, T.

    2004-01-01

    Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region

  7. Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH fusion to gfp (green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Bat-Erdene Jugder

    2016-07-01

    Full Text Available Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2. Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

  8. Effects of acid precipitation on embryonic mortality of Ambystoma salamanders in the Connecticut Valley of Massachusetts

    Energy Technology Data Exchange (ETDEWEB)

    Cook, R P

    1983-01-01

    An investigation of increased embryonic mortality of the spotted salamander Ambystoma maculatum concomitant with breeding pond acidification from acid rainfall in the Connecticut Valley of Massachusetts analyzes the pH and chemistry of rain and pond water and monitored embryonic mortality in 1976 and 1977. Although acid rain continues to occur in the area and Ambystoma breeding ponds are acidic, the average pH of six ponds dropped from 5.62 to 5.10 during the study. Pond pH decreased up to 0.75 pH units following heavy rainfall. Despite this, embryonic mortality of spotted and Jefferson salamanders was low, and no significant correlation between pond pH and percent embryonic mortality was found. The size of present populations and the embryonic acid tolerance exhibited by the salamander indicate that acid rain has not had an effect in this location. 22 references, 2 figures, 4 tables.

  9. Road deicing salt irreversibly disrupts osmoregulation of salamander egg clutches

    International Nuclear Information System (INIS)

    Karraker, Nancy E.; Gibbs, James P.

    2011-01-01

    It has been postulated that road deicing salts are sufficiently diluted by spring rains to ameliorate any physiological impacts to amphibians breeding in wetlands near roads. We tested this conjecture by exposing clutches of the spotted salamander (Ambystoma maculatum) to three chloride concentrations (1 mg/L, 145 mg/L, 945 mg/L) for nine days, then transferred clutches to control water for nine days, and measured change in mass at three-day intervals. We measured mass change because water uptake by clutches reduces risks to embryos associated with freezing, predation, and disease. Clutches in controls sequestered water asymptotically. Those in the moderate concentrations lost 18% mass initially and regained 14% after transfer to control water. Clutches in high concentration lost 33% mass and then lost an additional 8% after transfer. Our results suggest that spring rains do not ameliorate the effects of deicing salts in wetlands with extremely high chloride concentrations. - Road deicing salts irreversibly disrupts osmoregulation of salamander egg clutches.

  10. Road deicing salt irreversibly disrupts osmoregulation of salamander egg clutches

    Energy Technology Data Exchange (ETDEWEB)

    Karraker, Nancy E., E-mail: karraker@hku.hk [Department of Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, NY 13210 (United States); Gibbs, James P [Department of Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, NY 13210 (United States)

    2011-03-15

    It has been postulated that road deicing salts are sufficiently diluted by spring rains to ameliorate any physiological impacts to amphibians breeding in wetlands near roads. We tested this conjecture by exposing clutches of the spotted salamander (Ambystoma maculatum) to three chloride concentrations (1 mg/L, 145 mg/L, 945 mg/L) for nine days, then transferred clutches to control water for nine days, and measured change in mass at three-day intervals. We measured mass change because water uptake by clutches reduces risks to embryos associated with freezing, predation, and disease. Clutches in controls sequestered water asymptotically. Those in the moderate concentrations lost 18% mass initially and regained 14% after transfer to control water. Clutches in high concentration lost 33% mass and then lost an additional 8% after transfer. Our results suggest that spring rains do not ameliorate the effects of deicing salts in wetlands with extremely high chloride concentrations. - Road deicing salts irreversibly disrupts osmoregulation of salamander egg clutches.

  11. Distribution of the Sonora Tiger Salamander (Ambystoma mavortium stebbinsi) in Mexico

    Science.gov (United States)

    Hossack, Blake R.; Muths, Erin L.; Rorabaugh, James C.; Lemos Espinal, Julio A.; Sigafus, Brent H.; Chambert, Thierry A.; Carreon Arroyo, Gerardo; Hurtado Felix, David; Toyos Martinez, Daniel; Jones, Thomas R.

    2016-01-01

    The Sonoran Tiger Salamander (Ambystoma mavortium stebbinsi Lowe, 1954) was listed as federally endangered in the USA in 1997 (USFWS 1997). In the USA, the distribution of A. mavortium stebbinsi is limited to the San Rafael Valley (approximately 567 km2), between the Sierra San Antonio (called the Patagonia Mountains in Arizona) and Huachuca Mountains, and south of the Canelo Hills, Arizona (Fig. 1). The USA listing was triggered by loss of natural wetland habitats, threats from invasive predators, frequent die-offs from disease, introgression with the introduced Barred Tiger Salamander (A. mavortium mavortium), and small range and number of breeding sites that increases susceptibility to stochastic events (USFWS 1997). Small population sizes and limited gene flow have caused inbreeding, which may further reduce population viability and the potential for recovery (Jones et al. 1988; Storfer et al. 2014). 

  12. Proton transfer events in GFP.

    Science.gov (United States)

    Di Donato, Mariangela; van Wilderen, Luuk J G W; Van Stokkum, Ivo H M; Stuart, Thomas Cohen; Kennis, John T M; Hellingwerf, Klaas J; van Grondelle, Rienk; Groot, Marie Louise

    2011-09-28

    Proton transfer is one of the most important elementary processes in biology. Green fluorescent protein (GFP) serves as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. Illumination initiates proton transfer through a 'proton-wire', formed by the chromophore (the proton donor), water molecule W22, Ser205 and Glu222 (the acceptor), on a picosecond time scale. To obtain a more refined view of this process, we have used a combined approach of time resolved mid-infrared spectroscopy and visible pump-dump-probe spectroscopy to resolve with atomic resolution how and how fast protons move through this wire. Our results indicate that absorption of light by GFP induces in 3 ps (10 ps in D(2)O) a shift of the equilibrium positions of all protons in the H-bonded network, leading to a partial protonation of Glu222 and to a so-called low barrier hydrogen bond (LBHB) for the chromophore's proton, giving rise to dual emission at 475 and 508 nm. This state is followed by a repositioning of the protons on the wire in 10 ps (80 ps in D(2)O), ultimately forming the fully deprotonated chromophore and protonated Glu222.

  13. Egg predators of an endemic Italian salamander, Salamandrina perspicillata (Savi, 1821

    Directory of Open Access Journals (Sweden)

    Antonio Romano

    2008-05-01

    Full Text Available We report new aquatic predators feeding on Northern spectacled salamander eggs, Salamandrina perspicillata, an endemic Italian species. Eggs were preyed upon by the leech, Trocheta bykowskii, and the trichopteran larvae of Potamophylax cingulatus and Halesus appenninus.

  14. Stylophora pistillata in the Red Sea demonstrate higher GFP fluorescence under ocean acidification conditions

    Science.gov (United States)

    Grinblat, Mila; Fine, Maoz; Tikochinski, Yaron; Loya, Yossi

    2018-03-01

    Ocean acidification is thought to exert a major impact on calcifying organisms, including corals. While previous studies have reported changes in the physiological response of corals to environmental change, none have described changes in expression of the ubiquitous host pigments—fluorescent proteins (FPs)—to ocean acidification. The function of FPs in corals is controversial, with the most common consideration being that these primarily regulate the light environment in the coral tissue and protect the host from harmful UV radiation. Here, we provide for the first time experimental evidence that increased fluorescence of colonies of the coral Stylophora pistillata is independent of stress and can be regulated by a non-stressful decrease in pH. Stylophora pistillata is the most abundant and among the most resilient coral species in the northern Gulf of Eilat/Aqaba (GoE/A). Fragmented "sub-colonies" ( n = 72) incubated for 33 days under three pH treatments (ambient, 7.9, and 7.6), under ambient light, and running seawater showed no stress or adverse physiological performance, but did display significantly higher fluorescence, with lower pH. Neither the average number of planulae shed from the experimental sub-colonies nor planulae green fluorescent protein (GFP) expression changed significantly among pH treatments. Sub-colonies incubated under the lower-than-ambient pH conditions showed an increase in both total protein and GFP expression. Since extensive protein synthesis requires a high level of transcription, we suggest that GFP constitutes a UV protection mechanism against potential RNA as well as against DNA damage caused by UV exposure. Manipulating the regulation of FPs in adult corals and planulae, under controlled and combined effects of pH, light, and temperature, is crucial if we are to obtain a better understanding of the role played by this group of proteins in cnidarians.

  15. In search of critically endangered species: the current situation of two tiny salamander species in the Neotropical mountains of Mexico.

    Science.gov (United States)

    Sandoval-Comte, Adriana; Pineda, Eduardo; Aguilar-López, José L

    2012-01-01

    Worldwide, one in every three species of amphibian is endangered, 39 species have gone extinct in the last 500 years and another 130 species are suspected to have gone extinct in recent decades. Of the amphibians, salamanders have the highest portion of their species in one of the risk categories, even higher than the frogs. To date there have been few studies that have used recent field data to examine the status of populations of endangered salamanders. In this study we evaluate the current situation of two tiny salamanders, Parvimolge townsendi and Thorius pennatulus, both of which are distributed at intermediate elevations in the mountains of the northern Neotropics and are considered to be critically endangered; the first has been proposed as possibly extinct. By carrying out exhaustive surveys in both historical and potentially suitable sites for these two species, we evaluated their abundance and the characteristics of their habitats, and we estimated their potential geographic distribution. We visited 22 sites, investing 672 person-hours of sampling effort in the surveys, and found 201 P. townsendi salamanders in 11 sites and only 13 T. pennatulus salamanders in 5 sites. Both species were preferentially found in cloud forest fragments that were well conserved or only moderately transformed, and some of the salamanders were found in shade coffee plantations. The potential distribution area of both species is markedly fragmented and we estimate that it has decreased by more than 48%. The results of this study highlight the importance of carrying out exhaustive, systematic field surveys to obtain accurate information about the current situation of critically endangered species, and help us better understand the crisis that amphibians are facing worldwide.

  16. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    International Nuclear Information System (INIS)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

    2007-01-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

  17. Dose rate estimation of the Tohoku hynobiid salamander, Hynobius lichenatus, in Fukushima.

    Science.gov (United States)

    Fuma, Shoichi; Ihara, Sadao; Kawaguchi, Isao; Ishikawa, Takahiro; Watanabe, Yoshito; Kubota, Yoshihisa; Sato, Youji; Takahashi, Hiroyuki; Aono, Tatsuo; Ishii, Nobuyoshi; Soeda, Haruhi; Matsui, Kumi; Une, Yumi; Minamiya, Yukio; Yoshida, Satoshi

    2015-05-01

    The radiological risks to the Tohoku hynobiid salamanders (class Amphibia), Hynobius lichenatus due to the Fukushima Dai-ichi Nuclear Power Plant accident were assessed in Fukushima Prefecture, including evacuation areas. Aquatic egg clutches (n = 1 for each sampling date and site; n = 4 in total), overwintering larvae (n = 1-5 for each sampling date and site; n = 17 in total), and terrestrial juveniles or adults (n = 1 or 3 for each sampling date and site; n = 12 in total) of H. lichenatus were collected from the end of April 2011 to April 2013. Environmental media such as litter (n = 1-5 for each sampling date and site; n = 30 in total), soil (n = 1-8 for each sampling date and site; n = 31 in total), water (n = 1 for each sampling date and site; n = 17 in total), and sediment (n = 1 for each sampling date and site; n = 17 in total) were also collected. Activity concentrations of (134)Cs + (137)Cs were 1.9-2800, 0.13-320, and 0.51-220 kBq (dry kg) (-1) in the litter, soil, and sediment samples, respectively, and were 0.31-220 and <0.29-40 kBq (wet kg)(-1) in the adult and larval salamanders, respectively. External and internal absorbed dose rates to H. lichenatus were calculated from these activity concentration data, using the ERICA Assessment Tool methodology. External dose rates were also measured in situ with glass dosimeters. There was agreement within a factor of 2 between the calculated and measured external dose rates. In the most severely contaminated habitat of this salamander, a northern part of Abukuma Mountains, the highest total dose rates were estimated to be 50 and 15 μGy h(-1) for the adults and overwintering larvae, respectively. Growth and survival of H. lichenatus was not affected at a dose rate of up to 490 μGy h(-1) in the previous laboratory chronic gamma-irradiation experiment, and thus growth and survival of this salamander would not be affected, even in the most severely contaminated habitat in Fukushima Prefecture. However, further

  18. Mechanics of lung ventilation in a large aquatic salamander, siren lacertina

    Science.gov (United States)

    Brainerd; j

    1998-06-01

    Lung ventilation in Siren lacertina was studied using X-ray video, measurements of body cavity pressure and electromyography of hypaxial muscles. S. lacertina utilizes a two-stroke buccal pump in which mixing of expired and inspired gas is minimized by partial expansion of the buccal cavity during exhalation and then full expansion after exhalation is complete. Mixing is further reduced by the use of one or two accessory inspirations after the first, mixed-gas cycle. Exhalation occurs in two phases: a passive phase in which hydrostatic pressure and possibly lung elasticity force air out of the lungs, and an active phase in which contraction of the transverse abdominis (TA) muscle increases body cavity pressure and forces most of the remaining air out. In electromyograms of the lateral hypaxial musculature, the TA became active 200-400 ms before the rise in body cavity pressure, and activity ceased at peak pressure. The TA was not active during inspiration, and no consistent activity during breathing was noted in the external oblique, internal oblique and rectus abdominis muscles. The finding that the TA is the primary expiratory muscle in S. lacertina agrees with findings in a previous study of another salamander, Necturus maculosus. Together, these results indicate that the use of the TA for exhalation is a primitive character for salamanders and support the hypothesis that the breathing mechanism of salamanders represents an intermediate step in evolution between a buccal pump, in which only head muscles are used for ventilation, and an aspiration pump, in which axial muscles are used for both exhalation and inhalation.

  19. Precise control of miR-125b levels is required to create a regeneration-permissive environment after spinal cord injury: a cross-species comparison between salamander and rat

    Directory of Open Access Journals (Sweden)

    Juan Felipe Diaz Quiroz

    2014-06-01

    Full Text Available Most spinal cord injuries lead to permanent paralysis in mammals. By contrast, the remarkable regenerative abilities of salamanders enable full functional recovery even from complete spinal cord transections. The molecular differences underlying this evolutionary divergence between mammals and amphibians are poorly understood. We focused on upstream regulators of gene expression as primary entry points into this question. We identified a group of microRNAs (miRNAs that are conserved between the Mexican axolotl salamander (Ambystoma mexicanum and mammals but show marked cross-species differences in regulation patterns following spinal cord injury. We found that precise post-injury levels of one of these miRNAs (miR-125b is essential for functional recovery, and guides correct regeneration of axons through the lesion site in a process involving the direct downstream target Sema4D in axolotls. Translating these results to a mammalian model, we increased miR-125b levels in the rat through mimic treatments following spinal cord transection. These treatments downregulated Sema4D and other glial-scar-related genes, and enhanced the animal’s functional recovery. Our study identifies a key regulatory molecule conserved between salamander and mammal, and shows that the expression of miR-125b and Sema4D must be carefully controlled in the right cells at the correct level to promote regeneration. We also show that these molecular components of the salamander’s regeneration-permissive environment can be experimentally harnessed to improve treatment outcomes for mammalian spinal cord injuries.

  20. Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

    Directory of Open Access Journals (Sweden)

    Norazlina Ahmad

    2012-01-01

    Full Text Available Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

  1. In search of critically endangered species: the current situation of two tiny salamander species in the Neotropical mountains of Mexico.

    Directory of Open Access Journals (Sweden)

    Adriana Sandoval-Comte

    Full Text Available Worldwide, one in every three species of amphibian is endangered, 39 species have gone extinct in the last 500 years and another 130 species are suspected to have gone extinct in recent decades. Of the amphibians, salamanders have the highest portion of their species in one of the risk categories, even higher than the frogs. To date there have been few studies that have used recent field data to examine the status of populations of endangered salamanders. In this study we evaluate the current situation of two tiny salamanders, Parvimolge townsendi and Thorius pennatulus, both of which are distributed at intermediate elevations in the mountains of the northern Neotropics and are considered to be critically endangered; the first has been proposed as possibly extinct. By carrying out exhaustive surveys in both historical and potentially suitable sites for these two species, we evaluated their abundance and the characteristics of their habitats, and we estimated their potential geographic distribution. We visited 22 sites, investing 672 person-hours of sampling effort in the surveys, and found 201 P. townsendi salamanders in 11 sites and only 13 T. pennatulus salamanders in 5 sites. Both species were preferentially found in cloud forest fragments that were well conserved or only moderately transformed, and some of the salamanders were found in shade coffee plantations. The potential distribution area of both species is markedly fragmented and we estimate that it has decreased by more than 48%. The results of this study highlight the importance of carrying out exhaustive, systematic field surveys to obtain accurate information about the current situation of critically endangered species, and help us better understand the crisis that amphibians are facing worldwide.

  2. Ballistic tongue projection in a miniaturized salamander.

    Science.gov (United States)

    Deban, Stephen M; Bloom, Segall V

    2018-05-20

    Miniaturization of body size is often accompanied by peculiarities in morphology that can have functional consequences. We examined the feeding behavior and morphology of the miniaturized plethodontid salamander Thorius, one of the smallest vertebrates, to determine if its performance and biomechanics differ from those of its larger relatives. High-speed imaging and dynamics analysis of feeding at a range of temperatures show that tongue projection in Thorius macdougalli is ballistic and achieves accelerations of up to 600 G with low thermal sensitivity, indicating that tongue projection is powered by an elastic-recoil mechanism. Preceding ballistic projection is an unusual preparatory phase of tongue protrusion, which, like tongue retraction, shows lower performance and higher thermal sensitivity that are indicative of movement being powered directly by muscle shortening. The variability of tongue-projection kinematics and dynamics is comparable to larger ballistic-tongued plethodontids and reveals that Thorius is capable of modulating its tongue movements in response to prey distance. Morphological examination revealed that T. macdougalli possesses a reduced number of myofibers in the tongue muscles, a large projector muscle mass relative to tongue mass, and an unusual folding of the tongue skeleton, compared with larger relatives. Nonetheless, T. macdougalli retains the elaborated collagen aponeuroses in the projector muscle that store elastic energy and a tongue skeleton that is free of direct myofiber insertion, two features that appear to be essential for ballistic tongue projection in salamanders. © 2018 Wiley Periodicals, Inc.

  3. Function and structure of GFP-like proteins in the protein data bank.

    Science.gov (United States)

    Ong, Wayne J-H; Alvarez, Samuel; Leroux, Ivan E; Shahid, Ramza S; Samma, Alex A; Peshkepija, Paola; Morgan, Alicia L; Mulcahy, Shawn; Zimmer, Marc

    2011-04-01

    The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.

  4. Generation of chickens expressing Cre recombinase.

    Science.gov (United States)

    Leighton, Philip A; Pedersen, Darlene; Ching, Kathryn; Collarini, Ellen J; Izquierdo, Shelley; Jacob, Roy; van de Lavoir, Marie-Cecile

    2016-10-01

    Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.

  5. Conservation genetics of the endangered Shenandoah salamander (Plethodon shenandoah, Plethodontidae)

    Science.gov (United States)

    Carpenter, D.W.; Jung, R.E.; Sites, J.W.

    2001-01-01

    The Shenandoah salamander (Plethodon shenandoah) is restricted to three isolated talus outcrops in Shenandoah National Park, VA, USA and has one of the smallest ranges of any tetrapod vertebrate. This species was listed as endangered under the US Endangered Species Act in 1989 over concern that direct competition with the red-backed salamander (Plethodon cinereus), successional habitat changes, and human impacts may cause its decline and possible extinction. We address two issues herein: (1) whether extensive introgression (through long-term hybridization) is present between the two species and threatens the survival of P. shenandoah, and (2) the level of population structure within P. shenandoah. We provide evidence from mtDNA haplotypes that shows no genetic differentiation among the three isolates of P. shenandoah, suggesting that their fragmentation is a geologically recent event, and/or that the isolates are still connected by occasional gene flow. There is also no evidence for extensive introgression of alleles in either direction between P. cinereus and P. shenandoah, which suggests that P. shenandoah may not be in danger of being genetically swamped out through hybridization with P. cinereus.

  6. Taxonomy Icon Data: Japanese giant salamander [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Japanese giant salamander Andrias japonicus Chordata/Vertebrata/Amphibia Andrias_japonicus_L.png Andrias_jap...onicus_NL.png Andrias_japonicus_S.png Andrias_japonicus_NS.png http://biosciencedbc....jp/taxonomy_icon/icon.cgi?i=Andrias+japonicus&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Andrias+jap...onicus&t=NL http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Andrias+japonicus...&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Andrias+japonicus&t=NS ...

  7. An annotated review of the Salamander types described in the Fauna Japonica

    NARCIS (Netherlands)

    Hoogmoed, M.S.

    1978-01-01

    The whereabouts of the salamander types described by Temminck & Schlegel in the Fauna Japonica (1838) are discussed and lectotypes are selected from the syntypes for the following nominal species : Salamandra naevia Temminck & Schlegel, S. unguiculata Temminck & Schlegel, S. subcristata Temminck &

  8. Physical condition, sex, and age-class of eastern red-backed salamanders (Plethodon cinereus) in forested and open habitats of West Virginia, USA

    Science.gov (United States)

    Breanna L. Riedel; Kevin R. Russell; W. Mark Ford

    2012-01-01

    Nonforested habitats such as open fields and pastures have been considered unsuitable for desiccation-prone woodland salamanders such as the Eastern Red-backed Salamander (Plethodon cinereus). Recent research has suggested that Plethodon cinereus may not only disperse across but also reside within open habitats including fields,...

  9. Data congruence, paedomorphosis and salamanders

    Directory of Open Access Journals (Sweden)

    Struck Torsten H

    2007-10-01

    Full Text Available Abstract Background The retention of ancestral juvenile characters by adult stages of descendants is called paedomorphosis. However, this process can mislead phylogenetic analyses based on morphological data, even in combination with molecular data, because the assessment if a character is primary absent or secondary lost is difficult. Thus, the detection of incongruence between morphological and molecular data is necessary to investigate the reliability of simultaneous analyses. Different methods have been proposed to detect data congruence or incongruence. Five of them (PABA, PBS, NDI, LILD, DRI are used herein to assess incongruence between morphological and molecular data in a case study addressing salamander phylogeny, which comprises several supposedly paedomorphic taxa. Therefore, previously published data sets were compiled herein. Furthermore, two strategies ameliorating effects of paedomorphosis on phylogenetic studies were tested herein using a statistical rigor. Additionally, efficiency of the different methods to assess incongruence was analyzed using this empirical data set. Finally, a test statistic is presented for all these methods except DRI. Results The addition of morphological data to molecular data results in both different positions of three of the four paedomorphic taxa and strong incongruence, but treating the morphological data using different strategies ameliorating the negative impact of paedomorphosis revokes these changes and minimizes the conflict. Of these strategies the strategy to just exclude paedomorphic character traits seem to be most beneficial. Of the three molecular partitions analyzed herein the RAG1 partition seems to be the most suitable to resolve deep salamander phylogeny. The rRNA and mtDNA partition are either too conserved or too variable, respectively. Of the different methods to detect incongruence, the NDI and PABA approaches are more conservative in the indication of incongruence than LILD and

  10. Conservation genetics of extremely isolated urban populations of the northern dusky salamander (Desmognathus fuscus in New York City

    Directory of Open Access Journals (Sweden)

    Jason Munshi-South

    2013-04-01

    Full Text Available Urbanization is a major cause of amphibian decline. Stream-dwelling plethodontid salamanders are particularly susceptible to urbanization due to declining water quality and hydrological changes, but few studies have examined these taxa in cities. The northern dusky salamander (Desmognathus fuscus was once common in the New York City metropolitan area, but has substantially declined throughout the region in recent decades. We used five tetranucleotide microsatellite loci to examine population differentiation, genetic variation, and bottlenecks among five remnant urban populations of dusky salamanders in NYC. These genetic measures provide information on isolation, prevalence of inbreeding, long-term prospects for population persistence, and potential for evolutionary responses to future environmental change. All populations were genetically differentiated from each other, and the most isolated populations in Manhattan have maintained very little genetic variation (i.e. <20% heterozygosity. A majority of the populations also exhibited evidence of genetic bottlenecks. These findings contrast with published estimates of high genetic variation within and lack of structure between populations of other desmognathine salamanders sampled over similar or larger spatial scales. Declines in genetic variation likely resulted from population extirpations and the degradation of stream and terrestrial paths for dispersal in NYC. Loss of genetic variability in populations isolated by human development may be an underappreciated cause and/or consequence of the decline of this species in urbanized areas of the northeast USA.

  11. SPATIALLY AUTOCORRELATED DEMOGRAPHY AND INTERPOND MIGRATION IN THE CALIFORNIA TIGER SALAMANDER (AMBYSTOME CALIFORNIENSE)

    Science.gov (United States)

    We investigated the metapopulation structure of the California tiger salamander (Ambystoma californiense) using a combination of indirect and direct methods to evaluate two key requirements of modern metapopulation models: 1) that patches support somewhat independent populations ...

  12. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  13. Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

    Science.gov (United States)

    Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

    2006-11-15

    Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

  14. [Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

    Science.gov (United States)

    Shisha, E N; Korkhovoĭ, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

    2013-01-01

    The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind.

  15. Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.

    Science.gov (United States)

    Kakimoto, Yuriko; Tashiro, Shinya; Kojima, Rieko; Morozumi, Yuki; Endo, Toshiya; Tamura, Yasushi

    2018-04-18

    Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

  16. Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

    International Nuclear Information System (INIS)

    Wan Haiyan; Korzh, Svitlana; Li Zhen; Mudumana, Sudha Puttur; Korzh, Vladimir; Jiang Yunjin; Lin Shuo; Gong Zhiyuan

    2006-01-01

    In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development

  17. Comparing population patterns to processes: abundance and survival of a forest salamander following habitat degradation.

    Directory of Open Access Journals (Sweden)

    Clint R V Otto

    Full Text Available Habitat degradation resulting from anthropogenic activities poses immediate and prolonged threats to biodiversity, particularly among declining amphibians. Many studies infer amphibian response to habitat degradation by correlating patterns in species occupancy or abundance with environmental effects, often without regard to the demographic processes underlying these patterns. We evaluated how retention of vertical green trees (CANOPY and coarse woody debris (CWD influenced terrestrial salamander abundance and apparent survival in recently clearcut forests. Estimated abundance of unmarked salamanders was positively related to CANOPY (β Canopy  = 0.21 (0.02-1.19; 95% CI, but not CWD (β CWD  = 0.11 (-0.13-0.35 within 3,600 m2 sites, whereas estimated abundance of unmarked salamanders was not related to CANOPY (β Canopy  = -0.01 (-0.21-0.18 or CWD (β CWD  = -0.02 (-0.23-0.19 for 9 m2 enclosures. In contrast, apparent survival of marked salamanders within our enclosures over 1 month was positively influenced by both CANOPY and CWD retention (β Canopy  = 0.73 (0.27-1.19; 95% CI and β CWD  = 1.01 (0.53-1.50. Our results indicate that environmental correlates to abundance are scale dependent reflecting habitat selection processes and organism movements after a habitat disturbance event. Our study also provides a cautionary example of how scientific inference is conditional on the response variable(s, and scale(s of measure chosen by the investigator, which can have important implications for species conservation and management. Our research highlights the need for joint evaluation of population state variables, such as abundance, and population-level process, such as survival, when assessing anthropogenic impacts on forest biodiversity.

  18. Unexpected rarity of the pathogen Batrachochytrium dendrobatidis in Appalachian Plethodon Salamanders: 1957-2011.

    Directory of Open Access Journals (Sweden)

    Carly Muletz

    Full Text Available Widespread population declines in terrestrial Plethodon salamanders occurred by the 1980s throughout the Appalachian Mountains, the center of global salamander diversity, with no evident recovery. We tested the hypothesis that the historic introduction and spread of the pathogenic fungus Batrachochytrium dendrobatidis (Bd into the eastern US was followed by Plethodon population declines. We expected to detect elevated prevalence of Bd prior to population declines as observed for Central American plethodontids. We tested 1,498 Plethodon salamanders of 12 species (892 museum specimens, 606 wild individuals for the presence of Bd, and tested 94 of those for Batrachochytrium salamandrivorans (Bs and for ranavirus. Field samples were collected in 2011 from 48 field sites across a 767 km transect. Historic samples from museum specimens were collected at five sites with the greatest number and longest duration of collection (1957-987, four of which were sampled in the field in 2011. None of the museum specimens were positive for Bd, but four P. cinereus from field surveys were positive. The overall Bd prevalence from 1957-2011 for 12 Plethodon species sampled across a 757 km transect was 0.2% (95% CI 0.1-0.7%. All 94 samples were negative for Bs and ranavirus. We conclude that known amphibian pathogens are unlikely causes for declines in these Plethodon populations. Furthermore, these exceptionally low levels of Bd, in a region known to harbor Bd, may indicate that Plethodon specific traits limit Bd infection.

  19. A novel thermal decomposition approach to synthesize hydroxyapatite-silver nanocomposites and their antibacterial action against GFP-expressing antibiotic resistant E. coli.

    Science.gov (United States)

    Sahni, Geetika; Gopinath, P; Jeevanandam, P

    2013-03-01

    A novel thermal decomposition approach to synthesize hydroxyapatite-silver (Hap-Ag) nanocomposites has been reported. The nanocomposites were characterized by X-ray diffraction, field emission scanning electron microscopy coupled with energy dispersive X-ray analysis, transmission electron microscopy and diffuse reflectance spectroscopy techniques. Antibacterial activity studies for the nanocomposites were explored using a new rapid access method employing recombinant green fluorescent protein (GFP) expressing antibiotic resistant Escherichia coli (E. coli). The antibacterial activity was studied by visual turbidity analysis, optical density analysis, fluorescence spectroscopy and microscopy. The mechanism of bactericidal action of the nanocomposites on E. coli was investigated using atomic force microscopy, and TEM analysis. Excellent bactericidal activity at low concentration of the nanocomposites was observed which may allow their use in the production of microbial contamination free prosthetics. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    McCusker, Catherine D; Gardiner, David M

    2013-01-01

    The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

  1. Synthesis and Properties of the p-Sulfonamide Analogue of the Green Fluorescent Protein (GFP) Chromophore: The Mimic of GFP Chromophore with Very Strong N-H Photoacid Strength.

    Science.gov (United States)

    Chen, Yi-Hui; Sung, Robert; Sung, Kuangsen

    2018-04-06

    The para-sulfonamide analogue ( p-TsABDI) of a green fluorescent protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its S 1 excited-state p K a * value in dimethylsulfoxide (DMSO) is -1.5, which is strong enough to partially protonate dipolar aprotic solvents and causes excited-state proton transfer (ESPT), so it can partially mimic the GFP chromophore to further study the ESPT-related photophysics and the blinking phenomenon of GFP. In comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (p K a = 7.4, p K a * = 1.3 in water), p-TsABDI (p K a = 6.7, p K a * = -1.5 in DMSO) is a better photoacid for pH-jump studies.

  2. Identification of conservation units of the hynobiid salamander Pachyhynobius shangchengensis.

    Science.gov (United States)

    Su, L-N; Zhao, Y-Y; Wu, X-M; Zhang, H-F; Li, X-C

    2015-08-19

    The evolutionary significant units (ESUs) of the salamander Pachyhynobius shangchengensis (Hynobiidae) in the Dabieshan mountains, southeastern China, were identified based on mitochondrial DNA data. We used methods for detecting cryptic species, such as the minimum spanning tree, the automatic barcode gap discovery, and the generalized mixed Yule-coalescent model; geographical partitioning was also used to identify the ESUs. A total of four ESUs were identified.

  3. Prevalence of Batrachochytrium dendrobatidis in a Nicaraguan, micro-endemic Neotropical salamander, Bolitoglossa mombachoensis

    NARCIS (Netherlands)

    Stark, Tariq; Laurijssens, Carlijn; Weterings, Martijn; Martel, An; Köhler, Gunther; Pasmans, Frank

    2017-01-01

    Amphibians are the most threatened terrestrial vertebrates on the planet and are iconic in the global biodiversity crisis. Their global decline caused by the fungal agent Batrachochytrium dendrobatidis (Bd) is well known. Declines of Mesoamerican salamanders of the family Plethodontidae, mainly

  4. Transcriptomic analysis of endangered Chinese salamander: identification of immune, sex and reproduction-related genes and genetic markers.

    Directory of Open Access Journals (Sweden)

    Rongbo Che

    Full Text Available The Chinese salamander (Hynobius chinensis, an endangered amphibian species of salamander endemic to China, has attracted much attention because of its value of studying paleontology evolutionary history and decreasing population size. Despite increasing interest in the Hynobius chinensis genome, genomic resources for the species are still very limited. A comprehensive transcriptome of Hynobius chinensis, which will provide a resource for genome annotation, candidate genes identification and molecular marker development should be generated to supplement it.We performed a de novo assembly of Hynobius chinensis transcriptome by Illumina sequencing. A total of 148,510 nonredundant unigenes with an average length of approximately 580 bp were obtained. In all, 60,388 (40.66% unigenes showed homologous matches in at least one database and 33,537 (22.58% unigenes were annotated by all four databases. In total, 41,553 unigenes were categorized into 62 sub-categories by BLAST2GO search, and 19,468 transcripts were assigned to 140 KEGG pathways. A large number of unigenes involved in immune system, local adaptation, reproduction and sex determination were identified, as well as 31,982 simple sequence repeats (SSRs and 460,923 putative single nucleotide polymorphisms (SNPs.This dataset represents the first transcriptome analysis of the Chinese salamander (Hynobius chinensis, an endangered species, to be also the first time of hynobiidae. The transcriptome will provide valuable resource for further research in discovery of new genes, protection of population, adaptive evolution and survey of various pathways, as well as development of molecule markers in Chinese salamander; and reference information for closely related species.

  5. 3D bite modeling and feeding mechanics of the largest living amphibian, the Chinese giant salamander Andrias davidianus (Amphibia:Urodela.

    Directory of Open Access Journals (Sweden)

    Josep Fortuny

    Full Text Available Biting is an integral feature of the feeding mechanism for aquatic and terrestrial salamanders to capture, fix or immobilize elusive or struggling prey. However, little information is available on how it works and the functional implications of this biting system in amphibians although such approaches might be essential to understand feeding systems performed by early tetrapods. Herein, the skull biomechanics of the Chinese giant salamander, Andrias davidianus is investigated using 3D finite element analysis. The results reveal that the prey contact position is crucial for the structural performance of the skull, which is probably related to the lack of a bony bridge between the posterior end of the maxilla and the anterior quadrato-squamosal region. Giant salamanders perform asymmetrical strikes. These strikes are unusual and specialized behavior but might indeed be beneficial in such sit-and-wait or ambush-predators to capture laterally approaching prey. However, once captured by an asymmetrical strike, large, elusive and struggling prey have to be brought to the anterior jaw region to be subdued by a strong bite. Given their basal position within extant salamanders and their "conservative" morphology, cryptobranchids may be useful models to reconstruct the feeding ecology and biomechanics of different members of early tetrapods and amphibians, with similar osteological and myological constraints.

  6. Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

    Science.gov (United States)

    Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

    2018-05-01

    Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

  7. Larval salamanders and channel geomorphology are indicators of hydrologic permanence in forested headwater streams

    Science.gov (United States)

    Regulatory agencies need rapid indicators of hydrologic permanence for jurisdictional determinations of headwater streams. Our study objective was to assess the utility of larval salamander presence and assemblage structure and habitat variables for determining stream permanence ...

  8. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  9. A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

    Science.gov (United States)

    Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

    2006-05-01

    Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

  10. Differentiated egg size of the cannibalistic salamander Hynobius retardatus

    OpenAIRE

    Michimae, Hirofumi

    2007-01-01

    Larvae of the salamander, Hynobius retardatus, are carnivorous, and even though there are two morphs, a typical morph and a broad-headed or “cannibal” morph, both are cannibalistic. They also sometimes eat other large prey, for example larvae of the frog, Rana pirica. In natural habitats, use of both conspecific and R. pirica larvae as food may contribute more strongly to high survival and substantially to fitness when larval densities are higher, because early-stage H. retardatus larvae some...

  11. Macrohabitat models of occurrence for the threatened Cheat Mountain salamander, Plethodon nettingi

    Science.gov (United States)

    Lester O. Dillard; Kevin R. Russell; W. Mark Ford

    2008-01-01

    The federally threatened Cheat Mountain salamander (Plethodon nettingi; hereafter CMS) is known to occur at approximately 70 small, scattered sites in the Allegheny Mountains of eastern West Virginia. We used a comparative modeling approach to explain the landscape-level distribution and habitat relationships of CMS in relation to a suite of biotic...

  12. Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus.

    Science.gov (United States)

    Limon, Agenor; Reyes-Ruiz, Jorge Mauricio; Eusebi, Fabrizio; Miledi, Ricardo

    2007-09-25

    Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC(50) values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins.

  13. Telocytes in pancreas of the Chinese giant salamander (Andrias davidianus).

    Science.gov (United States)

    Zhang, Hui; Yu, Pengcheng; Zhong, Shengwei; Ge, Tingting; Peng, Shasha; Guo, Xiaoquan; Zhou, Zuohong

    2016-11-01

    Telocytes (TCs), novel interstitial cells, have been identified in various organs of many mammals. However, information about TCs of lower animals remains rare. Herein, pancreatic TCs of the Chinese giant salamanders (Andrias davidianus) were identified by CD34 immunohistochemistry (IHC) and transmission electron microscopy (TEM). The IHC micrographs revealed CD34 + TCs with long telopodes (Tps) that were located in the interstitium of the pancreas. CD34 + TCs/Tps were frequently observed between exocrine acinar cells and were close to blood vessels. The TEM micrographs also showed the existence of TCs in the interstitium of the pancreas. TCs had distinctive ultrastructural features, such as one to three very long and thin Tps with podoms and podomers, caveolae, dichotomous branching, neighbouring exosomes and vesicles. The Tps and exosomes were found in close proximity to exocrine acinar cells and α cells. It is suggested that TCs may play a role in the regeneration of acinar cells and α cells. In conclusion, our results demonstrated the presence of TCs in the pancreas of the Chinese giant salamander. This finding will assist us in a better understanding of TCs functions in the amphibian pancreas. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  14. Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons

    International Nuclear Information System (INIS)

    Jackson, Cheryl A.; Messinger, Jeff; Palmer, Matthew T.; Peduzzi, Jean D.; Morrow, Casey D.

    2003-01-01

    The spread of intramuscularly inoculated poliovirus to the central nervous system (CNS) has been documented in humans, monkeys, and mice transgenic for the human poliovirus receptor. Poliovirus spread is thought to be due to infection of the peripheral nerve and retrograde transport of poliovirus through the axon to the neuron cell body, where final virus uncoating occurs and translation/replication ensues. In previous studies, we have shown that polio-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. Using a replicon that encodes green fluorescent protein (GFP), we found that following intrathecal inoculation, GFP expression was confined to motorneurons of the spinal cord. To further characterize the gene expression of poliovirus in the periphery and CNS, we have intramuscularly inoculated transgenic mice with poliovirus replicons encoding GFP. Expression of GFP was demonstrated in the muscle, sciatic nerve, dorsal root ganglion, and the ventral horn motorneurons following intramuscular inoculation. There was no evidence of paralysis or behavioral abnormalities in the mice following intramuscular inoculation of the replicon encoding GFP. Injection of replicon RNA alone (naked RNA) into the muscle of transgenic mice or rats, which do not express the poliovirus receptor, also resulted in expression of GFP in the muscle, sciatic nerve, dorsal root ganglion, and ventral horn motorneurons, indicating that transport of the replicon RNA from the periphery to CNS had occurred. GFP expression was found in the muscles and sciatic nerve as early as 6 h after injection of replicons or replicon RNA, even after sciatic nerve section. Analysis at longer times postinjection revealed GFP expression similar to 6 h levels in the cut sciatic nerves and robust expression in the nerves of uncut animals. The infection and expression of GFP in the CNS following intramuscular inoculation of encapsidated replicons encoding GFP occurred in juvenile or

  15. Bacterially produced Pt-GFP as ratiometric dual-excitation sensor for in planta mapping of leaf apoplastic pH in intact Avena sativa and Vicia faba.

    Science.gov (United States)

    Geilfus, Christoph-Martin; Mühling, Karl H; Kaiser, Hartmut; Plieth, Christoph

    2014-01-01

    Ratiometric analysis with H(+)-sensitive fluorescent sensors is a suitable approach for monitoring apoplastic pH dynamics. For the acidic range, the acidotropic dual-excitation dye Oregon Green 488 is an excellent pH sensor. Long lasting (hours) recordings of apoplastic pH in the near neutral range, however, are more problematic because suitable pH indicators that combine a good pH responsiveness at a near neutral pH with a high photostability are lacking. The fluorescent pH reporter protein from Ptilosarcus gurneyi (Pt-GFP) comprises both properties. But, as a genetically encoded indicator and expressed by the plant itself, it can be used almost exclusively in readily transformed plants. In this study we present a novel approach and use purified recombinant indicators for measuring ion concentrations in the apoplast of crop plants such as Vicia faba L. and Avena sativa L. Pt-GFP was purified using a bacterial expression system and subsequently loaded through stomata into the leaf apoplast of intact plants. Imaging verified the apoplastic localization of Pt-GFP and excluded its presence in the symplast. The pH-dependent emission signal stood out clearly from the background. PtGFP is highly photostable, allowing ratiometric measurements over hours. By using this approach, a chloride-induced alkalinizations of the apoplast was demonstrated for the first in oat. Pt-GFP appears to be an excellent sensor for the quantification of leaf apoplastic pH in the neutral range. The presented approach encourages to also use other genetically encoded biosensors for spatiotemporal mapping of apoplastic ion dynamics.

  16. Effects of chronic γ-irradiation on growth and survival of the Tohoku hynobiid salamander, Hynobius lichenatus

    Energy Technology Data Exchange (ETDEWEB)

    Fuma, Shoichi [Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 Japan (Japan); Une, Yumi [Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201 (Japan); Ihara, Sadao [Hokkaido University of Education Kushiro Campus, 1-15-55 Shiroyama, Kushiro, Hokkaido 085-8580 Japan (Japan); Matsui, Kumi [Laboratory of Veterinary Physiology 1, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201 (Japan); Kudo, Tomoo; Tokiwa, Toshihiro [Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201 (Japan); Kubota, Yoshihisa; Soeda, Haruhi [Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 Japan (Japan); Ishikawa, Takahiro [Department of Technical Support and Development, Research, Development and Support Centre, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 Japan (Japan); Doi, Kazutaka [Project for Human Health, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 Japan (Japan); Watanabe, Yoshito; Yoshida, Satoshi [Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 Japan (Japan)

    2014-09-15

    The Tohoku hynobiid salamanders, Hynobius lichenatus, were chronically irradiated with γ-rays from embryonic to juvenile stages for 450 days. At 490 μGy h{sup −1} or lower dose rates, growth and survival were not significantly affected by irradiation, and any morphological aberrations and histological damages were not observed. At 4600 μGy h{sup −1}, growth was severely inhibited, and all the individuals died mostly at the juvenile stage. Chronic LD{sub 50} was 42 Gy as a total dose. In the liver, the number of hematopoietic cells was significantly reduced in the living juveniles, and these cells disappeared in the dead juveniles. In the spleen, mature lymphocytes were depleted in the living larvae, and almost all the heamtopoietic cells disappeared in the dead juveniles. These results suggest that this salamander died due to acute radiation syndrome, i.e., hematopoietic damage and subsequent sepsis caused by immune depression. The death would be also attributed to skin damage inducing infection. At 18,000 μGy h{sup −1}, morphological aberrations and severe growth inhibition were observed. All the individuals died at the larval stage due to a multiple organ failure. Chronic LD{sub 50} was 28 Gy as a total dose. Assuming that chronic LD{sub 50} was 42 Gy at lower dose rates than 4600 μGy h{sup −1}, a chronic median lethal dose rate could be estimated to be <340 μGy h{sup −1} for the whole life (>14 years). These results suggest that, among guidance dose rates, i.e., 4–400 μGy h{sup −1}, proposed by various organisations and research programmes for protection of amphibians and taxonomic groups or ecosystems including amphibians, most of them would protect this salamander but the highest value may not on the whole life scale. - Highlights: • The salamanders, Hynobius lichenatus, were chronically γ-irradiated for 450 days. • At 490 μGy h{sup −1} or lower, irradiation did not significantly affect growth and survival. • All the individuals

  17. Combined use of different Gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa

    DEFF Research Database (Denmark)

    Boldt, T.S.; Sørensen, J.; Karlsson, U.

    2004-01-01

    Single-cell localization and activity of Pseudomonas,fluorescens F113, colonizing alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1 ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of different stability. The monitoring systems permitted non...... of chlorinated biphenyl was constructed, using another gfp fusion with the meta-pathway Pin promoter from Pseudomonas putida (TOL plasmid). Expression of this promoter, which is strongly induced by the PCB-2 degradation product, 3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on alfalfa...... roots using the P. fluorescens F113rifpcb reporter. A small but distinct fraction of the introduced bacteria activated the Pm promoter and thus appeared to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading cells, which by design were identical to the sensing cells, were located...

  18. Proliferating neuronal progenitors in the postnatal hippocampus transiently express the proneural gene Ngn2.

    Science.gov (United States)

    Ozen, Ilknur; Galichet, Christophe; Watts, Colin; Parras, Carlos; Guillemot, François; Raineteau, Olivier

    2007-05-01

    Little is known of the transcription factors expressed by adult neural progenitors produced in the hippocampal neurogenic niche. Here, we study the expression of the proneural basic helix-loop-helix (bHLH) transcription factor Neurogenin-2 (Ngn2) in the adult hippocampus. We have characterized the pattern of expression of Ngn2 in the adult hippocampus using immunostaining for Ngn2 protein and a Ngn2-green fluorescent protein (GFP) reporter mouse strain. A significant proportion of Ngn2-expressing cells were mitotically active. Ngn2-GFP expression was restricted to the subgranular zone and declined with age. Neuronal markers were used to determine the phenotype of Ngn2-expressing cells. The vast majority of Ngn2-GFP-positive cells expressed the immature neuronal markers, doublecortin (DCX) and polysialic acid-neural cell adhesion molecule (PSA-NCAM). Finally, the pattern of Ngn2 expression was studied following seizure induction. Our data show an increase in neurogenesis, detected in these animals by bromodeoxyuridine (BrdU) and DCX staining that was contemporaneous with a marked increase in Ngn2-GFP-expression. Taken together, our results show that Ngn2-GFP represents a specific marker for neurogenesis and its modulation in the adult hippocampus. Ngn2 transient expression in proliferating neuronal progenitors supports the idea that it plays a significant role in adult neurogenesis.

  19. Salamander chytrid fungus (Batrachochytrium salamandrivorans) in the United States—Developing research, monitoring, and management strategies

    Science.gov (United States)

    Grant, Evan H. Campbell; Muths, Erin L.; Katz, Rachel A.; Canessa, Stefano; Adams, Michael J.; Ballard, Jennifer R.; Berger, Lee; Briggs, Cheryl J.; Coleman, Jeremy; Gray, Matthew J.; Harris, M. Camille; Harris, Reid N.; Hossack, Blake R.; Huyvaert, Kathryn P.; Kolby, Jonathan E.; Lips, Karen R.; Lovich, Robert E.; McCallum, Hamish I.; Mendelson, Joseph R.; Nanjappa, Priya; Olson, Deanna H.; Powers, Jenny G.; Richgels, Katherine L. D.; Russell, Robin E.; Schmidt, Benedikt R.; Spitzen-van der Sluijs, Annemarieke; Watry, Mary Kay; Woodhams, Douglas C.; White, C. LeAnn

    2016-01-20

    The recently (2013) identified pathogenic chytrid fungus, Batrachochytrium salamandrivorans (Bsal), poses a severe threat to the distribution and abundance of salamanders within the United States and Europe. Development of a response strategy for the potential, and likely, invasion of Bsal into the United States is crucial to protect global salamander biodiversity. A formal working group, led by Amphibian Research and Monitoring Initiative (ARMI) scientists from the U.S. Geological Survey (USGS) Patuxent Wildlife Research Center, Fort Collins Science Center, and Forest and Rangeland Ecosystem Science Center, was held at the USGS Powell Center for Analysis and Synthesis in Fort Collins, Colorado, United States from June 23 to June 25, 2015, to identify crucial Bsal research and monitoring needs that could inform conservation and management strategies for salamanders in the United States. Key findings of the workshop included the following: (1) the introduction of Bsal into the United States is highly probable, if not inevitable, thus requiring development of immediate short-term and long-term intervention strategies to prevent Bsal establishment and biodiversity decline; (2) management actions targeted towards pathogen containment may be ineffective in reducing the long-term spread of Bsal throughout the United States; and (3) early detection of Bsal through surveillance at key amphibian import locations, among high-risk wild populations, and through analysis of archived samples is necessary for developing management responses. Top research priorities during the preinvasion stage included the following: (1) deployment of qualified diagnostic methods for Bsal and establishment of standardized laboratory practices, (2) assessment of susceptibility for amphibian hosts (including anurans), and (3) development and evaluation of short- and long-term pathogen intervention and management strategies. Several outcomes were achieved during the workshop, including development

  20. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum.

    Directory of Open Access Journals (Sweden)

    Catherine D McCusker

    Full Text Available The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP, to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

  1. Impact of High-Level Expression of Heterologous Protein on Lactococcus lactis Host.

    Science.gov (United States)

    Kim, Mina; Jin, Yerin; An, Hyun-Joo; Kim, Jaehan

    2017-07-28

    The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N -acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

  2. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  3. The Dynamics of Two Hybrid Zones in Appalachian Salamanders of the Genus Plethodon

    Science.gov (United States)

    Nelson G. Hairston; R. Haven Wiley; Charles K. Smith; Kenneth A. Kneidel

    1992-01-01

    Two zones of intergradation between populations of Plethodon have been studied for 18 and 20 years, respectively. The data consist of systematic scores of colors, made at least twice annually. Near Heintooga Overlook in the Balsam Mountains (Great Smoky Mountains National Park), the salamanders' cheeks are gray. Proceeding north toward the...

  4. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  5. Heterologous expression of an algal hydrogenase in a heterocystous cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Thorsten Heidorn; Peter Lindblad [Dept. of Physiological Botany, Uppsala University, Villavogen 6, SE-752 36 Uppsala, (Sweden)

    2006-07-01

    For the expression of an active algal [FeFe] hydrogenase in the heterocystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyanobacteria cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  6. Heterologous expression of an algal hydrogenase in a heterocystous cyanobacterium

    International Nuclear Information System (INIS)

    Thorsten Heidorn; Peter Lindblad

    2006-01-01

    For the expression of an active algal [FeFe] hydrogenase in the heterocystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyanobacteria cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  7. Potential utility of eGFP-expressing NOG mice (NOG-EGFP as a high purity cancer sampling system

    Directory of Open Access Journals (Sweden)

    Shima Kentaro

    2012-06-01

    Full Text Available Abstract Purpose It is still technically difficult to collect high purity cancer cells from tumor tissues, which contain noncancerous cells. We hypothesized that xenograft models of NOG mice expressing enhanced green fluorescent protein (eGFP, referred to as NOG-EGFP mice, may be useful for obtaining such high purity cancer cells for detailed molecular and cellular analyses. Methods Pancreato-biliary cancer cell lines were implanted subcutaneously to compare the tumorigenicity between NOG-EGFP mice and nonobese diabetic/severe combined immunodeficiency (NOD/SCID mice. To obtain high purity cancer cells, the subcutaneous tumors were harvested from the mice and enzymatically dissociated into single-cell suspensions. Then, the cells were sorted by fluorescence-activated cell sorting (FACS for separation of the host cells and the cancer cells. Thereafter, the contamination rate of host cells in collected cancer cells was quantified by using FACS analysis. The viability of cancer cells after FACS sorting was evaluated by cell culture and subsequent subcutaneous reimplantation in NOG-EGFP mice. Results The tumorigenicity of NOG-EGFP mice was significantly better than that of NOD/SCID mice in all of the analyzed cell lines (p  Conclusions This method provides a novel cancer sampling system for molecular and cellular analysis with high accuracy and should contribute to the development of personalized medicine.

  8. HCV IRES-mediated core expression in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ye Zhao

    Full Text Available The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

  9. GFP-Mutant Human Tau Transgenic Mice Develop Tauopathy Following CNS Injections of Alzheimer's Brain-Derived Pathological Tau or Synthetic Mutant Human Tau Fibrils.

    Science.gov (United States)

    Gibbons, Garrett S; Banks, Rachel A; Kim, Bumjin; Xu, Hong; Changolkar, Lakshmi; Leight, Susan N; Riddle, Dawn M; Li, Chi; Gathagan, Ronald J; Brown, Hannah J; Zhang, Bin; Trojanowski, John Q; Lee, Virginia M-Y

    2017-11-22

    Neurodegenerative proteinopathies characterized by intracellular aggregates of tau proteins, termed tauopathies, include Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) with tau pathology (FTLD-tau), and related disorders. Pathological tau proteins derived from human AD brains (AD-tau) act as proteopathic seeds that initiate the templated aggregation of soluble tau upon intracerebral injection into tau transgenic (Tg) and wild-type mice, thereby modeling human tau pathology. In this study, we found that aged Tg mice of both sexes expressing human tau proteins harboring a pathogenic P301L MAPT mutation labeled with green fluorescent protein (T40PL-GFP Tg mouse line) exhibited hyperphosphorylated tau mislocalized to the somatodentritic domain of neurons, but these mice did not develop de novo insoluble tau aggregates, which are characteristic of human AD and related tauopathies. However, intracerebral injections of either T40PL preformed fibrils (PFFs) or AD-tau seeds into T40PL-GFP mice induced abundant intraneuronal pathological inclusions of hyperphosphorylated T40PL-GFP. These injections of pathological tau resulted in the propagation of tau pathology from the injection site to neuroanatomically connected brain regions, and these tau inclusions consisted of both T40PL-GFP and WT endogenous mouse tau. Primary neurons cultured from the brains of neonatal T40PL-GFP mice provided an informative in vitro model for examining the uptake and localization of tau PFFs. These findings demonstrate the seeded aggregation of T40PL-GFP in vivo by synthetic PFFs and human AD-tau and the utility of this system to study the neuropathological spread of tau aggregates. SIGNIFICANCE STATEMENT The stereotypical spread of pathological tau protein aggregates have recently been attributed to the transmission of proteopathic seeds. Despite the extensive use of transgenic mouse models to investigate the propagation of tau pathology in vivo , details of the aggregation

  10. Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras.

    Science.gov (United States)

    Künzel, Timo; Heiermann, Reinhard; Frank, Uri; Müller, Werner; Tilmann, Wido; Bause, Markus; Nonn, Anja; Helling, Matthias; Schwarz, Ryan S; Plickert, Günter

    2010-12-01

    To analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. Evaluation of the pH- and Thermal Stability of the Recombinant Green Fluorescent Protein (GFP) in the Presence of Sodium Chloride

    Science.gov (United States)

    Ishii, Marina; Kunimura, Juliana Sayuri; Jeng, Hélio Tallon; Vessoni Penna, Thereza Christina; Cholewa, Olivia

    The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95°C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84 ±0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94±0.60) was half that observed in phosphate buffer (pH 6.08±0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80°C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65±0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80°C. GFP pH-and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.

  12. Heterologous expression of an algal hydrogenase in a hetero-cystous cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Thorsten Heidorn; Peter Lindblad [Dept. of Physiological Botany, Uppsala University, V illavagen 6, SE-752 36 Uppsala, (Sweden)

    2006-07-01

    For the expression of an active algal [FeFe] hydrogenase in the hetero-cystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyano-bacterial cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  13. Heterologous expression of an algal hydrogenase in a hetero-cystous cyanobacterium

    International Nuclear Information System (INIS)

    Thorsten Heidorn; Peter Lindblad

    2006-01-01

    For the expression of an active algal [FeFe] hydrogenase in the hetero-cystous cyanobacterium Nostoc punctiforme A TCC 29133 the Chlamydomonas reinhardtii hydrogenase gene hydA1 and the accessory genes hydEF and hydG are to be introduced into the cyano-bacterial cells. The genes were amplified by PCR from EST clones, cloned into the cloning vector pBluescript SK+ and sequenced. An expression vector for multi-cistronic cloning, based on pSCR202, was constructed and for a functional test GFP was inserted as a reporter gene. The GFP construct was transformed into Nostoc punctiforme A TCC 29133 by electroporation and expression of GFP was visualized by fluorescence microscopy. (authors)

  14. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    Science.gov (United States)

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

  15. Modelling cross-scale relationships between climate, hydrology, and individual animals: Generating scenarios for stream salamanders

    Directory of Open Access Journals (Sweden)

    Philippe eGirard

    2015-07-01

    Full Text Available Hybrid modelling provides a unique opportunity to study cross-scale relationships in environmental systems by linking together models of global, regional, landscape, and local-scale processes, yet the approach is rarely applied to address conservation and management questions. Here, we demonstrate how a hybrid modelling approach can be used to assess the effect of cross-scale interactions on the survival of the Allegheny Mountain Dusky Salamander (Desmognathus ochrophaeus in response to changes in temperature and water availability induced by climate change at the northern limits of its distribution. To do so, we combine regional climate modelling with a landscape-scale integrated surface-groundwater flow model and an individual-based model of stream salamanders. On average, climate scenarios depict a warmer and wetter environment for the 2050 horizon. The increase in average annual temperature and extended hydrological activity time series in the future, combined with a better synchronization with the salamanders’ reproduction period, result in a significant increase in the long-term population viability of the salamanders. This indicates that climate change may not necessarily limit the survivability of small, stream-dwelling animals in headwater basins located in cold and humid regions. This new knowledge suggests that habitat conservation initiatives for amphibians with large latitudinal distributions in Eastern North America should be prioritized at the northern limits of their ranges to facilitate species migration and persistence in the face of climate change. This example demonstrates how hybrid models can serve as powerful tools for informing management and conservation decisions.

  16. Effect of thermal acclimation on locomotor energetics and locomotor performance in a lungless salamander, Desmognathus ochrophaeus.

    Science.gov (United States)

    Feder, M E

    1986-03-01

    To determine the effects of thermal acclimation upon locomotor performance and the rate of oxygen consumption (MO2) during activity, small (less than 3 g), lungless salamanders, Desmognathus ochrophaeus Cope, were acclimated to three temperatures (5, 13 and 21 degrees C) and exercised at various controlled speeds within an exercise wheel while their MO2 was measured. MO2 increased with speed at low speeds (less than 14 cm min-1). Although animals could sustain greater speeds, MO2 did not increase further. These small, exclusively skin-breathing salamanders could increase their MO2 9-11 times during exercise and could sustain nearly half of the oxygen flux expected across a similar surface area of the mammalian lung. However, their maximum aerobic speed was remarkably slow (14 cm min-1) and their net cost of transport remarkably large (15-17 ml O2 g-1 km-1). Thermal acclimation affected MO2 during activity, the maximum sustainable speed and locomotor stamina in different ways. During exercise at 13 degrees C, cold-acclimated animals had a significantly greater MO2 than warm-acclimated animals, but did not differ in stamina or the maximum sustainable speed. During exercise at 21 degrees C, cold acclimation did not affect the MO2 significantly, but it decreased the stamina and increased the rate of lactate accumulation. Thus, these results suggest that thermal acclimation of the MO2 is not tightly coupled to thermal acclimation of locomotor performance in salamanders.

  17. Remote sensing of gene expression in Planta: transgenic plants as monitors of exogenous stress perception in extraterrestrial environments

    Science.gov (United States)

    Manak, Michael S.; Paul, Anna-Lisa; Sehnke, Paul C.; Ferl, Robert J.

    2002-01-01

    Transgenic arabidopsis plants containing the alcohol dehydrogenase (Adh) gene promoter fused to the green fluorescent protein (GFP) reporter gene were developed as biological sensors for monitoring physiological responses to unique environments. Plants were monitored in vivo during exposure to hypoxia, high salt, cold, and abcissic acid in experiments designed to characterize the utility and responses of the Adh/GFP biosensors. Plants in the presence of environmental stimuli that induced the Adh promoter responded by expressing GFP, which in turn generated a detectable fluorescent signal. The GFP signal degraded when the inducing stimulus was removed. Digital imaging of the Adh/GFP plants exposed to each of the exogenous stresses demonstrated that the stress-induced gene expression could be followed in real time. The experimental results established the feasibility of using a digital monitoring system for collecting gene expression data in real time from Transgenic Arabidopsis Gene Expression System (TAGES) biosensor plants during space exploration experiments.

  18. Ambient ultraviolet radiation causes mortality in salamander eggs

    International Nuclear Information System (INIS)

    Blaustein, A.R.; Edmond, B.; Kiesecker, J.M.

    1995-01-01

    Previous research has shown that amphibian species have differential sensitivity to ultraviolet-B (UV-B) radiation. In some anuran species, ambient levels of UV-B cause mortality in embryonic stages and hatching success is significantly reduced. Projected increases in UV-B may affect an increasing number of species. The adverse effects of UV-B may eventually be manifested at the population level and may ultimately contribute to population declines. Using field experiments, we investigated the effects of ambient UV-B on salamander (Ambystoma gracile) embryos developing at natural oviposition sites. We show that the hatching success of eggs of A. gracile shielded from UV-B is significantly higher than those not shielded from UV-B. 27 refs., 1 fig

  19. 76 FR 44036 - Proposed Low-Effect Habitat Conservation Plan for the California Tiger Salamander, AT&T Portable...

    Science.gov (United States)

    2011-07-22

    ... developed portion of the AT&T facility on October 25 and 29, 2010. In addition, several salamander larvae... were rejected due to the presence of other Federally listed species, including vernal pool fairy shrimp...

  20. Fire salamander (Salamandra salamandra in Larzac plateau: low occurrence, pond-breeding and cohabitation of larvae with paedomorphic palmate newts (Lissotriton helveticus

    Directory of Open Access Journals (Sweden)

    Mathieu Denoël

    2014-06-01

    Full Text Available Alternative reproductive strategies are widespread in caudate amphibians. Among them, fire salamanders (Salamandra salamandra usually rely on streams to give birth to aquatic larvae but also use ponds, whereas palmate newt larvae (Lissotriton helveticus typically metamorphose into terrestrial juveniles, but can also reproduce in retaining their gills, a process known as paedomorphosis. Here we report repeated observations of an unusual case of coexistence of these two alternative traits in the same pond (Larzac, France. The prevalence of fire salamanders in Southern Larzac was very low (pond occupancy: 0.36%. The observed abundance of fire salamander larvae and paedomorphic newts was also low in the studied pond. On one hand, the rarity of this coexistence pattern may suggest that habitat characteristics may not be optimal or that competition or predation processes might be operating. However, these hypotheses remain to be tested. On the other hand, as this is the only known case of breeding in Southern Larzac, it could be considered to be at a high risk of extirpation.

  1. Geographic variation, genetic structure, and conservation unit designation in the Larch Mountain salamander (Plethodon larselli).

    Science.gov (United States)

    R. Steven Wagner; Mark P. Miller; Charles M. Crisafulli; Susan M. Haig

    2005-01-01

    The Larch Mountain salamander (Plethodon larselli Burns, 1954) is an endemic species in the Pacific northwestern United States facing threats related to habitat destruction. To facilitate development of conservation strategies, we used DNA sequences and RAPDs (random amplified polymorphic DNA) to examine differences among populations of this...

  2. Projected loss of a salamander diversity hotspot as a consequence of projected global climate change

    Science.gov (United States)

    Joseph R. Milanovich; William E. Peterman; Nathan P. Nibbelink; John C. Maerz

    2010-01-01

    Background: Significant shifts in climate are considered a threat to plants and animals with significant physiological limitations and limited dispersal abilities. The southern Appalachian Mountains are a global hotspot for plethodontid salamander diversity. Plethodontids are lungless ectotherms, so their ecology is strongly governed by temperature and precipitation....

  3. Overexpression of LOV KELCH protein 2 confers dehydration tolerance and is associated with enhanced expression of dehydration-inducible genes in Arabidopsis thaliana.

    Science.gov (United States)

    Miyazaki, Yuji; Abe, Hiroshi; Takase, Tomoyuki; Kobayashi, Masatomo; Kiyosue, Tomohiro

    2015-05-01

    The overexpression of LKP2 confers dehydration tolerance in Arabidopsis thaliana ; this is likely due to enhanced expression of dehydration-inducible genes and reduced stomatal opening. LOV KELCH protein 2 (LKP2) modulates the circadian rhythm and flowering time in plants. In this study, we observed that LKP2 overexpression enhanced dehydration tolerance in Arabidopsis. Microarray analysis demonstrated that expression of water deprivation-responsive genes was higher in the absence of dehydration stress in transgenic Arabidopsis plants expressing green fluorescent protein-tagged LKP2 (GFP-LKP2) than in control transgenic plants expressing GFP. After dehydration followed by rehydration, GFP-LKP2 plants developed more leaves and roots and exhibited higher survival rates than control plants. In the absence of dehydration stress, four dehydration-inducible genes, namely DREB1A, DREB1B, DREB1C, and RD29A, were expressed in GFP-LKP2 plants, whereas they were not expressed or were expressed at low levels in control plants. Under dehydration stress, the expression of DREB2B and RD29A peaked faster in the GFP-LKP2 plants than in control plants. The stomatal aperture of GFP-LKP2 plants was smaller than that of control plants. These results suggest that the dehydration tolerance of GFP-LKP2 plants is caused by upregulation of DREB1A-C/CBF1-3 and their downstream targets; restricted stomatal opening in the absence of dehydration stress also appears to contribute to the phenotype. The rapid and high expression of DREB2B and its downstream target genes also likely accounts for some features of the GFP-LKP2 phenotype. Our results suggest that LKP2 can be used for biotechnological applications not only to adjust the flowering time control but also to enhance dehydration tolerance.

  4. Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

    Science.gov (United States)

    Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

    2015-01-01

    To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

  5. Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

    Science.gov (United States)

    Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

    2015-12-30

    Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

  6. Male pheromone protein components activate female vomeronasal neurons in the salamander Plethodon shermani

    Directory of Open Access Journals (Sweden)

    Feldhoff Pamela W

    2006-03-01

    Full Text Available Abstract Background The mental gland pheromone of male Plethodon salamanders contains two main protein components: a 22 kDa protein named Plethodon Receptivity Factor (PRF and a 7 kDa protein named Plethodon Modulating Factor (PMF, respectively. Each protein component individually has opposing effects on female courtship behavior, with PRF shortening and PMF lengthening courtship. In this study, we test the hypothesis that PRF or PMF individually activate vomeronasal neurons. The agmatine-uptake technique was used to visualize chemosensory neurons that were activated by each protein component individually. Results Vomeronasal neurons exposed to agmatine in saline did not demonstrate significant labeling. However, a population of vomeronasal neurons was labeled following exposure to either PRF or PMF. When expressed as a percent of control level labeled cells, PRF labeled more neurons than did PMF. These percentages for PRF and PMF, added together, parallel the percentage of labeled vomeronasal neurons when females are exposed to the whole pheromone. Conclusion This study suggests that two specific populations of female vomeronasal neurons are responsible for responding to each of the two components of the male pheromone mixture. These two neural populations, therefore, could express different receptors which, in turn, transmit different information to the brain, thus accounting for the different female behavior elicited by each pheromone component.

  7. Phylogenetic analysis of Common Garter Snake (Thamnophis sirtalis) stomach contents detects cryptic range of a secretive salamander (Ensatina eschscholtzii oregonensis) Herpetological Conservation and Biology 5(3):395–402

    Science.gov (United States)

    Sean B. Reilly; Andrew D Gottsho; Justin M. Garwood; Bryan. Jennings

    2010-01-01

    Given the current global amphibian decline, it is crucial to obtain accurate and current information regarding species distributions. Secretive amphibians such as plethodontid salamanders can be difficult to detect in many cases, especially in remote, high elevation areas. We used molecular phylogenetic analyses to identify three partially digested salamanders palped...

  8. Elaboration and quality control of the inoculum of the experimental vaccine Brucella S19-tn7-GFP for use in white animals and associated serological test for the detection of anti-GFP antibodies

    International Nuclear Information System (INIS)

    Salas Alfaro, Dariana

    2014-01-01

    The preparation of the inoculum of the experimental vaccine Brucella S19-Tn7-GFP is optimized for application in white animals. An associated serological test has allowed differentiating infected animals from those vaccinated with the experimental strain. The same bacteriological and biological properties of the B. abortus S19-Tn7-GFP strain have maintained in the parental vaccine strain S19 and is stable over time. A protocol for the inoculums of strain S19-Tn7-GFP is established for its preparation and use in white animals and quality control. The inoculum stability is evaluated through the simulation of conditions that can be presented in the transportation and application process in the field. An enzyme immunoassay ELISA is optimized for the detection of anti-GFP antibodies in cattle [es

  9. Can the eastern red-backed salamander (Plethodon cinereus) persist in an acidified landscape?

    Science.gov (United States)

    Bondi, Cheryl A; Beier, Colin M.; Ducey, Peter K; Lawrence, Gregory B.; Bailey, Scott W.

    2016-01-01

    Hardwood forests of eastern North America have experienced decades of acidic deposition, leading to soil acidification where base cation supply was insufficient to neutralize acid inputs. Negative impacts of soil acidity on amphibians include disrupted embryonic development, lower growth rates, and habitat loss. However, some amphibians exhibit intraspecific variation in acid tolerance, suggesting the potential for local adaptation in areas where soils are naturally acidic. The eastern red-backed salamander (Plethodon cinereus) is a highly abundant top predator of the northern hardwood forest floor. Early research found that P. cinereus was sensitive to acidic soils, avoiding substrates with pH habitats. However, recent studies have documented P. cinereus populations in lower pH conditions than previously observed, suggesting some populations may persist in acidic conditions. Here, we evaluated relationships between organic horizon soil pH and P. cinereus abundance, adult health (body size and condition), and microhabitat selection, based on surveys of 34 hardwood forests in northeastern United States that encompass a regional soil pH gradient. We found no associations between soil pH and P. cinereus abundance or health, and observed that this salamander used substrates with pH similar to that available, suggesting that pH does not mediate their fine-scale distributions. The strongest negative predictor of P. cinereus abundance was the presence of dusky salamanders (Desmognathus spp.), which were most abundant in the western Adirondacks. Our results indicate that P. cinereus occupies a wider range of soil pH than has been previously thought, which has implications for their functional role in forest food webs and nutrient cycles in acid-impaired ecosystems. Tolerance of P. cinereus for more acidic habitats, including anthropogenically acidified forests, may be due to local adaptation in reproductively isolated populations and/or generalist

  10. Effects of pH on embryo tolerance and adult behavior in the tiger salamander, Ambystoma tigrinum tigrinum

    Energy Technology Data Exchange (ETDEWEB)

    Whiteman, H H; Howard, R D; Whitten, K A [Purdue Univ., Lafayette, IN (United States). Dept. of Biological Sciences

    1995-08-01

    Adult discrimination ability and embryo performance was examined under different pH conditions in the eastern tiger salamander. Individuals from three populations were collected in habitats that differed naturally in pH. Two pH treatments were used to determine adult pH discrimination ability, and eight pH treatments to evaluate embryo performance. Results suggested that the pH of the source-population habitat could influence breeding-habitat discrimination by adults. Decreasing pH produced similar patterns of lethal and sublethal effects on embryos from the three populations, with reduced performance at low pH. The pH at which 50% mortality occurs was estimated at 4.2, suggesting that tiger salamanders were relatively acid tolerant. The study suggested that adult behavior patterns could influence the success of population reintroductions to previously acidified areas. 78 refs., 2 tabs., 4 figs.

  11. [A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

    Science.gov (United States)

    Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

    2012-06-01

    To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

  12. Green Fluorescent Protein (GFP-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Shengdi Fan

    2013-09-01

    Full Text Available Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC and size exclusion chromatography (SEC at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23-lauryl-ether (Brij-35 was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

  13. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity.

    Science.gov (United States)

    Shagin, Dmitry A; Barsova, Ekaterina V; Yanushevich, Yurii G; Fradkov, Arkady F; Lukyanov, Konstantin A; Labas, Yulii A; Semenova, Tatiana N; Ugalde, Juan A; Meyers, Ann; Nunez, Jose M; Widder, Edith A; Lukyanov, Sergey A; Matz, Mikhail V

    2004-05-01

    Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

  14. Patterning protein complexes on DNA nanostructures using a GFP nanobody.

    Science.gov (United States)

    Sommese, R F; Hariadi, R F; Kim, K; Liu, M; Tyska, M J; Sivaramakrishnan, S

    2016-11-01

    DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies. © 2016 The Protein Society.

  15. A new species of salamander (Bolitoglossa: Plethodontidae from the Cordillera Oriental of the Colombian Andes

    Directory of Open Access Journals (Sweden)

    Andrés R. Acosta-Galvis

    2012-01-01

    Full Text Available Eight species of salamanders are recognized to Cordillera Oriental of Colombia. Here we describe a new species of the genus Bolitoglossa, named Bolitoglossa guaneae sp. nov. The highest number of species of this genus is found in the cloud forests located in the western flank of the Cordillera Oriental.

  16. Woodland salamanders as metrics of forest ecosystem recovery: a case study from California’s redwoods

    Science.gov (United States)

    Hart Welsh; Garth Hodgson

    2013-01-01

    Woodland (Plethodontid) salamanders occur in huge numbers in healthy forests in North America where the abundances of many species vary along successional gradients. Their high numbers and trophic role as predators on shredder and decomposer arthropods influence nutrient and carbon pathways at the leaf litter/soil interface. Their extreme niche conservatism and low...

  17. A multiplexed miRNA and transgene expression platform for simultaneous repression and expression of protein coding sequences.

    Science.gov (United States)

    Seyhan, Attila A

    2016-01-01

    Knockdown of single or multiple gene targets by RNA interference (RNAi) is necessary to overcome escape mutants or isoform redundancy. It is also necessary to use multiple RNAi reagents to knockdown multiple targets. It is also desirable to express a transgene or positive regulatory elements and inhibit a target gene in a coordinated fashion. This study reports a flexible multiplexed RNAi and transgene platform using endogenous intronic primary microRNAs (pri-miRNAs) as a scaffold located in the green fluorescent protein (GFP) as a model for any functional transgene. The multiplexed intronic miRNA - GFP transgene platform was designed to co-express multiple small RNAs within the polycistronic cluster from a Pol II promoter at more moderate levels to reduce potential vector toxicity. The native intronic miRNAs are co-transcribed with a precursor GFP mRNA as a single transcript and presumably cleaved out of the precursor-(pre) mRNA by the RNA splicing machinery, spliceosome. The spliced intron with miRNA hairpins will be further processed into mature miRNAs or small interfering RNAs (siRNAs) capable of triggering RNAi effects, while the ligated exons become a mature messenger RNA for the translation of the functional GFP protein. Data show that this approach led to robust RNAi-mediated silencing of multiple Renilla Luciferase (R-Luc)-tagged target genes and coordinated expression of functional GFP from a single transcript in transiently transfected HeLa cells. The results demonstrated that this design facilitates the coordinated expression of all mature miRNAs either as individual miRNAs or as multiple miRNAs and the associated protein. The data suggest that, it is possible to simultaneously deliver multiple negative (miRNA or shRNA) and positive (transgene) regulatory elements. Because many cellular processes require simultaneous repression and activation of downstream pathways, this approach offers a platform technology to achieve that dual manipulation efficiently

  18. Histological and MS spectrometric analyses of the modified tissue of bulgy form tadpoles induced by salamander predation

    Directory of Open Access Journals (Sweden)

    Tsukasa Mori

    2012-02-01

    The rapid induction of a defensive morphology by a prey species in face of a predation risk is an intriguing in ecological context; however, the physiological mechanisms that underlie this phenotypic plasticity remain uncertain. Here we investigated the phenotypic changes shown by Rana pirica tadpoles in response to a predation threat by larvae of the salamander Hynobius retardatus. One such response is the bulgy morph phenotype, a relatively rapid swelling in size by the tadpoles that begins within 4 days and reaches a maximum at 8 to 10 days. We found that although the total volume of bodily fluid increased significantly (P<0.01 in bulgy morph tadpoles, osmotic pressure was maintained at the same level as control tadpoles by a significant increase (P<0.01 in Na and Cl ion concentrations. In our previous report, we identified a novel frog gene named pirica that affects the waterproofing of the skin membrane in tadpoles. Our results support the hypothesis that predator-induced expression of pirica on the skin membrane causes retention of absorbed water. Midline sections of bulgy morph tadpoles showed the presence of swollen connective tissue beneath the skin that was sparsely composed of cells containing hyaluronic acid. Mass spectrographic (LC-MS/MS analysis identified histone H3 and 14-3-3 zeta as the most abundant constituents in the liquid aspirated from the connective tissue of bulgy tadpoles. Immunohistochemistry using antibodies against these proteins showed the presence of non-chromatin associated histone H3 in the swollen connective tissue. Histones and 14-3-3 proteins are also involved in antimicrobial activity and secretion of antibacterial proteins, respectively. Bulgy tadpoles have a larger surface area than controls, and their skin often has bite wounds inflicted by the larval salamanders. Thus, formation of the bulgy morph may also require and be supported by activation of innate immune systems.

  19. Evaluation of the effects of ethinylestradiol on sexual differentiation in the olvas-GFP/STII-YI medaka (transgenic Oryzias latipes) strain as estimated by proliferative activity of germ cells

    International Nuclear Information System (INIS)

    Hano, Takeshi; Oshima, Yuji; Kinoshita, Masato; Tanaka, Minoru; Mishima, Noriko; Wakamatsu, Yuko; Ozato, Kenjiro; Shimasaki, Yohei; Honjo, Tsuneo

    2011-01-01

    We evaluated the effects of 17(-ethinylestradiol (EE 2 ) on sexual differentiation in transgenic olvas-GFP/STII-YI medaka (Oryzias latipes) in terms of the proliferative activity of germ cells. This strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ cell-specific expression of GFP can be visualized in living (transparent) individuals. From 0 days post-hatch (0 dph) onwards, juveniles were exposed to graded concentrations of EE 2 (25.2-1710 ng/L) for 35 days. The gonads of live specimens were monitored by measuring their size and calculating their GFP-fluorescence area. GFP-fluorescent area in control females was about 10 times that in control males at 10 days posthatch (dph) whereas the gonadal size of 10 dph males that had been exposed to 158 ng/L of EE 2 significantly increased up to twice the size of control males, indicating that abnormal sexual differentiation towards female might occur in these individuals. Histological examination and identification of the sex-linked marker SL1 indicated that male to female sex reversal occurred at EE 2 exposure ≥45.1 ng/L at 35 dph. These results suggest that observation of proliferative activity of germ cells in the olvas-GFP/STII-YI strain could be applied to facilitated screening fish model to detect adverse effects on sexual differentiation as early as 10 dph juveniles.

  20. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

    Science.gov (United States)

    Bach, Thomas J

    2013-01-01

    We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

  1. Cutaneous mastocytomas in the neotenic caudate amphibians Ambystoma mexicanum (axolotl) and Ambystoma tigrinun (tiger salamander)

    Science.gov (United States)

    Harshbarger, J.C.; Chang, S.C.; DeLanney, L.E.; Rose, F.L.; Green, D.E.

    1999-01-01

    Spontaneous mastocytomas studied in 18 axolotls (Ambystoma mexicanum) and six tiger salamanders (Ambystoma tigrinum) were gray-white, uni- to multilobular cutaneous protrusions from 2mm to 2cm in diameter. Tumors were moderately cellular unencapsulated masses that usually infiltrated the dermis and hypodermis with the destruction of intervening tissues. Some tumors were invading superficial bundles of the underlying skeletal muscle. Tumors consisted of mitotically active cells derived from a single lineage but showing a range of differentiation. Immature cells had nearly smooth to lightly cleft or folded basophilic nuclei bordered by a band of cytoplasm with few cytoplasmic processes and containing a few small uniform eccentric granules. Mature cells had basophilic nuclei with deep clefts or folds and abundant eosinophilic cytoplasm with multiple long intertwining cytoplasmic extensions packed with metachromatic granules. The axolotls were old individuals from an inbred laboratory colony. The tiger salamanders were wild animals from a single polluted pond. They could have been old and inbred. Both groups were neotenic. These are the first mastocytomas discovered in cold-blooded animals.

  2. Fostering Children’s Connection to Nature Through Authentic Situations: The Case of Saving Salamanders at School

    Directory of Open Access Journals (Sweden)

    Stephan Barthel

    2018-06-01

    Full Text Available The aim of this paper is to explore how children learn to form new relationships with nature. It draws on a longitudinal case study of children participating in a stewardship project involving the conservation of salamanders during the school day in Stockholm, Sweden. The qualitative method includes two waves of data collection: when a group of 10-year-old children participated in the project (2015 and 2 years after they participated (2017. We conducted 49 interviews with children as well as using participant observations and questionnaires. We found indications that children developed sympathy for salamanders and increased concern and care for nature, and that such relationships persisted 2 years after participation. Our rich qualitative data suggest that whole situations of sufficient unpredictability triggering free exploration of the area, direct sensory contact and significant experiences of interacting with a species were important for children’s development of affective relationships with the salamander species and with nature in an open-ended sense. Saving the lives of trapped animals enabled direct sensory interaction, feedback, increased understanding, and development of new skills for dynamically exploring further ways of saving species in an interactive process experienced as deeply meaningful, enjoyable and connecting. The behavioral setting instilled a sense of pride and commitment, and the high degree of responsibility given to the children while exploring the habitat during authentic situations enriched children’s enjoyment. The study has implications for the design of education programs that aim to connect children with nature and for a child-sensitive urban policy that supports authentic nature situations in close spatial proximity to preschools and schools.

  3. Transcriptional Profiling of Cholinergic Neurons From Basal Forebrain Identifies Changes in Expression of Genes Between Sleep and Wake.

    Science.gov (United States)

    Nikonova, Elena V; Gilliland, Jason DA; Tanis, Keith Q; Podtelezhnikov, Alexei A; Rigby, Alison M; Galante, Raymond J; Finney, Eva M; Stone, David J; Renger, John J; Pack, Allan I; Winrow, Christopher J

    2017-06-01

    To assess differences in gene expression in cholinergic basal forebrain cells between sleeping and sleep-deprived mice sacrificed at the same time of day. Tg(ChAT-eGFP)86Gsat mice expressing enhanced green fluorescent protein (eGFP) under control of the choline acetyltransferase (Chat) promoter were utilized to guide laser capture of cholinergic cells in basal forebrain. Messenger RNA expression levels in these cells were profiled using microarrays. Gene expression in eGFP(+) neurons was compared (1) to that in eGFP(-) neurons and to adjacent white matter, (2) between 7:00 am (lights on) and 7:00 pm (lights off), (3) between sleep-deprived and sleeping animals at 0, 3, 6, and 9 hours from lights on. There was a marked enrichment of ChAT and other markers of cholinergic neurons in eGFP(+) cells. Comparison of gene expression in these eGFP(+) neurons between 7:00 am and 7:00 pm revealed expected differences in the expression of clock genes (Arntl2, Per1, Per2, Dbp, Nr1d1) as well as mGluR3. Comparison of expression between spontaneous sleep and sleep-deprived groups sacrificed at the same time of day revealed a number of transcripts (n = 55) that had higher expression in sleep deprivation compared to sleep. Genes upregulated in sleep deprivation predominantly were from the protein folding pathway (25 transcripts, including chaperones). Among 42 transcripts upregulated in sleep was the cold-inducible RNA-binding protein. Cholinergic cell signatures were characterized. Whether the identified genes are changing as a consequence of differences in behavioral state or as part of the molecular regulatory mechanism remains to be determined. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  4. Efficacy of chemical disinfectants for the containment of the salamander chytrid fungus Batrachochytrium salamandrivorans

    OpenAIRE

    Van Rooij, Pascale; Pasmans, Frank; Coen, Yanaïka; Martel, An

    2017-01-01

    The recently emerged chytrid fungus Batrachochytrium salamandrivorans (Bsal) causes European salamander declines. Proper hygiene protocols including disinfection procedures are crucial to prevent disease transmission. Here, the efficacy of chemical disinfectants in killing Bsal was evaluated. At all tested conditions, Biocidal (R), Chloramine-T (R), Dettol medical (R), Disolol (R), ethanol, F10 (R), Hibiscrub (R), potassium permanganate, Safe4 (R), sodium hypochlorite, and Virkon S (R), were ...

  5. Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts

    Czech Academy of Sciences Publication Activity Database

    Kolesová, H.; Čapek, Martin; Radochová, Barbora; Janáček, Jiří; Sedmera, David

    2016-01-01

    Roč. 146, č. 2 (2016), s. 142-152 ISSN 0948-6143 R&D Projects: GA ČR(CZ) GA13-12412S; GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : green fluorescent protein (GFP) * confocal microscopy * optical projection tomography * tissue transparency * heart * embryo Subject RIV: EA - Cell Biology Impact factor: 2.553, year: 2016

  6. Two types of Tet-On transgenic lines for doxycycline-inducible gene expression in zebrafish rod photoreceptors and a gateway-based tet-on toolkit.

    Directory of Open Access Journals (Sweden)

    Leah J Campbell

    Full Text Available The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA, one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.

  7. Cardiac-specific activation of Cre expression at late fetal development

    International Nuclear Information System (INIS)

    Opherk, Jan P.; Yampolsky, Peter; Hardt, Stefan E.; Schoels, Wolfgang; Katus, Hugo A.; Koenen, Michael; Zehelein, Joerg

    2007-01-01

    In a first step towards dissecting molecular mechanisms that contribute to the development of cardiac diseases, we have generated transgenic mice that express a Cre-GFP fusion protein under the transcriptional control of a 4.3 kb murine cardiac Troponin I gene (cTnI) promoter. Cre-GFP expression, similar in three transgenic lines, is described in one line. In mouse embryos, transgenic for the Cre-GFP and ROSA lacZ reporter allele, first Cre-mediated recombination appeared at 16.5 dpc selectively at the heart. Like the endogenous cTnI gene, transgenic Cre expression showed a slow rise through fetal development that increased neonatally. Bitransgenic hearts, stained at 30 days of age, showed intense signals in ventricular and atrial myocytes while no recombination occurred in other tissues. The delayed onset of Cre activity in cTnI-Cre mice could provide a useful genetic tool to evaluate the function of loxP targeted cardiac genes without interference of recombination during early heart development

  8. Differential diagnosis of feline leukemia virus subgroups using pseudotype viruses expressing green fluorescent protein.

    Science.gov (United States)

    Nakamura, Megumi; Sato, Eiji; Miura, Tomoyuki; Baba, Kenji; Shimoda, Tetsuya; Miyazawa, Takayuki

    2010-06-01

    Feline leukemia virus (FeLV) is classified into three receptor interference subgroups, A, B and C. In this study, to differentiate FeLV subgroups, we developed a simple assay system using pseudotype viruses expressing green fluorescent protein (GFP). We prepared gfp pseudotype viruses, named gfp(FeLV-A), gfp(FeLV-B) and gfp(FeLV-C) harboring envelopes of FeLV-A, B and C, respectively. The gfp pseudotype viruses completely interfered with the same subgroups of FeLV reference strains on FEA cells (a feline embryonic fibroblast cell line). We also confirmed that the pseudotype viruses could differentiate FeLV subgroups in field isolates. The assay will be useful for differential diagnosis of FeLV subgroups in veterinary diagnostic laboratories in the future.

  9. The trophic role of a forest salamander: impacts on invertebrates, leaf litter retention, and the humification process

    Science.gov (United States)

    M. L. Best; H. H. Welsh

    2014-01-01

    Woodland (Plethodontid) salamanders are the most abundant vertebrates in North American forests, functioning as predators on invertebrates and prey for higher trophic levels. We investigated the role of Ensatina (Ensatina eschscholtzii) in regulating invertebrate numbers and leaf litter retention in a northern California forest. Our objective was...

  10. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

    Science.gov (United States)

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2006-05-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

  11. Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

    International Nuclear Information System (INIS)

    Pearson, John; Godinho, Susana A.; Tavares, Alvaro; Glover, David M.

    2006-01-01

    Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells

  12. Evidence of green fluorescent protein and growth hormone expression in red abalone (Haliotis rufescens larvae

    Directory of Open Access Journals (Sweden)

    Mancilla-Sánchez Edgar

    2017-02-01

    Full Text Available The red abalone Haliotis rufescens is a highly appreciated mollusk in the national and international markets. Due to its natural over-exploitation and low growth rate, several genetic improvements were made, however special efforts are needed to increase its production. This study presents transgenic abalone’s larvae expressing green fluorescent protein (GFP fused to Cobia (Rachycentron canadum Growth Hormone (GH using sperm media transgenesis technique (SMT, pAcGFP1-N vector under the control of cytomegalovirus (CMV promoter. Sperms were exposed to three voltages (0.5, 0.75 and 1.0 Kv using a micropulser electroporator (Bio-Rad®. The highest GFP-GH expression average (40% was obtained in abalone larvae at 0.75 v. GFP and GH transgenes were positively detected by PCR, western blot and confocal microscope, respectively.

  13. New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Ana Isabel Garcia Diaz

    2016-04-01

    Full Text Available The Wistar Kyoto (WKY rat and the spontaneously hypertensive (SHR rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN and metabolic syndrome, respectively. Novel transgenic (Tg strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP under the rat elongation factor 1 alpha (EF1a promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

  14. Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

    Science.gov (United States)

    Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin Simon; Williams, Melissa; Zaveri, Nurulain T; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L; Toll, Lawrence

    2015-08-19

    The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These

  15. Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells

    Directory of Open Access Journals (Sweden)

    Verena Staudacher

    2018-04-01

    Full Text Available Redox-sensitive green fluorescent protein 2 (roGFP2 is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis. Keywords: Peroxiredoxin, Redox sensor, roGFP2, H2O2, Plasmodium falciparum

  16. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

    Science.gov (United States)

    Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

    2013-03-25

    Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally

  17. Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

    Science.gov (United States)

    Smith, E. G.; D'Angelo, C.; Salih, A.; Wiedenmann, J.

    2013-06-01

    Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.

  18. Sensory feedback plays a significant role in generating walking gait and in gait transition in salamanders: A simulation study

    Directory of Open Access Journals (Sweden)

    Nalin eHarischandra

    2011-11-01

    Full Text Available Here, we use a three-dimensional, neuro-musculo-mechanical model of a salamander with realistic physical parameters in order to investigate the role of sensory feedback in gait generation and transition. Activation of limb and axial muscles were driven by neural output patterns obtained from a central pattern generator (CPG which is composed of simulated spiking neurons with adaptation. The CPG consists of a body CPG and four limb CPGs that are interconnected via synapses both ipsilateraly and contralaterally. We use the model both with and without sensory modulation and for different combinations of ipsilateral and contralateral coupling between the limb CPGs. We found that the proprioceptive sensory inputs are essential in obtaining a coordinated walking gait. The sensory feedback includes the signals coming from the stretch receptor like intraspinal neurons located in the girdle regions and the limb stretch receptors residing in the hip and scapula regions of the salamander. On the other hand, coordinated motor output patterns for the trotting gait were obtainable without the sensory inputs. We found that the gait transition from walking to trotting can be induced by increased activity of the descending drive coming from the mesencephalic locomotor region (MLR and is helped by the sensory inputs at the hip and scapula regions detecting the late stance phase. More neurophysiological experiments are required to identify the precise type of mechanoreceptors in the salamander and the neural mechanisms mediating the sensory modulation.

  19. Expression pattern of Ccr2 and Cx3cr1 in inherited retinal degeneration.

    Science.gov (United States)

    Kohno, Hideo; Koso, Hideto; Okano, Kiichiro; Sundermeier, Thomas R; Saito, Saburo; Watanabe, Sumiko; Tsuneoka, Hiroshi; Sakai, Tsutomu

    2015-10-12

    Though accumulating evidence suggests that microglia, resident macrophages in the retina, and bone marrow-derived macrophages can cause retinal inflammation which accelerates photoreceptor cell death, the details of how these cells are activated during retinal degeneration (RD) remain uncertain. Therefore, it is important to clarify which cells play a dominant role in fueling retinal inflammation. However, distinguishing between microglia and macrophages is difficult using conventional techniques such as cell markers (e.g., Iba-1). Recently, two mouse models for visualizing chemokine receptors were established, Cx3cr1 (GFP/GFP) and Ccr2 (RFP/RFP) mice. As Cx3cr1 is expressed in microglia and Ccr2 is reportedly expressed in activated macrophages, these mice have the potential to distinguish microglia and macrophages, yielding novel information about the activation of these inflammatory cells and their individual roles in retinal inflammation. In this study, c-mer proto-oncogene tyrosine kinase (Mertk) (-/-) mice, which show photoreceptor cell death due to defective retinal pigment epithelium phagocytosis, were employed as an animal model of RD. Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were established by breeding Mertk (-/-) , Cx3cr1 (GFP/GFP) , and Ccr2 (RFP/RFP) mice. The retinal morphology and pattern of inflammatory cell activation and invasion of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were evaluated using retina and retinal pigment epithelium (RPE) flat mounts, retinal sections, and flow cytometry. Four-week-old Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice showed Cx3cr1-GFP-positive microglia in the inner retina. Cx3cr1-GFP and Ccr2-RFP dual positive activated microglia were observed in the outer retina and subretinal space of 6- and 8-week-old animals. Ccr2-RFP single positive bone marrow-derived macrophages were observed to migrate into the retina of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice. These invading cells were still observed in the

  20. Osteological Variation among Extreme Morphological Forms in the Mexican Salamander Genus Chiropterotriton (Amphibia: Plethodontidae: Morphological Evolution And Homoplasy.

    Directory of Open Access Journals (Sweden)

    David M Darda

    Full Text Available Osteological variation is recorded among and within four of the most distinctive species of the Mexican salamander genus Chiropterotriton. Analysis of the data is consistent with the monophyletic status of the genus and documents previously unrecorded intraspecific and interspecific variation. Most of the recorded variation involves qualitative and quantitative proportional differences, but four fixed differences constitute autapomorphic states that affirm and diagnose some species (C. dimidiatus, C. magnipes. Osteological variation in 15 characters is analyzed with respect to predictions generated from four hypotheses: 1 phylogeny, 2 adaptation to specific habitats (the four species include cave-dwelling, terrestrial, and arboreal forms, 3 size-free shape, and 4 size. High levels of intraspecific variation suggest that the characters studied are not subject to rigid functional constraints in salamanders, regardless of size. The pattern predicted by the hypothesis based on size differences seen among these four Chiropterotriton species matches most closely the observed pattern of relative skull robustness. Since size change and heterochrony are often associated in plethodontid evolution, it is likely that changes in developmental timing play a role in the morphological transitions among these morphologically diverse taxa. Webbed feet, miniaturization, body shape, and an unusual tarsal arrangement are morphologies exhibited in species of Chiropterotrition that are shown to be homoplastic with other clades of tropical plethodontids. Although extensive homoplasy in salamanders might be seen as a roadblock to unraveling phylogenetic hypotheses, the homologous developmental systems that appear to underlie such homoplasy may reveal common and consistent evolutionary processes at work.

  1. Solution structure and phylogenetics of Prod1, a member of the three-finger protein superfamily implicated in salamander limb regeneration.

    Directory of Open Access Journals (Sweden)

    Acely Garza-Garcia

    Full Text Available BACKGROUND: Following the amputation of a limb, newts and salamanders have the capability to regenerate the lost tissues via a complex process that takes place at the site of injury. Initially these cells undergo dedifferentiation to a state competent to regenerate the missing limb structures. Crucially, dedifferentiated cells have memory of their level of origin along the proximodistal (PD axis of the limb, a property known as positional identity. Notophthalmus viridescens Prod1 is a cell-surface molecule of the three-finger protein (TFP superfamily involved in the specification of newt limb PD identity. The TFP superfamily is a highly diverse group of metazoan proteins that includes snake venom toxins, mammalian transmembrane receptors and miscellaneous signaling molecules. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of identifying potential orthologs of Prod1, we have solved its 3D structure and compared it to other known TFPs using phylogenetic techniques. The analysis shows that TFP 3D structures group in different categories according to function. Prod1 clusters with other cell surface protein TFP domains including the complement regulator CD59 and the C-terminal domain of urokinase-type plasminogen activator. To infer orthology, a structure-based multiple sequence alignment of representative TFP family members was built and analyzed by phylogenetic methods. Prod1 has been proposed to be the salamander CD59 but our analysis fails to support this association. Prod1 is not a good match for any of the TFP families present in mammals and this result was further supported by the identification of the putative orthologs of both CD59 and N. viridescens Prod1 in sequence data for the salamander Ambystoma tigrinum. CONCLUSIONS/SIGNIFICANCE: The available data suggest that Prod1, and thereby its role in encoding PD identity, is restricted to salamanders. The lack of comparable limb-regenerative capability in other adult vertebrates could be

  2. Nicotinic Receptor Alpha7 Expression during Tooth Morphogenesis Reveals Functional Pleiotropy

    Science.gov (United States)

    Rogers, Scott W.; Gahring, Lorise C.

    2012-01-01

    The expression of nicotinic acetylcholine receptor (nAChR) subtype, alpha7, was investigated in the developing teeth of mice that were modified through homologous recombination to express a bi-cistronic IRES-driven tau-enhanced green fluorescent protein (GFP); alpha7GFP) or IRES-Cre (alpha7Cre). The expression of alpha7GFP was detected first in cells of the condensing mesenchyme at embryonic (E) day E13.5 where it intensifies through E14.5. This expression ends abruptly at E15.5, but was again observed in ameloblasts of incisors at E16.5 or molar ameloblasts by E17.5–E18.5. This expression remains detectable until molar enamel deposition is completed or throughout life as in the constantly erupting mouse incisors. The expression of alpha7GFP also identifies all stages of innervation of the tooth organ. Ablation of the alpha7-cell lineage using a conditional alpha7Cre×ROSA26-LoxP(diphtheria toxin A) strategy substantially reduced the mesenchyme and this corresponded with excessive epithelium overgrowth consistent with an instructive role by these cells during ectoderm patterning. However, alpha7knock-out (KO) mice exhibited normal tooth size and shape indicating that under normal conditions alpha7 expression is dispensable to this process. The function of ameloblasts in alpha7KO mice is altered relative to controls. High resolution micro-computed tomography analysis of adult mandibular incisors revealed enamel volume of the alpha7KO was significantly reduced and the organization of enamel rods was altered relative to controls. These results demonstrate distinct and varied spatiotemporal expression of alpha7 during tooth development, and they suggest that dysfunction of this receptor would have diverse impacts upon the adult organ. PMID:22666322

  3. Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland.

    Science.gov (United States)

    Syaidah, Rahimi; Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

    2013-11-01

    The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

  4. The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

    Science.gov (United States)

    Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

    2013-03-01

    Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

  5. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...... constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli...

  6. A little bit is better than nothing: the incomplete parthenogenesis of salamanders, frogs and fish

    Directory of Open Access Journals (Sweden)

    Schartl Manfred

    2010-08-01

    Full Text Available Abstract A re-examination of the mitochondrial genomes of unisexual salamander lineages, published in BMC Evolutionary Biology, shows them to be the oldest unisexual vertebrates known, having been around for 5 million years. This presents a challenge to the prediction that lack of genetic recombination is a fast track to extinction. See research article http://www.biomedcentral.com/1471-2148/10/238

  7. TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

    Czech Academy of Sciences Publication Activity Database

    Brejchová, Jana; Sýkora, Jan; Ostašov, Pavel; Merta, Ladislav; Roubalová, Lenka; Janáček, Jiří; Hof, Martin; Svoboda, Petr

    2015-01-01

    Roč. 1848, č. 3 (2015), s. 781-796 ISSN 0005-2736 R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 ; RVO:61388955 Keywords : cholesterol * TRH-R-eGFP mobility * FRAP * RICS * DPH fluorescence * G protein coupling Subject RIV: CE - Biochemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.687, year: 2015

  8. Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

    Science.gov (United States)

    König, Alexander; Glebe, Dieter

    2017-01-01

    To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

  9. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

    Science.gov (United States)

    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  10. [Isolation and purification of BMScs of GFP transgenic mouse using the method of adhering to cuture plastic in different time].

    Science.gov (United States)

    Li, Fu-Qiang; Zhou, Hong-Ying; Yang, Hui-Lun; Xiang, Tao; Mei, Yan; Hu, Huo-Zhen; Wang, Ting-Hua

    2006-03-01

    To adopt the method of adhering to culture plastic in different time for cultivating and purifying BMSCs of green fluorescent protein (GFP) transgenic mice. Bone marrow cells isolated from GFP transgenic mice are directly planted in culture flask and an exchange of the total volume medium is made at different time. Then the cells adhering to culture plastic are differently counted according to the cell types and are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54 in three days. Moreover, the cells after the exchange of the total volume medium in 4 hours, 8 hours and 24 hours are selected and successively subcultured down to the fifth passage. Then the result of amplification is calculated and the cells are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54. With the extending of the time for the first exchange of medium, the density of cells adhering to culture plastic increased accordingly, but the BMSCs proportion decreased. The cells after first exchange of medium in 4 hours had high BMSCs proportion but low BMSCs density, and the cells in 24 hours had high BMSCs density and low BMSCs proportion. However, the cells in 8-10 hours had high BMSCs density and also high BMSCs proportion. The subcultured BMSCs could stably express GFP. The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to make the first exchange of total volume medium in 8-10 hours. The subcultured cell has the capacity for amplification and will probably be a seed cell for the research of tissue engineering and gene therapy.

  11. Using PCR to Target Misconceptions about Gene Expression

    Directory of Open Access Journals (Sweden)

    Leslie K. Wright

    2013-02-01

    Full Text Available We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA and gene expression (mRNA/protein and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression.

  12. Effects of atrazine on egg masses of the yellow-spotted salamander (Ambystoma maculatum) and its endosymbiotic alga (Oophila amblystomatis)

    International Nuclear Information System (INIS)

    Baxter, Leilan; Brain, Richard A.; Hosmer, Alan J.; Nema, Mohini; Müller, Kirsten M.; Solomon, Keith R.; Hanson, Mark L.

    2015-01-01

    Embryonic growth of the yellow-spotted salamander (Ambystoma maculatum) is enhanced by the presence of the green alga Oophila amblystomatis, in the egg capsule. To further assess potential impacts of herbicides on this relationship, A. maculatum egg masses were exposed to atrazine (0–338 μg/L) until hatching (up to 66 days). Exposure to atrazine reduced PSII yield of the symbiotic algae in a concentration-dependent manner, but did not significantly affect visible algal growth or any metrics associated with salamander development. Algal cells were also cultured in the laboratory for toxicity testing. In the 96-h growth inhibition test (0–680 μg/L), ECx values were generally greater than those reported for standard algal test species. Complete recovery of growth rates occurred within 96-h of transferring cells to untreated media. Overall, development of A. maculatum embryos was not affected by exposure to atrazine at concentrations and durations exceeding those found in the environment. - Highlights: • The yellow-spotted salamander produces eggs that are colonized by a symbiotic green alga. • We tested the sensitivity of this system to the herbicide atrazine. • Embryo development was not significantly affected by exposure at up to 300 μg/L. • The alga was isolated and 96-h growth tests were performed in the laboratory. • EC50s for Oophila sp. were >100 μg/L. - Development of Ambystoma maculatum embryos in egg masses was not impacted by exposure to atrazine at concentrations and durations exceeding those commonly found in the environment.

  13. RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.

    Science.gov (United States)

    Dean, Kimberly M; Grayhack, Elizabeth J

    2012-12-01

    We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

  14. Response of stream-breeding salamander larvae to sediment deposition in southern Appalachian (U.S.A.) headwater streams

    Science.gov (United States)

    S. Conner Keitzer; Reuben. Goforth

    2012-01-01

    Summary 1. Increased fine sediment deposition is a prevalent threat to stream biodiversity and has been shown to impact stream-breeding salamanders negatively. However, their complex life histories make it difficult to determine which stage is affected. 2. We conducted field experiments from 26 August to 11 September 2010 and 11 October to 11...

  15. A population of Pax7-expressing muscle progenitor cells show differential responses to muscle injury dependent on developmental stage and injury extent

    Directory of Open Access Journals (Sweden)

    Stefanie eKnappe

    2015-08-01

    Full Text Available Muscle regeneration in vertebrates occurs by the activation of quiescent progenitor cells that express pax7 and replace and repair damaged fibers. We have developed a mechanical injury paradigm in zebrafish to determine whether developmental stage and injury size affect the regeneration dynamics of damaged muscle. We found that both small, focal injuries and large injuries affecting the entire myotome lead to the expression of myf5 and myogenin. Their expression was prolonged in older larvae, indicating a slower process of regeneration. We characterized the endogenous behavior of a population of muscle-resident Pax7-expressing cells using a pax7a:eGFP transgenic line and found that GFP+ cell migration in the myotome dramatically declined between 5 and 7 days post fertilization (dpf. Following a small injury, we observed that GFP+ cells responded by extending processes, before migrating to the injured fibers. Furthermore, these cells responded more rapidly to injury in 4dpf larvae compared to 7dpf. Interestingly, we did not see GFP+ fibers after repair of small injuries, indicating that pax7a-expressing cells did not contribute to fiber formation in this injury context. On the contrary, numerous GFP+ fibers could be observed after a large single myotome injury. Both injury models were accompanied by an increased number of proliferating GFP+ cells, which was more pronounced in larvae injured at 4dpf than 7dpf, This indicates intriguing developmental differences, even at these relatively early ages. Our data also suggests an interesting disparity in the role that pax7a-expressing muscle progenitor cells play during muscle regeneration, which may reflect the extent of muscle damage.

  16. Estimating occurrence and detection probabilities for stream-breeding salamanders in the Gulf Coastal Plain

    Science.gov (United States)

    Lamb, Jennifer Y.; Waddle, J. Hardin; Qualls, Carl P.

    2017-01-01

    Large gaps exist in our knowledge of the ecology of stream-breeding plethodontid salamanders in the Gulf Coastal Plain. Data describing where these salamanders are likely to occur along environmental gradients, as well as their likelihood of detection, are important for the prevention and management of amphibian declines. We used presence/absence data from leaf litter bag surveys and a hierarchical Bayesian multispecies single-season occupancy model to estimate the occurrence of five species of plethodontids across reaches in headwater streams in the Gulf Coastal Plain. Average detection probabilities were high (range = 0.432–0.942) and unaffected by sampling covariates specific to the use of litter bags (i.e., bag submergence, sampling season, in-stream cover). Estimates of occurrence probabilities differed substantially between species (range = 0.092–0.703) and were influenced by the size of the upstream drainage area and by the maximum proportion of the reach that dried. The effects of these two factors were not equivalent across species. Our results demonstrate that hierarchical multispecies models successfully estimate occurrence parameters for both rare and common stream-breeding plethodontids. The resulting models clarify how species are distributed within stream networks, and they provide baseline values that will be useful in evaluating the conservation statuses of plethodontid species within lotic systems in the Gulf Coastal Plain.

  17. Growth, survival, longevity, and population size of the Big Mouth Cave salamander (Gyrinophilus palleucus necturoides) from the type locality in Grundy County, Tennessee, USA

    Science.gov (United States)

    Niemiller, Matthew L.; Glorioso, Brad M.; Fenolio, Dante B.; Reynolds, R. Graham; Taylor, Steven J.; Miller, Brian T.

    2016-01-01

    Salamander species that live entirely in subterranean habitats have evolved adaptations that allow them to cope with perpetual darkness and limited energy resources. We conducted a 26-month mark–recapture study to better understand the individual growth and demography of a population of the Big Mouth Cave Salamander (Gyrinophilus palleucus necturoides). We employed a growth model to estimate growth rates, age at sexual maturity, and longevity, and an open population model to estimate population size, density, detectability, and survival rates. Furthermore, we examined cover use and evidence of potential predation. Individuals probably reach sexual maturity in 3–5 years and live at least nine years. Survival rates were generally high (>75%) but declined during the study. More than 30% of captured salamanders had regenerating tails or tail damage, which presumably represent predation attempts by conspecifics or crayfishes. Most salamanders (>90%) were found under cover (e.g., rocks, trash, decaying plant material). Based on 11 surveys during the study, population size estimates ranged from 21 to 104 individuals in the ca. 710 m2 study area. Previous surveys indicated that this population experienced a significant decline from the early 1970s through the 1990s, perhaps related to silvicultural and agricultural practices. However, our data suggest that this population has either recovered or stabilized during the past 20 years. Differences in relative abundance between early surveys and our survey could be associated with differences in survey methods or sampling conditions rather than an increase in population size. Regardless, our study demonstrates that this population is larger than previously thought and is in no immediate risk of extirpation, though it does appear to exhibit higher rates of predation than expected for a species believed to be an apex predator of subterranean food webs.

  18. A vector carrying the GFP gene (Green fluorescent protein as a yeast marker for fermentation processes Um vetor com o gene da GFP (Green fluorescent protein para a marcação de leveduras em processos fermentativos

    Directory of Open Access Journals (Sweden)

    Luiz Humberto Gomes

    2000-12-01

    Full Text Available Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3. Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.Leveduras contaminantes podem causar grandes perdas em processos fermentativos quando infectam culturas puras e degradam a qualidade do produto final. Estas leveduras podem ser detectadas por diversos métodos mas nenhum deles oferece resultados com a exatidão e precisão necessárias, quando os contaminantes estão em baixa freqüência. Culturas puras contendo um gene marcador podem ser utilizadas para a direta identificação de células e colônias contaminantes. Detecção rápida e fácil é desejada e marcadores morfológicos podem auxiliar na visualização da cultura marcada. O gene da GFP (green fluorescent protein extraído da Aequorea victoria

  19. Normal distribution and medullary-to-cortical shift of Nestin-expressing cells in acute renal ischemia.

    Science.gov (United States)

    Patschan, D; Michurina, T; Shi, H K; Dolff, S; Brodsky, S V; Vasilieva, T; Cohen-Gould, L; Winaver, J; Chander, P N; Enikolopov, G; Goligorsky, M S

    2007-04-01

    Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 microm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration.

  20. Rapid-response flood mapping during Hurricanes Harvey, Irma and Maria by the Global Flood Partnership (GFP)

    Science.gov (United States)

    Cohen, S.; Alfieri, L.; Brakenridge, G. R.; Coughlan, E.; Galantowicz, J. F.; Hong, Y.; Kettner, A.; Nghiem, S. V.; Prados, A. I.; Rudari, R.; Salamon, P.; Trigg, M.; Weerts, A.

    2017-12-01

    The Global Flood Partnership (GFP; https://gfp.jrc.ec.europa.eu) is a multi-disciplinary group of scientists, operational agencies and flood risk managers focused on developing efficient and effective global flood management tools. Launched in 2014, its aim is to establish a partnership for global flood forecasting, monitoring and impact assessment to strengthen preparedness and response and to reduce global disaster losses. International organizations, the private sector, national authorities, universities and research agencies contribute to the GFP on a voluntary basis and benefit from a global network focused on flood risk reduction. At the onset of Hurricane Harvey, GFP was `activated' using email requests via its mailing service. Soon after, flood inundation maps, based on remote sensing analysis and modeling, were shared by different agencies, institutions, and individuals. These products were disseminated, to varying degrees of effectiveness, to federal, state and local agencies via emails and data-sharing services. This generated a broad data-sharing network which was utilized at the early stages of Hurricane Irma's impact, just two weeks after Harvey. In this presentation, we will describe the extent and chronology of the GFP response to both Hurricanes Harvey, Irma and Maria. We will assess the potential usefulness of this effort for event managers in various types of organizations and discuss future improvements to be implemented.

  1. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

    Directory of Open Access Journals (Sweden)

    Margaret Rohrbaugh

    Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

  2. Expression of cholera toxin B–proinsulin fusion protein in lettuce and tobacco chloroplasts – oral administration protects against development of insulitis in non-obese diabetic mice

    OpenAIRE

    Ruhlman, Tracey; Ahangari, Raheleh; Devine, Andrew; Samsam, Mohtahsem; Daniell, Henry

    2007-01-01

    Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit–human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB–green fluorescent protein (CTB-GFP) or interferon–GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to ...

  3. Olfactory effects of a hypervariable multicomponent pheromone in the red-legged salamander, Plethodon shermani.

    Directory of Open Access Journals (Sweden)

    Damien B Wilburn

    Full Text Available Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF, is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males-likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex. Courtship trials with females receiving different PMF isoform mixtures had variable effects on female mating receptivity, with only the most complex mixtures increasing receptivity, such that we believe that sufficient isoform diversity allows males to improve their reproductive success with any female in the mating population. The aim of this study was to test the effects of isoform variability on VNE neuron activation using the agmatine uptake assay. All isoform mixtures activated a similar number of neurons (>200% over background except for a single purified PMF isoform (+17%. These data further support the hypothesis that PMF isoforms act synergistically in order to regulate female receptivity, and different putative mechanisms are discussed.

  4. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  5. Multiple upstream modules regulate zebrafish myf5 expression

    Directory of Open Access Journals (Sweden)

    Weng Chih-Wei

    2007-01-01

    Full Text Available Abstract Background Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood. Results We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1 the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2 the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3 the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4 the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5 the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm. Conclusion We suggest

  6. The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages.

    Directory of Open Access Journals (Sweden)

    Meret Cepero Malo

    Full Text Available Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers.

  7. Antibacterial activity and mechanism of Ag-ZnO nanocomposite on S. aureus and GFP-expressing antibiotic resistant E. coli.

    Science.gov (United States)

    Matai, Ishita; Sachdev, Abhay; Dubey, Poornima; Kumar, S Uday; Bhushan, Bharat; Gopinath, P

    2014-03-01

    Emergence of multi-resistant organisms (MROs) leads to ineffective treatment with the currently available medications which pose a great threat to public health and food technology sectors. In this regard, there is an urgent need to strengthen the present therapies or to look over for other potential alternatives like use of "metal nanocomposites". Thus, the present study focuses on synthesis of silver-zinc oxide (Ag-ZnO) nanocomposites which will have a broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria. Ag-ZnO nanocomposites of varied molar ratios were synthesized by simple microwave assisted reactions in the absence of surfactants. The crystalline behavior, composition and morphological analysis of the prepared powders were evaluated by X-ray diffraction, infrared spectroscopy, field emission scanning electron microscopy (FE-SEM) and atomic absorption spectrophotometry (AAS). Particle size measurements were carried out by transmission electron microscopy (TEM). Staphylococcus aureus and recombinant green fluorescent protein (GFP) expressing antibiotic resistant Escherichia coli were selected as Gram-positive and Gram-negative model systems respectively and the bactericidal activity of Ag-ZnO nanocomposite was studied. The minimum inhibitory concentration (MIC) and minimum killing concentration (MKC) of the nanocomposite against the model systems were determined by visual turbidity analysis and optical density analysis. Qualitative and quantitative assessments of its antibacterial effects were performed by fluorescent microscopy, fluorescent spectroscopy and Gram staining measurements. Changes in cellular morphology were examined by atomic force microscopy (AFM), FE-SEM and TEM. Finally, on the basis of the present investigation and previously published reports, a plausible antibacterial mechanism of Ag-ZnO nanocomposites was proposed. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Tongue and taste organ development in the ontogeny of direct-developing salamander Plethodon cinereus (Lissamphibia: Plethodontidae).

    Science.gov (United States)

    Budzik, Karolina A; Żuwała, Krystyna; Kerney, Ryan

    2016-07-01

    The latest research on direct developing caecilian and anuran species indicate presence of only one generation of taste organs during their ontogeny. This is distinct from indirect developing batrachians studied thus far, which possess taste buds in larvae and anatomically distinct taste discs in metamorphs. This study is a description of the tongue and taste organ morphology and development in direct developing salamander Plethodon cinereus (Plethodontidae) using histology and electron microscopy techniques. The results reveal two distinct stages tongue morphology (primary and secondary), similar to metamorphic urodeles, although only one stage of taste organ morphology. Taste disc sensory zones emerge on the surface of the oropharyngeal epithelium by the end of embryonic development, which coincides with maturation of the soft tongue. Taste organs occur in the epithelium of the tongue pad (where they are situated on the dermal papillae), the palate and the inner surface of the mandible and the maxilla. Plethodon cinereus embryos only possess taste disc type taste organs. Similar to the direct developing anuran Eleutherodactylus coqui (Eleutherodactylidae), these salamanders do not recapitulate larval taste bud morphology as an embryo. The lack of taste bud formation is probably a broadly distributed feature characteristic to direct developing batrachians. J. Morphol. 277:906-915, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes

    2004-02-09

    Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

  10. Computer-assisted photo identification outperforms visible implant elastomers in an endangered salamander, Eurycea tonkawae.

    Directory of Open Access Journals (Sweden)

    Nathan F Bendik

    Full Text Available Despite recognition that nearly one-third of the 6300 amphibian species are threatened with extinction, our understanding of the general ecology and population status of many amphibians is relatively poor. A widely-used method for monitoring amphibians involves injecting captured individuals with unique combinations of colored visible implant elastomer (VIE. We compared VIE identification to a less-invasive method - computer-assisted photographic identification (photoID - in endangered Jollyville Plateau salamanders (Eurycea tonkawae, a species with a known range limited to eight stream drainages in central Texas. We based photoID on the unique pigmentation patterns on the dorsal head region of 1215 individual salamanders using identification software Wild-ID. We compared the performance of photoID methods to VIEs using both 'high-quality' and 'low-quality' images, which were taken using two different camera types and technologies. For high-quality images, the photoID method had a false rejection rate of 0.76% compared to 1.90% for VIEs. Using a comparable dataset of lower-quality images, the false rejection rate was much higher (15.9%. Photo matching scores were negatively correlated with time between captures, suggesting that evolving natural marks could increase misidentification rates in longer term capture-recapture studies. Our study demonstrates the utility of large-scale capture-recapture using photo identification methods for Eurycea and other species with stable natural marks that can be reliably photographed.

  11. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death

    DEFF Research Database (Denmark)

    Nørholm, Morten Helge Hauberg; Nour-Eldin, Hussam H; Brodersen, Peter

    2006-01-01

    GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP...

  12. Establishment of Lactobacillus plantarum strain in honey bee digestive tract monitored using gfp fluorescence.

    Science.gov (United States)

    Javorský, P; Fecskeová, L Kolesár; Hrehová, L; Sabo, R; Legáth, J; Pristas, P

    2017-04-26

    Lactic acid bacteria are symbiotic bacteria that naturally reside in the gastrointestinal tract of honey bees. They serve a multitude of functions and are considered beneficial and completely harmless. In our experiments Lactobacillus plantarum strain B35, isolated from honey bee digestive tract, was modified using pAD43-25 plasmid carrying a functional GFP gene sequence (gfpmut3a) and used as a model for monitoring and optimisation of the mode of application. The establishment of this strain in honey bee digestive tract was monitored using GFP fluorescence. Three different modes of oral application of this strain were tested: water suspension of lyophilised bacteria, aerosol application of these bacteria and consumption of sugar honey paste containing the lyophilised lactobacilli. Two days after administration the L. plantarum B35-gfp was present throughout the honey bee digestive tract with 10 4 -10 5 cfu/bee with highest count observed for aerosol application.

  13. Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy

    Science.gov (United States)

    Glazachev, Yu I.; Orlova, D. Y.; Řezníčková, P.; Bártová, E.

    2018-05-01

    We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1β-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.

  14. TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm.

    Directory of Open Access Journals (Sweden)

    Nicole A J Krentz

    Full Text Available Human embryonic stem cells (hESCs have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-Associated protein (Cas to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.

  15. Quantitative analysis of lentiviral transgene expression in mice over seven generations.

    Science.gov (United States)

    Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

    2010-10-01

    Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken

  16. A 3D musculo-mechanical model of the salamander for the study of different gaits and modes of locomotion

    Directory of Open Access Journals (Sweden)

    Nalin eHarischandra

    2010-12-01

    Full Text Available Computer simulation has been used to investigate several aspectsof locomotion in salamanders. Here we introduce a three-dimensionalforward dynamics mechanical model of a salamander, with physicallyrealistic weight and size parameters. Movements of the four limbs and ofthe trunk and tail are generated by sets of linearly modeled skeletalmuscles. In this study, activation of these muscles were driven byprescribed neural output patterns. The model was successfully used tomimic locomotion on level ground and in water. We compare thewalking gait where a wave of activity in the axial muscles travelsbetween the girdles, with the trotting gait in simulations usingthe musculo-mechanical model. In a separate experiment, the model is usedto compare different strategies for turning while stepping; either bybending the trunk or by using side-stepping in the front legs. We foundthat for turning, the use of side-stepping alone or in combination withtrunk bending, was more effective than the use of trunk bending alone. Weconclude that the musculo-mechanical model described here together with aproper neural controller is useful for neuro-physiological experiments insilico.

  17. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

    International Nuclear Information System (INIS)

    Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O.

    2010-01-01

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties

  18. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus.

    Science.gov (United States)

    Flannery, J G; Zolotukhin, S; Vaquero, M I; LaVail, M M; Muzyczka, N; Hauswirth, W W

    1997-06-24

    We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-microl injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

  19. Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

    Science.gov (United States)

    Guan, Chengran; Cui, Wenjing; Cheng, Jintao; Zhou, Li; Guo, Junling; Hu, Xu; Xiao, Guoping; Zhou, Zhemin

    2015-09-21

    Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the -10 and -35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression

  20. The impact of intragenic CpG content on gene expression.

    Science.gov (United States)

    Bauer, Asli Petra; Leikam, Doris; Krinner, Simone; Notka, Frank; Ludwig, Christine; Längst, Gernot; Wagner, Ralf

    2010-07-01

    The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1 alpha). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

  1. Pathological analysis, detection of antigens, FasL expression analysis and leucocytes survival analysis in tilapia (Oreochromis niloticus) after infection with green fluorescent protein labeled Streptococcus agalactiae.

    Science.gov (United States)

    Wang, Jingyuan; Wu, Jinying; Yi, Liyuan; Hou, Zengxin; Li, Wensheng

    2017-03-01

    The pathogenesis of Streptococcus agalactiae infection in tilapia has not been fully described. To understand this, we investigated the clinic-pathological features of acute experimental septicemia in tilapia (Oreochromis niloticus) after receiving an intra-peritoneal injection with S. agalactiae THN-1901GFP. Immunohistochemistry and sections of pathological tissues were used to estimate the level of damage in the head-kidney, liver, spleen and trunk-kidney. The expression of FasL was analyzed by western blotting in these samples based on their damage levels. Leucocytes were isolated from the head-kidney and incubated with S. agalactiae THN-1901GFP. Then, phagocytosis, programmed cell death and the expression of FasL were analyzed. The infected tissues showed varying degrees of necrosis and histolysis. The serous membrane of the intestine was dissolved by S. agalactiae THN-1901GFP. Antigens of S. agalactiae THN-1901GFP accumulated in different parts of the infected organs. In the head-kidney and spleen, the expression of FasL was up-regulated in parallel with increased tissue damage. After being incubated with S. agalactiae THN-1901GFP, the phagocytic capacity and ability were both very high and the expression of FasL remained high in leucocytes. S. agalactiae THN-1901GFP was able to survive for a long period of time after being engulfed by phagocytic cells. These findings offer insight into the pathogenesis of S. agalactiae infection in tilapia. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Circadian Activators Are Expressed Days before They Initiate Clock Function in Late Pacemaker Neurons from Drosophila.

    Science.gov (United States)

    Liu, Tianxin; Mahesh, Guruswamy; Houl, Jerry H; Hardin, Paul E

    2015-06-03

    Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. We were surprised to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis. Copyright © 2015 the authors 0270-6474/15/358662-10$15.00/0.

  3. The fluorescence lifetime of BRI1-GFP as probe for the noninvasive determination of the membrane potential in living cells

    Science.gov (United States)

    Elgass, K.; Caesar, K.; Schleifenbaum, F.; Meixner, A. J.; Harter, K.

    2010-02-01

    As the excited state lifetime of a fluorescent molecule depends on its environment, it is possible to use it as a probe for physico-chemical parameters of the surrounding medium. Whereas this is well known for many solid guest/host systems, only few reports of quantitative, temporal resolved in vivo studies to monitor the nano-environment for a protein-coupled chromophore such as GFP are known from literature. Here we present a novel approach to determine the membrane potential of living (plant) cells based on the fluorescence lifetime (FLT) analysis of membrane-located GFP. By using confocal sample scanning microscopy (CSSM) combined with fluorescence lifetime imaging microscopy, we recently showed that the phytohormone brassinolide (BL) induces cell wall expansion and a decrease in the FLT of the BRI1-GFP in living cells of Arabidopsis thaliana seedlings. BRI1 is the dominant functional receptor for BL in Arabidopsis and locates to the plasma membrane. Although the dependence of the FLT of GFP on its physico-chemical environment such as pH-value, refractive index and pressure has been reported, the observed FLT decrease of BRI1-GFP in response to BL application could not be explained by these parameters. However, our in vivo FLT and CSSM analyses indicate that the BLinduced change in the FLT of BRI1-GFP is caused by hyperpolarisation of the plasma membrane (Em). Thus, our results indicate that BRI1-GFP serves as sensitive and non-invasive probe for recording the Em of the plasma membrane in living plant cells with high spatio-temporal resolution.

  4. Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

    Science.gov (United States)

    Masullo, Tiziana; Armata, Nerina; Pendolino, Flavio; Colombo, Paolo; Lo Celso, Fabrizio; Mazzola, Salvatore; Cuttitta, Angela

    2016-02-01

    The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.

  5. The influence of a water current on the larval deposition pattern of females of a diverging fire salamander population (Salamandra salamandra)

    NARCIS (Netherlands)

    Krause, E.T.; Caspers, B.A.

    2015-01-01

    Fire salamanders are amphibians that exhibit a highly specific reproductive mode termed ovo-viviparity. The eggs develop inside their mothers, and the females give birth to fully developed larvae. The larvae in our study area cluster in two distinct genetic groups that can be linked directly to the

  6. Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD

    Science.gov (United States)

    Granger, C. L.; Cyr, R. J.

    2001-01-01

    Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

  7. Effect-directed analysis for estrogenic compounds in a fluvial sediment sample using transgenic cyp19a1b-GFP zebrafish embryos.

    Science.gov (United States)

    Fetter, Eva; Krauss, Martin; Brion, François; Kah, Olivier; Scholz, Stefan; Brack, Werner

    2014-09-01

    Xenoestrogens may persist in the environment by binding to sediments or suspended particulate matter serving as long-term reservoir and source of exposure, particularly for organisms living in or in contact with sediments. In this study, we present for the first time an effect-directed analysis (EDA) for identifying estrogenic compounds in a sediment sample using embryos of a transgenic reporter fish strain. In the tg(cyp19a1b-GFP) transgenic zebrafish strain, the expression of GFP (green fluorescent protein) in the brain is driven by an oestrogen responsive element in the promoter of the cyp19a1b (aromatase) gene. The selected sediment sample of the Czech river Bilina had already been analysed in a previous EDA using the yeast oestrogen screening assay and had revealed fractions containing estrogenic compounds. When normal phase HPLC (high performance liquid chromatography) fractionation was used for the separation of the sediment sample, the biotest with transgenic fish embryos revealed two estrogenic fractions. Chemical analysis of candidate compounds in these sediment fractions suggested alkylphenols and estrone as candidate compounds responsible for the observed estrogenic effect. Alkylphenol concentrations could partially explain the estrogenicity of the fractions. However, xenoestrogens below the analytical detection limit or non-targeted estrogenic compounds have probably also contributed to the sample's estrogenic potency. The results indicated the suitability of the tg(cyp19a1b-GFP) fish embryo for an integrated chemical-biological analysis of estrogenic effects. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Heinrich B

    2017-05-01

    Full Text Available B Heinrich,1 J Klein,1 M Delic,1 K Goepfert,1 V Engel,1 L Geberzahn,1 M Lusky,2 P Erbs,2 X Preville,3 M Moehler1 1First Department of Internal Medicine, University Medical Center Mainz, Mainz, Germany; 2Transgene SA, Illkirch-Graffenstaden, 3Amoneta Diagnostics, Huningue, France Abstract: Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF or transforming 5-fluorocytosine (5-FC into 5-fluorouracil (5-FU. We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs and the interaction with the autologous cytotoxic T lymphocyte (CTL clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1, markers of immunogenic cell death (ICD, could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse

  9. Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration

    Science.gov (United States)

    Zhu, Wei; Kuo, Dwight; Nathanson, Jason; Satoh, Akira; Pao, Gerald M.; Yeo, Gene W.; Bryant, Susan V.; Voss, S. Randal; Gardiner, David M.; Hunter, Tony

    2012-01-01

    Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration. PMID:22913491

  10. Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats

    NARCIS (Netherlands)

    Fitzgerald, A.; Kan, van J.A.L.; Plummer, K.M.

    2004-01-01

    RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing

  11. Imaging Herpes Simplex Virus Type 1 Amplicon Vector–Mediated Gene Expression in Human Glioma Spheroids

    Directory of Open Access Journals (Sweden)

    Christine Kaestle

    2011-05-01

    Full Text Available Vectors derived from herpes simplex virus type 1 (HSV-1 have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector–mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP. After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector–mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 μm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application—injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

  12. A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.

    Science.gov (United States)

    Wendel, Sofie; Fischer, Emil C; Martínez, Virginia; Seppälä, Susanna; Nørholm, Morten H H

    2016-05-03

    Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing. Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency. We have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech.

  13. Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

    Science.gov (United States)

    Holman, Edna C.; Campbell, Leah J.; Hines, John; Crews, Craig M.

    2012-01-01

    Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes. PMID:23028429

  14. Microarray analysis of microRNA expression during axolotl limb regeneration.

    Directory of Open Access Journals (Sweden)

    Edna C Holman

    Full Text Available Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum ("Amex" miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3'UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.

  15. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    Science.gov (United States)

    Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2016-01-01

    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

  16. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    Directory of Open Access Journals (Sweden)

    Fauziah Abu Bakar

    2016-04-01

    Full Text Available Bacterial toxin-antitoxin (TA systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.

  17. Prolonged peritoneal gene expression using a helper-dependent adenovirus.

    Science.gov (United States)

    Liu, Limin; Shi, Chang-Xin; Ghayur, Ayesha; Zhang, Claire; Su, Je Yen; Hoff, Catherine M; Margetts, Peter J

    2009-01-01

    Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis. The causes of EPS are not well defined and are likely multifactorial. A suitable animal model would facilitate research into the pathophysiology and treatment of EPS. We developed a helper-dependent adenovirus that expresses both green fluorescent protein (GFP) and active transforming growth factor-beta (TGF-beta1; HDAdTGF-beta1). Mice were administered HDAdTGF-beta1 via intraperitoneal injection and the response was compared with mice administered either first-generation adenovirus expressing TGF-beta1 (AdTGF-beta1) or control adenovirus (AdGFP). HDAdTGF-beta1-treated mice continued to express the GFP reporter transgene to day 74, the end of the observation period. Transgene expression lasted less than 28 days in the animals treated with first-generation adenoviruses. Animals treated with first-generation AdTGF-beta1 demonstrated submesothelial thickening and angiogenesis at day 7, with almost complete resolution by day 28. The HDAdTGF-beta1-treated mice demonstrated progressive peritoneal fibrosis with adhesion formation and encapsulation of bowels. Weight gain was significantly reduced in animals treated with HDAdTGF-beta1 compared to both the control-treated animals and the AdTGF-beta1-treated animals. Inflammation was not a major component of the fibroproliferative response. Peritoneal administration of a first-generation AdTGF-beta1 leads to transient gene expression, resulting in a resolving fibrotic response and histology similar to that seen in simple peritoneal sclerosis. Prolonged TGF-beta1 expression induced by the helper-dependent HDAdTGF-beta1 led to changes in peritoneal morphology resembling EPS. This suggests that TGF-beta1 may be a contributing factor in both simple peritoneal sclerosis and EPS. This model will be useful for elucidation of the mechanism of EPS and evaluation of potential treatment.

  18. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

    Science.gov (United States)

    Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.

    2016-01-01

    The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves

  19. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine.

    Directory of Open Access Journals (Sweden)

    Daiane P Oldiges

    2016-12-01

    Full Text Available The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST. The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein-blasticidin deaminase, and HlGST fused to the MSA-1 (merozoite surface antigen 1 signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on Hl

  20. Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication

    International Nuclear Information System (INIS)

    DeWire, Scott M.; Money, Eric S.; Krall, Stuart P.; Damania, Blossom

    2003-01-01

    Rhesus monkey rhadinovirus (RRV) is a γ-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Lack of an efficient culture system to grow high titers of virus, and the lack of an in vivo animal model system, has hampered the study of KSHV replication and pathogenesis. RRV is capable of replicating to high titers on fibroblasts, thus facilitating the construction of recombinant rhadinoviruses. In addition, the ability to experimentally infect naieve rhesus macaques with RRV makes it an excellent model system to study γ-herpesvirus replication. Our study describes, for the first time, the construction of a GFP-expressing RRV recombinant virus using a traditional homologous recombination strategy. We have also developed two new methods for determining viral titers of RRV including a traditional viral plaque assay and a quantitative real-time PCR assay. We have compared the replication of wild-type RRV with that of the RRV-GFP recombinant virus in one-step growth curves. We have also measured the sensitivity of RRV to a small panel of antiviral drugs. The development of both the recombination strategy and the viral quantitation assays for RRV will lay the foundation for future studies to evaluate the contribution of individual genes to viral replication both in vitro and in vivo

  1. Chloride equilibrium potential in salamander cones

    Directory of Open Access Journals (Sweden)

    Bryson Eric J

    2004-12-01

    Full Text Available Abstract Background GABAergic inhibition and effects of intracellular chloride ions on calcium channel activity have been proposed to regulate neurotransmission from photoreceptors. To assess the impact of these and other chloride-dependent mechanisms on release from cones, the chloride equilibrium potential (ECl was determined in red-sensitive, large single cones from the tiger salamander retinal slice. Results Whole cell recordings were done using gramicidin perforated patch techniques to maintain endogenous Cl- levels. Membrane potentials were corrected for liquid junction potentials. Cone resting potentials were found to average -46 mV. To measure ECl, we applied long depolarizing steps to activate the calcium-activated chloride current (ICl(Ca and then determined the reversal potential for the current component that was inhibited by the Cl- channel blocker, niflumic acid. With this method, ECl was found to average -46 mV. In a complementary approach, we used a Cl-sensitive dye, MEQ, to measure the Cl- flux produced by depolarization with elevated concentrations of K+. The membrane potentials produced by the various high K+ solutions were measured in separate current clamp experiments. Consistent with electrophysiological experiments, MEQ fluorescence measurements indicated that ECl was below -36 mV. Conclusions The results of this study indicate that ECl is close to the dark resting potential. This will minimize the impact of chloride-dependent presynaptic mechanisms in cone terminals involving GABAa receptors, glutamate transporters and ICl(Ca.

  2. [Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

    Science.gov (United States)

    Zhu, Jing; Chen, Bo-Jiang; Huang, Na; Li, Wei-Min

    2014-03-01

    To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells. Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot. AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot. The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.

  3. Developing landscape habitat models for rare amphibians with small geographic ranges: a case study of Siskiyou Mountains salamanders in the western USA

    Science.gov (United States)

    Nobuya Suzuki; Deanna H. Olson; Edward C. Reilly

    2007-01-01

    To advance the development of conservation planning for rare species with small geographic ranges, we determined habitat associations of Siskiyou Mountains salamanders (Plethodon stormi) and developed habitat suitability models at fine (10 ha), medium (40 ha), and broad (202 ha) spatial scales using available geographic information systems data and...

  4. Quantitative assessment of fibroblast growth factor receptor 1 expression in neurons and glia

    Directory of Open Access Journals (Sweden)

    Lisha Choubey

    2017-04-01

    Full Text Available Background Fibroblast growth factors (FGFs and their receptors (FGFRs have numerous functions in the developing and adult central nervous system (CNS. For example, the FGFR1 receptor is important for proliferation and fate specification of radial glial cells in the cortex and hippocampus, oligodendrocyte proliferation and regeneration, midline glia morphology and soma translocation, Bergmann glia morphology, and cerebellar morphogenesis. In addition, FGFR1 signaling in astrocytes is required for postnatal maturation of interneurons expressing parvalbumin (PV. FGFR1 is implicated in synapse formation in the hippocampus, and alterations in the expression of Fgfr1 and its ligand, Fgf2 accompany major depression. Understanding which cell types express Fgfr1 during development may elucidate its roles in normal development of the brain as well as illuminate possible causes of certain neuropsychiatric disorders. Methods Here, we used a BAC transgenic reporter line to trace Fgfr1 expression in the developing postnatal murine CNS. The specific transgenic line employed was created by the GENSAT project, tgFGFR1-EGFPGP338Gsat, and includes a gene encoding enhanced green fluorescent protein (EGFP under the regulation of the Fgfr1 promoter, to trace Fgfr1 expression in the developing CNS. Unbiased stereological counts were performed for several cell types in the cortex and hippocampus. Results This model reveals that Fgfr1 is primarily expressed in glial cells, in both astrocytes and oligodendrocytes, along with some neurons. Dual labeling experiments indicate that the proportion of GFP+ (Fgfr1+ cells that are also GFAP+ increases from postnatal day 7 (P7 to 1 month, illuminating dynamic changes in Fgfr1 expression during postnatal development of the cortex. In postnatal neurogenic areas, GFP expression was also observed in SOX2, doublecortin (DCX, and brain lipid-binding protein (BLBP expressing cells. Fgfr1 is also highly expressed in DCX positive cells of

  5. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

    Directory of Open Access Journals (Sweden)

    Michael Hartmann

    2013-08-01

    Full Text Available We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL. By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1, shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

  6. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Hao; Suslov, Nikolai B.; Li, Nan-Sheng; Shelke, Sandip A.; Evans, Molly E.; Koldobskaya, Yelena; Rice, Phoebe A.; Piccirilli, Joseph A. [UC

    2014-08-21

    Spinach is an in vitro–selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

  7. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    International Nuclear Information System (INIS)

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O'Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed

  8. Plethodontid salamander mitochondrial genomics: A parsimonyevaluation of character conflict and implications for historicalbiogeography

    Energy Technology Data Exchange (ETDEWEB)

    Macey, J. Robert

    2005-01-19

    A new parsimony analysis of 27 complete mitochondrial genomic sequences is conducted to investigate the phylogenetic relationships of plethodontid salamanders. This analysis focuses on the amount of character conflict between phylogenetic trees recovered from newly conducted parsimony searches and the Bayesian and maximum likelihood topology reported by Mueller et al. (2004, PNAS, 101, 13820-13825). Strong support for Hemidactylium as the sister taxon to all other plethodontids is recovered from parsimony analyses. Plotting area relationships on the most parsimonious phylogenetic tree suggests that eastern North America is the origin of the family Plethodontidae supporting the ''Out of Appalachia'' hypothesis. A new taxonomy that recognizes clades recovered from phylogenetic analyses is proposed.

  9. Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ito, Keisuke; Sugawara, Taishi; Shiroishi, Mitsunori; Tokuda, Natsuko; Kurokawa, Azusa; Misaka, Takumi; Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Nomura, Norimichi; Murata, Takeshi; Abe, Keiko; Iwata, So

    2008-01-01

    Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination

  10. Plasmid stability in dried cells of the desert cyanobacterium Chroococcidiopsis and its potential for GFP imaging of survivors on Earth and in space.

    Science.gov (United States)

    Billi, Daniela

    2012-06-01

    Two GFP-based plasmids, namely pTTQ18-GFP-pDU1(mini) and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1(mini)-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecA(Syn) distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecA(Syn) structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

  11. Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

    Science.gov (United States)

    Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

    2017-07-25

    the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 10 4 to 32 × 10 4 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.

  12. Road deicing salt irreversibly disrupts osmoregulation of salamander egg clutches.

    Science.gov (United States)

    Karraker, Nancy E; Gibbs, James P

    2011-03-01

    It has been postulated that road deicing salts are sufficiently diluted by spring rains to ameliorate any physiological impacts to amphibians breeding in wetlands near roads. We tested this conjecture by exposing clutches of the spotted salamander (Ambystoma maculatum) to three chloride concentrations (1 mg/L, 145 mg/L, 945 mg/L) for nine days, then transferred clutches to control water for nine days, and measured change in mass at three-day intervals. We measured mass change because water uptake by clutches reduces risks to embryos associated with freezing, predation, and disease. Clutches in controls sequestered water asymptotically. Those in the moderate concentrations lost 18% mass initially and regained 14% after transfer to control water. Clutches in high concentration lost 33% mass and then lost an additional 8% after transfer. Our results suggest that spring rains do not ameliorate the effects of deicing salts in wetlands with extremely high chloride concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. Selection of antigenic markers on a GFP-Cκ fusion scaffold with high sensitivity by eukaryotic ribosome display

    International Nuclear Information System (INIS)

    Yang Yongmin; Barankiewicz, Teresa J.; He Mingyue; Taussig, Michael J.; Chen, Swey-Shen

    2007-01-01

    Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (Cκ) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or Cκ (3') were selected by anti-GFP or anti-Cκ antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins

  14. Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3' untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos.

    Science.gov (United States)

    Kawakami, Y; Ishihara, M; Saito, T; Fujimoto, T; Adachi, S; Arai, K; Yamaha, E

    2012-12-01

    Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.

  15. Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

    Science.gov (United States)

    Zhao, Yanhong; Liao, Xiaofang; Huang, Zhipeng; Chen, Peng; Zhou, Bujin; Liu, Dongmei; Kong, Xiangjun; Zhou, Ruiyang

    2015-08-01

    Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In

  16. Pseudo-immunolabelling with the avidin-biotin-peroxidase complex (ABC) due to the presence of endogenous biotin in retinal Müller cells of goldfish and salamander

    NARCIS (Netherlands)

    Bhattacharjee, J.; Nunes Cardozo, B.; Kamphuis, W.; Kamermans, M.; Vrensen, G. F.

    1997-01-01

    Immunodetection techniques are dependent on enzyme-protein conjugates for the visualisation of antigen-antibody complexes. One of the most widely used is the avidin-biotin-peroxidase complex (ABC) method. The present study demonstrates that direct treatment of goldfish and salamander retinal

  17. Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

    Science.gov (United States)

    Nocarova, Eva; Fischer, Lukas

    2009-04-22

    Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with

  18. Site-level habitat models for the endemic, threatened Cheat Mountain salamander (Plethodon nettingi): the importance of geophysical and biotic attributes for predicting occurrence

    Science.gov (United States)

    Lester O. Dillard; Kevin R. Russell; W. Mark Ford

    2008-01-01

    The federally threatened Cheat Mountain salamander (Plethodon nettingi; hereafter CMS) is known to occur in approximately 70 small, scattered populations in the Allegheny Mountains of eastern West Virginia, USA. Current conservation and management efforts on federal, state, and private lands involving CMS largely rely on small scale, largely...

  19. Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils.

    Science.gov (United States)

    Anbalagan, Charumathi; Lafayette, Ivan; Antoniou-Kourounioti, Melissa; Gutierrez, Carmen; Martin, Jose Rodriguez; Chowdhuri, Debapratim K; De Pomerai, David I

    2013-01-01

    As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439-455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We

  20. Development of expression vectors for Escherichia coli based on the pCR2 replicon

    Directory of Open Access Journals (Sweden)

    Deb J K

    2007-05-01

    Full Text Available Abstract Background Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. Results We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E.coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. Conclusion We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

  1. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peddie, Christopher J.; Blight, Ken; Wilson, Emma [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Melia, Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden (Netherlands); Marrison, Jo [Department of Biology, The University of York, Heslington, York (United Kingdom); Carzaniga, Raffaella [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Domart, Marie-Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); O' Toole, Peter [Department of Biology, The University of York, Heslington, York (United Kingdom); Larijani, Banafshe [Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa (Spain); IKERBASQUE, Basque Foundation for Science, Bilbao (Spain); Collinson, Lucy M. [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom)

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. - Highlights: • GFP and mCherry fluorescence are preserved in heavy-metal stained mammalian cells embedded in resin • Fluorophores are stable and intensity is sufficient for detection in ultrathin sections • Overlay of separate LM and EM images from the same ultrathin section improves CLEM protein localisation precision • GFP is stable and active in the vacuum of an integrated light and scanning EM • Integrated light and electron microscopy shows new subcellular locations of the lipid diacylglycerol.

  2. The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

    Science.gov (United States)

    Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

    2016-10-15

    Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Diet of larval Ambystoma rivulare (Caudata: Ambystomatidae, a threatened salamander from the Volcán Nevado de Toluca, Mexico

    Directory of Open Access Journals (Sweden)

    Julio A. Lemos-Espinal

    2015-06-01

    Full Text Available Several species of salamander in the genus Ambystoma occur in the mountains surrounding Mexico City and are considered at risk of extinction. However, little is known about their ecology and natural history. The Toluca Stream Siredon (Ambystoma rivulare is classified as “Data Deficient” by the IUCN, and considered “Threatened” under Mexican law. From October 2013 to September 2014, we examined the diet of larval A. rivulare from a stream on the Volcán Nevado de Toluca in Mexico to provide insight into the suitability of the habitat to support this population of salamanders. Ostracods accounted for approximately 90% of all prey items consumed by larval A. rivulare. The number of ostracods found in stomachs increased with individual body size, but the proportion of ostracods in stomachs did not vary with body size. Nematodes were observed in approximately one third of the stomachs we examined. The diversity of prey in the diet of A. rivulare in the stream we studied is low and dominated by a single prey taxon, ostracods. Our results suggest that if environmental conditions in the stream change such that ostracods are negatively affected then the long-term persistence of this population of A. rivulare might be in jeopardy.

  4. Functional and neurochemical characterization of angiotensin type 1A receptor-expressing neurons in the nucleus of the solitary tract of the mouse.

    Science.gov (United States)

    Carter, D A; Choong, Y-T; Connelly, A A; Bassi, J K; Hunter, N O; Thongsepee, N; Llewellyn-Smith, I J; Fong, A Y; McDougall, S J; Allen, A M

    2017-10-01

    Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT 1A R) most widely expressed in adult neurons. Activation of the AT 1 R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT 1 R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT 1A R-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT 1A R promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT 1A R-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT 1A R-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT 1A R-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT 1A R-GFP neurons and TH-GFP neurons showed similar AT 1A R-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT 1A R-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing. Copyright © 2017 the American Physiological Society.

  5. The Importance of Maintaining Upland Forest Habitat Surrounding Salamander Breeding Ponds: Case Study of the Eastern Tiger Salamander in New York, USA

    Directory of Open Access Journals (Sweden)

    Valorie Titus

    2014-12-01

    Full Text Available Most amphibians use both wetland and upland habitats, but the extent of their movement in forested habitats is poorly known. We used radiotelemetry to observe the movements of adult and juvenile eastern tiger salamanders over a 4-year period. Females tended to move farther from the breeding ponds into upland forested habitat than males, while the distance a juvenile moved appeared to be related to body size, with the largest individuals moving as far as the adult females. Individuals chose refugia in native pitch pine—oak forested habitat and avoided open fields, roads, and developed areas. We also observed a difference in potential predation pressures in relation to the distance an individual moved from the edge of the pond. Our results support delineating forested wetland buffer zones on a case-by-case basis to reduce the impacts of concentrated predation, to increase and protect the availability of pitch pine—oak forests near the breeding pond, and to focus primarily on the habitat needs of the adult females and larger juveniles, which in turn will encompass habitat needs of adult males and smaller juveniles.

  6. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    Directory of Open Access Journals (Sweden)

    D’Urzo Nunzia

    2013-02-01

    Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

  7. Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

    Energy Technology Data Exchange (ETDEWEB)

    Yongmin, Yang [Institute of Genetics, San Diego, CA 92121-2233 (United States); IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States); Barankiewicz, Teresa J [Institute of Genetics, San Diego, CA 92121-2233 (United States); IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States); Mingyue, He [Babraham Institute, Cambridge CB2 4AT (United Kingdom); Taussig, Michael J [Babraham Institute, Cambridge CB2 4AT (United Kingdom); Chen, Swey-Shen [Institute of Genetics, San Diego, CA 92121-2233 (United States) and IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)

    2007-07-27

    Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.

  8. Habitat adaptation rather than genetic distance correlates with female preference in fire salamanders (Salamandra salamandra

    Directory of Open Access Journals (Sweden)

    Weitere Markus

    2009-06-01

    Full Text Available Abstract Background Although some mechanisms of habitat adaptation of conspecific populations have been recently elucidated, the evolution of female preference has rarely been addressed as a force driving habitat adaptation in natural settings. Habitat adaptation of fire salamanders (Salamandra salamandra, as found in Middle Europe (Germany, can be framed in an explicit phylogeographic framework that allows for the evolution of habitat adaptation between distinct populations to be traced. Typically, females of S. salamandra only deposit their larvae in small permanent streams. However, some populations of the western post-glacial recolonization lineage use small temporary ponds as larval habitats. Pond larvae display several habitat-specific adaptations that are absent in stream-adapted larvae. We conducted mate preference tests with females from three distinct German populations in order to determine the influence of habitat adaptation versus neutral genetic distance on female mate choice. Two populations that we tested belong to the western post-glacial recolonization group, but are adapted to either stream or pond habitats. The third population is adapted to streams but represents the eastern recolonization lineage. Results Despite large genetic distances with FST values around 0.5, the stream-adapted females preferred males from the same habitat type regardless of genetic distance. Conversely, pond-adapted females did not prefer males from their own population when compared to stream-adapted individuals of either lineage. Conclusion A comparative analysis of our data showed that habitat adaptation rather than neutral genetic distance correlates with female preference in these salamanders, and that habitat-dependent female preference of a specific pond-reproducing population may have been lost during adaptation to the novel environmental conditions of ponds.

  9. Effects of metamorphosis on the aquatic escape response of the two-lined salamander (Eurycea bislineata).

    Science.gov (United States)

    Azizi, Emanuel; Landberg, Tobias

    2002-03-01

    Although numerous studies have described the escape kinematics of fishes, little is known about the aquatic escape responses of salamanders. We compare the escape kinematics of larval and adult Eurycea bislineata, the two-lined salamander, to examine the effects of metamorphosis on aquatic escape performance. We hypothesize that shape changes associated with resorption of the larval tail fin at metamorphosis will affect aquatic locomotor performance. Escape responses were recorded using high-speed video, and the effects of life stage and total length on escape kinematics were analyzed statistically using analysis of covariance. Our results show that both larval and adult E. bislineata use a two-stage escape response (similar to the C-starts of fishes) that consists of a preparatory (stage 1) and a propulsive (stage 2) stroke. The duration of both kinematic stages and the distance traveled during stage 2 increased with total length. Both larval and adult E. bislineata had final escape trajectories that were directed away from the stimulus. The main kinematic difference between larvae and adults is that adults exhibit significantly greater maximum curvature during stage 1. Total escape duration and the distance traveled during stage 2 did not differ significantly between larvae and adults. Despite the significantly lower tail aspect ratio of adults, we found no significant decrease in the overall escape performance of adult E. bislineata. Our results suggest that adults may compensate for the decrease in tail aspect ratio by increasing their maximum curvature. These findings do not support the hypothesis that larvae exhibit better locomotor performance than adults as a result of stronger selective pressures on early life stages.

  10. Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

    Science.gov (United States)

    Wilburn, Damien B; Bowen, Kathleen E; Doty, Kari A; Arumugam, Sengodagounder; Lane, Andrew N; Feldhoff, Pamela W; Feldhoff, Richard C

    2014-01-01

    In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP) superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF) is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s) without the immediate need for complementary mutations. Consequently, this unique

  11. Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

    Directory of Open Access Journals (Sweden)

    Damien B Wilburn

    Full Text Available In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s without the immediate need for complementary mutations. Consequently

  12. Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens

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    Manuel Hiss

    2017-10-01

    Full Text Available The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

  13. Manipulation of gene expression by infrared laser heat shock and its application to the study of tracheal development in Drosophila.

    Science.gov (United States)

    Miao, Guangxia; Hayashi, Shigeo

    2015-03-01

    Induction of gene expression in a specific cell and a defined time window is desirable to investigate gene function at the cellular level during morphogenesis. To achieve this, we attempted to introduce the infrared laser-evoked gene operator system (IR-LEGO, Kamei et al., 2009) in the Drosophila embryo. In this technique, infrared laser light illumination induces genes to be expressed under the control of heat shock promoters at the single cell level. We applied IR-LEGO to a transgenic fly stock, HS-eGFP, in which the enhanced green fluorescent protein (eGFP) gene is placed under the control of heat shock protein 70 promoter, and showed that eGFP expression can be induced in single cells within 1-2 hr after IR illumination. Furthermore, induction of HS-Branchless transgene encoding the Drosophila fibroblast growth factor (FGF) effectively altered the migration and branching patterns of the tracheal system. Our results indicated that IR-LEGO is a promising choice for the timely control of gene expression in a small group of cells in the Drosophila embryo. By using IR-LEGO, we further demonstrated that the tracheal terminal branching program is sensitive to localized expression of exogenous FGF. © 2014 Wiley Periodicals, Inc.

  14. Comparison of expression vectors in Lactobacillus reuteri strains.

    Science.gov (United States)

    Lizier, Michela; Sarra, Pier G; Cauda, Roberto; Lucchini, Franco

    2010-07-01

    The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

  15. Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus.

    Science.gov (United States)

    Moreau, Morgane J J; Schaeffer, Patrick M

    2013-12-01

    The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.

  16. Defining POMC neurons using transgenic reagents: impact of transient Pomc expression in diverse immature neuronal populations.

    Science.gov (United States)

    Padilla, Stephanie L; Reef, Daniel; Zeltser, Lori M

    2012-03-01

    Melanocortin signaling plays a central role in the regulation of phenotypes related to body weight and energy homeostasis. To specifically target and study the function of proopiomelanocortin (POMC) neurons, Pomc promoter elements have been utilized to generate reporter and Cre recombinase transgenic reagents. Across gestation, we find that Pomc is dynamically expressed in many sites in the developing mouse forebrain, midbrain, hindbrain, spinal cord, and retina. Although Pomc expression in most embryonic brain regions is transient, it is sufficient to direct Cre-mediated recombination of floxed alleles. We visualize the populations affected by this transgene by crossing Pomc-Cre mice to ROSA reporter strains and identify 62 sites of recombination throughout the adult brain, including several nuclei implicated in energy homeostasis regulation. To compare the relationship between acute Pomc promoter activity and Pomc-Cre-mediated recombination at the single cell level, we crossed Pomc-enhanced green fluorescent protein (eGFP) and Pomc-Cre;ROSA-tdTomato lines. We detect the highest concentration of Pomc-eGFP+ cells in the arcuate nucleus of the hypothalamus and dentate gyrus but also observe smaller populations of labeled cells in the nucleus of the solitary tract, periventricular zone of the third ventricle, and cerebellum. Consistent with the dynamic nature of Pomc expression in the embryo, the vast majority of neurons marked with the tdTomato reporter do not express eGFP in the adult. Thus, recombination in off-target sites could contribute to physiological phenotypes using Pomc-Cre transgenics. For example, we find that approximately 83% of the cells in the arcuate nucleus of the hypothalamus immunoreactive for leptin-induced phosphorylated signal transducer and activator of transcription 3 are marked with Pomc-Cre;ROSA-tdTomato; only 13% of these are eGFP+ POMC neurons.

  17. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

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    Lee Okhyun

    2012-06-01

    Full Text Available Abstract Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38 in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff, contains three copies of oestrogen response elements (3ERE that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein. Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2, the synthetic oestrogen 17α- ethinyloestradiol (EE2, and the relatively weak environmental oestrogen nonylphenol (NP, and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures. For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish. We

  18. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  19. Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds.

    Science.gov (United States)

    Wang, Yunfeng; Sun, Yu; Chang, Qing; Ahmad, Shoeb; Zhou, Binfei; Kim, Yeunjung; Li, Huawei; Lin, Xi

    2013-01-01

    Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages. We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing. Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested. Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention. Copyright © 2013 John Wiley & Sons, Ltd.

  20. Interaction of PLP with GFP-MAL2 in the human oligodendroglial cell line HOG.

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    Raquel Bello-Morales

    Full Text Available The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP, the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested.

  1. Differential Structural Development of Adult-Born Septal Hippocampal Granule Cells in the Thy1-GFP Mouse, Nuclear Size as a New Index of Maturation.

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    Tijana Radic

    Full Text Available Adult neurogenesis is frequently studied in the mouse hippocampus. We examined the morphological development of adult-born, immature granule cells in the suprapyramidal blade of the septal dentate gyrus over the period of 7-77 days after mitosis with BrdU-labeling in 6-weeks-old male Thy1-GFP mice. As Thy1-GFP expression was restricted to maturated granule cells, it was combined with doublecortin-immunolabeling of immature granule cells. We developed a novel classification system that is easily applicable and enables objective and direct categorization of newborn granule cells based on the degree of dendritic development in relation to the layer specificity of the dentate gyrus. The structural development of adult-generated granule cells was correlated with age, albeit with notable differences in the time course of development between individual cells. In addition, the size of the nucleus, immunolabeled with the granule cell specific marker Prospero-related homeobox 1 gene, was a stable indicator of the degree of a cell's structural maturation and could be used as a straightforward parameter of granule cell development. Therefore, further studies could employ our doublecortin-staging system and nuclear size measurement to perform investigations of morphological development in combination with functional studies of adult-born granule cells. Furthermore, the Thy1-GFP transgenic mouse model can be used as an additional investigation tool because the reporter gene labels granule cells that are 4 weeks or older, while very young cells could be visualized through the immature marker doublecortin. This will enable comparison studies regarding the structure and function between young immature and older matured granule cells.

  2. Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

    Science.gov (United States)

    Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

    2012-08-01

    Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

  3. High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana.

    Science.gov (United States)

    Girke, Christopher; Arutyunova, Elena; Syed, Maria; Traub, Michaela; Möhlmann, Torsten; Lemieux, M Joanne

    2015-09-01

    Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein. The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST). AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7. The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter

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    Amrita Salvi

    2017-03-01

    Full Text Available Cancer cell migration and invasion involves temporal and spatial regulation of actin cytoskeleton reorganization, which is regulated by the WASP family of proteins such as N-WASP (Neural- Wiskott Aldrich Syndrome Protein. We have previously shown that expression of N-WASP was increased under hypoxic conditions. In order to characterize the regulation of N-WASP expression, we constructed an N-WASP promoter driven GFP reporter construct, N-WASPpro-GFP. Transfection of N-WASPpro-GFP construct and plasmid expressing HiF1α (Hypoxia Inducible factor 1α enhanced the expression of GFP suggesting that increased expression of N-WASP under hypoxic conditions is mediated by HiF1α. Sequence analysis of the N-WASP promoter revealed the presence of two hypoxia response elements (HREs characterized by the consensus sequence 5′-GCGTG-3′ at -132 bp(HRE1 and at -662 bp(HRE2 relative to transcription start site (TSS. Site-directed mutagenesis of HRE1(-132 but not HRE2(-662 abolished the HiF1α induced activation of N-WASP promoter. Similarly ChIP assay demonstrated that HiF1α bound to HRE1(-132 but not HRE2(-662 under hypoxic condition. MDA-MB-231 cells but not MDA-MB-231KD cells treated with hypoxia mimicking agent, DMOG showed enhanced gelatin degradation. Similarly MDA-MB-231KD(N-WASPpro-N-WASPR cells expressing N-WASPR under the transcriptional regulation of WT N-WASPpro but not MDA-MB-231KD(N-WASPproHRE1-N-WASPR cells expressing N-WASPR under the transcriptional regulation of N-WASPproHRE1 showed enhanced gelatin degradation when treated with DMOG. Thus indicating the importance of N-WASP in hypoxia induced invadopodia formation. Thus, our data demonstrates that hypoxia-induced activation of N-WASP expression is mediated by interaction of HiF1α with the HRE1(-132 and explains the role of N-WASP in hypoxia induced invadopodia formation.

  5. The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Jafari, Gholamali; Appleford, Peter J; Seago, Julian

    2011-01-01

    , an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat...

  6. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

    Science.gov (United States)

    Conway, J E; Rhys, C M; Zolotukhin, I; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1999-06-01

    Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

  7. Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

    DEFF Research Database (Denmark)

    Delvigne, Frank; Brognaux, Alison; Francis, Frédéric

    2011-01-01

    Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down......-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose...... fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great...

  8. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

    Science.gov (United States)

    Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

    2009-12-30

    Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements

  9. Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

    Science.gov (United States)

    2009-01-01

    Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

  10. Nutritional and medicinal characteristics of Chinese giant salamander (Andrias davidianus for applications in healthcare industry by artificial cultivation: A review

    Directory of Open Access Journals (Sweden)

    Dong He

    2018-03-01

    Full Text Available Andrias davidianus, i. e. Chinese giant salamander (CGS, is one of the largest and oldest amphibians existing in the world and is also one of the valuable biological resources of China. Wild CGS has been threatened with extinction in the past decades due to over capturing, deterioration of natural environment, the slow breeding and growth of the wild species in nature. However, in the past twenty years, with the breakthrough and progress of artificial breeding technology by artificial insemination, the number of artificially cultivated CGS has increased rapidly. Artificially cultivated CGS can either be released to the CGS living environment to increase the population in nature or legally applied in food and medicinal industry as a feedstock due to the unique nutritional and medicinal values of CGS as recorded historically. In this review, the nutritional components, bioactive components and medicinal activities of the artificially cultivated CGS will be summarized. The mucus, skin, meat and bone of CGS contain many different bioactive substances thereby having various medicinal activities including anti-aging, anti-fatigue, anti-tumor, therapy of burn and anti-infection and other physiological functions. This paper will further discuss the potential applications of the artificially cultivated CGS in healthcare industry and prospects of future technological development. Keywords: Andrias davidianus, Artificial breeding, Chinese giant salamander, Functional foods, Medicinal activity, Natural resource protection, Nutrition

  11. Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide.

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    Beth A Rasala

    Full Text Available Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly 'cleaved' at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (~100-fold increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.

  12. Population Structure and Evolution after Speciation of the Hokkaido Salamander (Hynobius retardatus.

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    Masatoshi Matsunami

    Full Text Available The Hokkaido salamander (Hynobius retardatus is endemic to Hokkaido Island, Japan, and shows intriguing flexible phenotypic plasticity and regional morphological diversity. However, to date, allozymes and partial mitochondria DNA sequences have provided only an outline of its demographic histories and the pattern of its genetic diversification. To understand the finer details of the population structure of this species and its evolution since speciation, we genotyped five regional populations by using 12 recently developed microsatellite polymorphic markers. We found a clear population structure with low gene flow among the five populations, but a close genetic relationship between the Teshio and Kitami populations. Our demographic analysis suggested that Teshio and Erimo had the largest effective population sizes among the five populations. These findings regarding the population structure and demography of H. retardatus improve our understanding of the faunal phylogeography on Hokkaido Island and also provide fundamental genetic information that will be useful for future studies.

  13. A High-Throughput Oxidative Stress Biosensor Based on Escherichia coli roGFP2 Cells Immobilized in a k-Carrageenan Matrix

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    Lia Ooi

    2015-01-01

    Full Text Available Biosensors fabricated with whole-cell bacteria appear to be suitable for detecting bioavailability and toxicity effects of the chemical(s of concern, but they are usually reported to have drawbacks like long response times (ranging from hours to days, narrow dynamic range and instability during long term storage. Our aim is to fabricate a sensitive whole-cell oxidative stress biosensor which has improved properties that address the mentioned weaknesses. In this paper, we report a novel high-throughput whole-cell biosensor fabricated by immobilizing roGFP2 expressing Escherichia coli cells in a k-carrageenan matrix, for the detection of oxidative stress challenged by metalloid compounds. The E. coli roGFP2 oxidative stress biosensor shows high sensitivity towards arsenite and selenite, with wide linear range and low detection limit (arsenite: 1.0 × 10−3–1.0 × 101 mg·L−1, LOD: 2.0 × 10−4 mg·L−1; selenite: 1.0 × 10−5–1.0 × 102 mg·L−1, LOD: 5.8 × 10−6 mg·L−1, short response times (0–9 min, high stability and reproducibility. This research is expected to provide a new direction in performing high-throughput environmental toxicity screening with living bacterial cells which is capable of measuring the bioavailability and toxicity of environmental stressors in a friction of a second.

  14. The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola.

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    Scott A C Godfrey

    2011-03-01

    Full Text Available Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1, which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1, revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

  15. Comprehensive analysis of gene expression patterns of hedgehog-related genes

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    Baillie David

    2006-10-01

    Full Text Available Abstract Background The Caenorhabditis elegans genome encodes ten proteins that share sequence similarity with the Hedgehog signaling molecule through their C-terminal autoprocessing Hint/Hog domain. These proteins contain novel N-terminal domains, and C. elegans encodes dozens of additional proteins containing only these N-terminal domains. These gene families are called warthog, groundhog, ground-like and quahog, collectively called hedgehog (hh-related genes. Previously, the expression pattern of seventeen genes was examined, which showed that they are primarily expressed in the ectoderm. Results With the completion of the C. elegans genome sequence in November 2002, we reexamined and identified 61 hh-related ORFs. Further, we identified 49 hh-related ORFs in C. briggsae. ORF analysis revealed that 30% of the genes still had errors in their predictions and we improved these predictions here. We performed a comprehensive expression analysis using GFP fusions of the putative intergenic regulatory sequence with one or two transgenic lines for most genes. The hh-related genes are expressed in one or a few of the following tissues: hypodermis, seam cells, excretory duct and pore cells, vulval epithelial cells, rectal epithelial cells, pharyngeal muscle or marginal cells, arcade cells, support cells of sensory organs, and neuronal cells. Using time-lapse recordings, we discovered that some hh-related genes are expressed in a cyclical fashion in phase with molting during larval development. We also generated several translational GFP fusions, but they did not show any subcellular localization. In addition, we also studied the expression patterns of two genes with similarity to Drosophila frizzled, T23D8.1 and F27E11.3A, and the ortholog of the Drosophila gene dally-like, gpn-1, which is a heparan sulfate proteoglycan. The two frizzled homologs are expressed in a few neurons in the head, and gpn-1 is expressed in the pharynx. Finally, we compare the

  16. Rare codons effect on expression of recombinant gene cassette in Escherichia coli BL21(DE3

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    Aghil Esmaeili-Bandboni

    2017-11-01

    Full Text Available Objective: To demonstrate the sensitivity of expression of fusion genes to existence of a large number of rare codons in recombinant gene sequenced. Methods: Primers for amplification of cholera toxin B, Shiga toxin B and gfp genes were designed by Primer3 software and synthesized. All of these 3 genes were cloned. Then the genes were fused together by restriction sites and enzymatic method. Two linkers were used as a flexible bridge in connection of these genes. Results: Cloning and fusion of cholera toxin B, Shiga toxin B and gfp genes were done correctly. After that, expression of the recombinant gene construction was surveyed. Conclusions: According to what was seen, because of the accumulation of 12 rare codons of Shiga toxin B and 19 rare codons of cholera toxin B in this gene cassette, the expression of the recombinant gene cassette, in Escherichia coli BL21, failed.

  17. Deep divergences and extensive phylogeographic structure in a clade of lowland tropical salamanders

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    Rovito Sean M

    2012-12-01

    Full Text Available Abstract Background The complex geological history of Mesoamerica provides the opportunity to study the impact of multiple biogeographic barriers on population differentiation. We examine phylogeographic patterns in a clade of lowland salamanders (Bolitoglossa subgenus Nanotriton using two mitochondrial genes and one nuclear gene. We use several phylogeographic analyses to infer the history of this clade and test hypotheses regarding the geographic origin of species and location of genetic breaks within species. We compare our results to those for other taxa to determine if historical events impacted different species in a similar manner. Results Deep genetic divergence between species indicates that they are relatively old, and two of the three widespread species show strong phylogeographic structure. Comparison of mtDNA and nuclear gene trees shows no evidence of hybridization or introgression between species. Isolated populations of Bolitoglossa rufescens from Los Tuxtlas region constitute a separate lineage based on molecular data and morphology, and divergence between Los Tuxtlas and other areas appears to predate the arrival of B. rufescens in other areas west of the Isthmus of Tehuantepec. The Isthmus appears responsible for Pliocene vicariance within B. rufescens, as has been shown for other taxa. The Motagua-Polochic fault system does not appear to have caused population vicariance, unlike in other systems. Conclusions Species of Nanotriton have responded to some major geological events in the same manner as other taxa, particularly in the case of the Isthmus of Tehuantepec. The deep divergence of the Los Tuxtlas populations of B. rufescens from other populations highlights the contribution of this volcanic system to patterns of regional endemism, and morphological differences observed in the Los Tuxtlas populations suggests that they may represent an undescribed species of Bolitoglossa. The absence of phylogeographic structure in B

  18. Behavioral Evaluation of hMSC-GFP+ Transplantation in an Hemiparkinson Experimental Model in Wistar Rat

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    Jéssica Paola Alcázar Arzuza

    2017-05-01

    Full Text Available The effect of hMSCs-GFP+ transplantation was evaluated in an experimental model of Parkinson's disease (PD in 27 Wistar rats, or in three experimental groups: control (CON  n=7, injured (LES n=10 and transplanted (LES+T n=10. In order to evaluate the influence of the transplantation on the motor behavior, one month after the injury, rotation behavior induced by apomorphine, neurological test, transversal bar and SNpc cells positive to TH were developed. Using the Anova test, there was a decrease in the number of turns in transplanted animals (p=0.005 as well as in the neurological test (p=0.0004 and in the transverse bar that lead to this group in an intermediate position regarding LES and CON groups. There is a possible recovery of the transplantation-mediated nigroestriatal pathway of hMSC-GFP +.

  19. Expression of Slug in S100β-protein-positive cells of postnatal developing rat anterior pituitary gland.

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    Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Yako, Hideji; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Yashiro, Takashi; Kato, Takako; Kato, Yukio

    2016-02-01

    Among heterogeneous S100β-protein-positive (S100β-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100β-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100β-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100β-promoter has allowed us to observe living S100β-positive cells. In the present study, we first confirmed that living S100β-positive cells in tissue cultures of S100β-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100β-positive cells. Interestingly, we detected Slug expression in S100β-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100β-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100β-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

  20. A new moss salamander, genus Nototriton (Caudata: Plethodontidae), from the Cordillera de Talamanca, in the Costa Rica-Panama border region.

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    Arias, Erick; Kubicki, Brian

    2018-01-07

    A new salamander belonging to the genus Nototriton, subgenus Nototriton, is described from the Caribbean slopes of the southeastern Cordillera de Talamanca in Costa Rica, within Parque Internacional La Amistad, at an elevation ca. 1500 m a.s.l. This new taxon is distinguished from its congeners by its morphological characteristics and by its differentiation in DNA sequences of the 16S rRNA, cytochrome oxidase subunit I (COI), and cytochrome b mitochondrial genes. This new species represents the southernmost extension known for the genus Nototriton.

  1. The effect of HCV Core protein on the expression of miR-150

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    Sayad Khanizadeh

    2016-09-01

    Full Text Available Background : Hepatitis C virus (HCV is considered as one of the major pathogenic agents of chronic liver diseases. Previous studies have shown that HCV proteins can interaction with gene regulatory networks such as microRNAs. The aim of this study was to investigate the effect of HCV core protein on the expression of miR-150 in a cell culture model. Materials and Methods: Plasmids expressing full HCV core protein was transfected into Huh7 cell lines while a GFP expressing plasmid employed as negative control. Subsequently, total RNA extracted and Real-Time PCR performed to measure the expression level of miR-150 expression. Moreover, trypan blue exclusion assay was performed to investigate the effect of core protein on cell viability. Results: The gene expression analysis of miR-150 in Huh7 cells showed that endogenous HCV core protein could significantly down regulation of miR-150 when compared to GFP control plasmid and normal cells (P<0.01. Beside, core protein induced no significant proliferative or cytotoxic effects on hepatic cells as determined by trypan blue exclusion assay (P<0.05. Conclusion: Our study suggests that HCV core protein can led to down regulation of miR-150 expression. This data revealed that HCV protein interactions with cell regulatory machinery may contribute to pathogenesis of chronic liver diseases.

  2. Response of Red-Backed Salamanders (Plethodon Cinereus to Changes in Hemlock Forest Soil Driven by Invasive Hemlock Woolly Adelgid (Adelges Tsugae

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    Alison Ochs

    2017-01-01

    Full Text Available Hemlock forests of the northeastern United States are declining due to the invasive hemlock woolly adelgid (HWA (Adelges tsugae. Hardwood species replace these forests, which affects soil properties that may influence other communities, such as red-backed salamanders (red-backs (Plethodon cinereus. This study examined the effects of HWA invasion on soil properties and how this affects red-backs at the Hemlock Removal Experiment at Harvard Forest, which consists of eight 0.8 ha plots treated with girdling to simulate HWA invasion, logging to simulate common management practices, or hemlock- or hardwood-dominated controls. Coverboard surveys were used to determine the relative abundance of red-backs between plots during June and July 2014 and soil cores were collected from which the bulk density, moisture, pH, temperature, leaf litter, and carbon-nitrogen ratio were measured. Ordination provided a soil quality index based on temperature, pH, and carbon-to-nitrogen ratio, which was significantly different between plot treatments (p < 0.05 and showed a significant negative correlation with the red-back relative abundance (p < 0.05. The findings support the hypothesis that red-backs are affected by soil quality, which is affected by plot treatment and thus HWA invasion. Further studies should explore how salamanders react in the long term towards changing environments and consider the use of red-backs as indicator species.

  3. Status of some populations of Mexican salamanders (Amphibia: Plethodontidae

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    Gabriela Parra-Olea

    1999-06-01

    Full Text Available Populations of Mexican plethodontid salamanders have been surveyed non-systematically over the last 25 years. In light of many reports of disappearance of amphibians around the world, we checked for persistence of reported species at ten of these sites. All of the commoner species persist (we observed individuals representing a total of 30 species. While observed densities of many species of Mexican plethodontids are lower to much lower than was the case 20 to 25 years ago, evidence for recent extinctions, such as has been reported for amphibian taxa elsewhere, is equivocal or lacking. Habitat modification has contributed to difficulties in finding certain species.Poblaciones de varias especies de salamandras pletodóntidas en México han sido monitoreadas de manera no sistemática durante los últimos 25 años. Diez de éstas poblaciones fueran visitadas recientemente con el propósito de verificar la persistencia de las especies reportadas para dichas localidades. Nuestras observaciones confirman la persistencia local de más de 30 especies cuyo estatus era desconocido, aunque la frecuencia de observación de estas especies es en general menor que en fechas anteriores. Estas observaciones son particularmente relevantes dada la situación actual de preocupación por la disminución mundial de anfibios.

  4. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media

    NARCIS (Netherlands)

    Veening, J. -W.; Smits, W. K.; Hamoen, L. W.; Kuipers, O. P.

    Aim: Understanding the basis for the heterogeneous (or bistable) expression patterns of competence development and sporulation in Bacillus subtilis. Methods and Results: Using flow cytometric analyses of various promoter-GFP fusions, we have determined the single-cell gene expression patterns of

  6. Hindcasting Historical Breeding Conditions for an Endangered Salamander in Ephemeral Wetlands of the Southeastern USA: Implications of Climate Change.

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    Houston C Chandler

    Full Text Available The hydroperiod of ephemeral wetlands is often the most important characteristic determining amphibian breeding success, especially for species with long development times. In mesic and wet pine flatwoods of the southeastern United States, ephemeral wetlands were a common landscape feature. Reticulated flatwoods salamanders (Ambystoma bishopi, a federally endangered species, depend exclusively on ephemeral wetlands and require at least 11 weeks to successfully metamorphose into terrestrial adults. We empirically modeled hydroperiod of 17 A. bishopi breeding wetlands by combining downscaled historical climate-model data with a recent 9-year record (2006-2014 of observed water levels. Empirical models were subsequently used to reconstruct wetland hydrologic conditions from 1896-2014 using the downscaled historical climate datasets. Reconstructed hydroperiods for the 17 wetlands were highly variable through time but were frequently unfavorable for A. bishopi reproduction (e.g., only 61% of years, using a conservative estimate of development time [12 weeks], were conducive to larval development and metamorphosis. Using change-point analysis, we identified significant shifts in average hydroperiod over the last century in all 17 wetlands. Mean hydroperiods were shorter in recent years than at any other point since 1896, and thus less suitable for A. bishopi reproduction. We suggest that climate change will continue to impact the reproductive success of flatwoods salamanders and other ephemeral wetland breeders by reducing the number of years these wetlands have suitable hydroperiods. Consequently, we emphasize the importance of conservation and management for mitigating other forms of habitat degradation, especially maintenance of high quality breeding sites where reproduction can occur during appropriate environmental conditions.

  7. A part toolbox to tune genetic expression in Bacillus subtilis

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    Guiziou, Sarah; Sauveplane, Vincent; Chang, Hung-Ju; Clerté, Caroline; Declerck, Nathalie; Jules, Matthieu; Bonnet, Jerome

    2016-01-01

    Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis. We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis. PMID:27402159

  8. Green Fluorescent Protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum

    Science.gov (United States)

    Marko Riedel; Gautier Calmin; Lassaad Belbahri; Francois Lefort; Monika Gotz; Stefan Wagner; Sabine. Werres

    2009-01-01

    Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen...

  9. Batrachochytrium salamandrivorans is the predominant chytrid fungus in Vietnamese salamanders

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    Laking, Alexandra E.; Ngo, Hai Ngoc; Pasmans, Frank; Martel, An; Nguyen, Tao Thien

    2017-01-01

    The amphibian chytrid fungi, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), pose a major threat to amphibian biodiversity. Recent evidence suggests Southeast Asia as a potential cradle for both fungi, which likely resulted in widespread host-pathogen co-existence. We sampled 583 salamanders from 8 species across Vietnam in 55 locations for Bsal and Bd, determined scaled mass index as a proxy for fitness and collected environmental data. Bsal was found within 14 of the 55 habitats (2 of which it was detected in 2013), in 5 salamandrid species, with a prevalence of 2.92%. The globalized pandemic lineage of Bd was found within one pond on one species with a prevalence of 0.69%. Combined with a complete lack of correlation between infection and individual body condition and absence of indication of associated disease, this suggests low level pathogen endemism and Bsal and Bd co-existence with Vietnamese salamandrid populations. Bsal was more widespread than Bd, and occurs at temperatures higher than tolerated by the type strain, suggesting a wider thermal niche than currently known. Therefore, this study provides support for the hypothesis that these chytrid fungi may be endemic to Asia and that species within this region may act as a disease reservoir. PMID:28287614

  10. Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes

    International Nuclear Information System (INIS)

    Biacchesi, Stephane; Skiadopoulos, Mario H.; Tran, Kim C.; Murphy, Brian R.; Collins, Peter L.; Buchholz, Ursula J.

    2004-01-01

    Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups (Biacchesi et al., Virology 315 (1) (2003) 1). We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was

  11. Wetland and microhabitat use by nesting four-toed salamanders in Maine

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    Chalmers, R.J.; Loftin, C.S.

    2006-01-01

    Little is known of Four-Toed Salamander (Hemidactylium scutatum) habitat use, despite the species' extensive range and elevated conservation status. We investigated species-habitat relationships that predict H. scutatum nesting presence in Maine at wetland and microhabitat scales by comparing microhabitats with and without nests. We created logistic regression models, selected models with AIC, and evaluated models with reserve data. Wetlands with nests were best predicted by shoreline microhabitat of Sphagnum spp., wood substrate, water flow, blue-joint reed grass (Calamagrostis canadensis), meadowsweet (Spiraea alba), steeplebush (Spiraea tomentosa), sensitive fern (Onoclea sensibilis), and absence of sheep laurel (Kalmia angustifolia) or deciduous forest canopy. Within occupied wetlands, shoreline microhabitat where nests occurred was best distinguished from available, unoccupied shoreline microhabitat by steeper shore, greater near-shore and basin water depth, deeper nesting vegetation, presence of moss spp. and winterberry (Ilex verticillata), and a negative association with S. alba, leatherleaf (Chamaedaphne calyculata), and K. angustifolia. These models of wetland and microhabitat use by H. scutatum may assist ecologists and managers in detecting and conserving this species. Copyright 2006 Society for the Study of Amphibians and Reptiles.

  12. Gonadotropin-Releasing Hormone Modulates Vomeronasal Neuron Response to Male Salamander Pheromone

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    Celeste R. Wirsig-Wiechmann

    2012-01-01

    Full Text Available Electrophysiological studies have shown that gonadotropin-releasing hormone (GnRH modifies chemosensory neurons responses to odors. We have previously demonstrated that male Plethodon shermani pheromone stimulates vomeronasal neurons in the female conspecific. In the present study we used agmatine uptake as a relative measure of the effects of GnRH on this pheromone-induced neural activation of vomeronasal neurons. Whole male pheromone extract containing 3 millimolar agmatine with or without 10 micromolar GnRH was applied to the nasolabial groove of female salamanders for 45 minutes. Immunocytochemical procedures were conducted to visualize and quantify relative agmatine uptake as measured by labeling density of activated vomeronasal neurons. The relative number of labeled neurons did not differ between the two groups: pheromone alone or pheromone-GnRH. However, vomeronasal neurons exposed to pheromone-GnRH collectively demonstrated higher labeling intensity, as a percentage above background (75% as compared with neurons exposed to pheromone alone (63%, P < 0.018. Since the labeling intensity of agmatine within neurons signifies the relative activity levels of the neurons, these results suggest that GnRH increases the response of female vomeronasal neurons to male pheromone.

  13. Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.

    Science.gov (United States)

    Yeh, H H; Tian, T; Medina, V; Falk, B W

    2001-10-10

    Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1. Copyright 2001 Academic Press.

  14. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

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    Yeon Sook Cho

    Full Text Available Interleukin-7 (IL-7 is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease.

  15. Historical dominance of low-severity fire in dry and wet mixed-conifer forest habitats of the endangered terrestrial Jemez Mountains salamander (Plethodon neomexicanus)

    Science.gov (United States)

    Margolis, Ellis; Malevich, Steven B.

    2016-01-01

    Anthropogenic alteration of ecosystem processes confounds forest management and conservation of rare, declining species. Restoration of forest structure and fire hazard reduction are central goals of forest management policy in the western United States, but restoration priorities and treatments have become increasingly contentious. Numerous studies have documented changes in fire regimes, forest stand structure and species composition following a century of fire exclusion in dry, frequent-fire forests of the western U.S. (e.g., ponderosa pine and dry mixed-conifer). In contrast, wet mixed-conifer forests are thought to have historically burned infrequently with mixed- or high-severity fire—resulting in reduced impacts from fire exclusion and low restoration need—but data are limited. In this study we quantified the current forest habitat of the federally endangered, terrestrial Jemez Mountains salamander (Plethodon neomexicanus) and compared it to dendroecological reconstructions of historical habitat (e.g., stand structure and composition), and fire regime parameters along a gradient from upper ponderosa pine to wet mixed-conifer forests. We found that current fire-free intervals in Jemez Mountains salamander habitat (116–165 years) are significantly longer than historical intervals, even in wet mixed-conifer forests. Historical mean fire intervals ranged from 10 to 42 years along the forest gradient. Low-severity fires were historically dominant across all forest types (92 of 102 fires). Although some mixed- or highseverity fire historically occurred at 67% of the plots over the last four centuries, complete mortality within 1.0 ha plots was rare, and asynchronous within and among sites. Climate was an important driver of temporal variability in fire severity, such that mixed- and high-severity fires were associated with more extreme drought than low-severity fires. Tree density in dry conifer forests historically ranged from open (90 trees/ha) to

  16. Highly tissue specific expression of Sphinx supports its male courtship related role in Drosophila melanogaster.

    Science.gov (United States)

    Chen, Ying; Dai, Hongzheng; Chen, Sidi; Zhang, Luoying; Long, Manyuan

    2011-04-26

    Sphinx is a lineage-specific non-coding RNA gene involved in regulating courtship behavior in Drosophila melanogaster. The 5' flanking region of the gene is conserved across Drosophila species, with the proximal 300 bp being conserved out to D. virilis and a further 600 bp region being conserved amongst the melanogaster subgroup (D. melanogaster, D. simulans, D. sechellia, D. yakuba, and D. erecta). Using a green fluorescence protein transformation system, we demonstrated that a 253 bp region of the highly conserved segment was sufficient to drive sphinx expression in male accessory gland. GFP signals were also observed in brain, wing hairs and leg bristles. An additional ∼800 bp upstream region was able to enhance expression specifically in proboscis, suggesting the existence of enhancer elements. Using anti-GFP staining, we identified putative sphinx expression signal in the brain antennal lobe and inner antennocerebral tract, suggesting that sphinx might be involved in olfactory neuron mediated regulation of male courtship behavior. Whole genome expression profiling of the sphinx knockout mutation identified significant up-regulated gene categories related to accessory gland protein function and odor perception, suggesting sphinx might be a negative regulator of its target genes.

  17. Highly tissue specific expression of Sphinx supports its male courtship related role in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Ying Chen

    2011-04-01

    Full Text Available Sphinx is a lineage-specific non-coding RNA gene involved in regulating courtship behavior in Drosophila melanogaster. The 5' flanking region of the gene is conserved across Drosophila species, with the proximal 300 bp being conserved out to D. virilis and a further 600 bp region being conserved amongst the melanogaster subgroup (D. melanogaster, D. simulans, D. sechellia, D. yakuba, and D. erecta. Using a green fluorescence protein transformation system, we demonstrated that a 253 bp region of the highly conserved segment was sufficient to drive sphinx expression in male accessory gland. GFP signals were also observed in brain, wing hairs and leg bristles. An additional ∼800 bp upstream region was able to enhance expression specifically in proboscis, suggesting the existence of enhancer elements. Using anti-GFP staining, we identified putative sphinx expression signal in the brain antennal lobe and inner antennocerebral tract, suggesting that sphinx might be involved in olfactory neuron mediated regulation of male courtship behavior. Whole genome expression profiling of the sphinx knockout mutation identified significant up-regulated gene categories related to accessory gland protein function and odor perception, suggesting sphinx might be a negative regulator of its target genes.

  18. Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors

    Directory of Open Access Journals (Sweden)

    Maria R. Mendoza

    2017-11-01

    Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

  19. Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

    Science.gov (United States)

    Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B

    2011-01-01

    Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  20. Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.

    Science.gov (United States)

    Li, Tengfei; Bourgeois, Jean-Pierre; Celli, Susanna; Glacial, Fabienne; Le Sourd, Anne-Marie; Mecheri, Salah; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Rougeon, François; Lafaye, Pierre

    2012-10-01

    Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

  1. Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection

    Science.gov (United States)

    Chang, Ming-Fong; Hsieh, Jung-Hsien; Chiang, Hao; Kan, Hung-Wei; Huang, Cho-Min; Chellis, Luke; Lin, Bo-Shiou; Miaw, Shi-Chuen; Pan, Chun-Liang; Chao, Chi-Chao; Hsieh, Sung-Tsang

    2016-01-01

    Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection. PMID:27748450

  2. Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5.

    NARCIS (Netherlands)

    Dekker, E. den; Schoeber, J.P.H.; Topala, C.N.; Graaf, S.F.J. van de; Hoenderop, J.G.J.; Bindels, R.J.M.

    2005-01-01

    To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of

  3. Changes in gravitational force affect gene expression in developing organ systems at different developmental times

    Directory of Open Access Journals (Sweden)

    Moorman Stephen J

    2005-05-01

    Full Text Available Abstract Background Little is known about the affect of microgravity on gene expression, particularly in vivo during embryonic development. Using transgenic zebrafish that express the gfp gene under the influence of a β-actin promoter, we examined the affect of simulated-microgravity on GFP expression in the heart, notochord, eye, somites, and rohon beard neurons. We exposed transgenic zebrafish to simulated-microgravity for different durations at a variety of developmental times in an attempt to determine periods of susceptibility for the different developing organ systems. Results The developing heart had a period of maximum susceptibility between 32 and 56 hours after fertilization when there was an approximately 30% increase in gene expression. The notochord, eye, somites, and rohon beard neurons all showed periods of susceptibility occurring between 24 and 72 hours after fertilization. In addition, the notochord showed a second period of susceptibility between 8 and 32 hours after fertilization. Interestingly, all organs appeared to be recovering by 80 hours after fertilization despite continued exposure to simulated-microgravity. Conclusion These results support the idea that exposure to microgravity can cause changes in gene expression in a variety of developing organ systems in live embryos and that there are periods of maximum susceptibility to the effects.

  4. Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

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    Shahrooz Ghaderi

    2018-03-01

    Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

  5. Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: A study of DNAJB6 chaperone

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    Rasha Mohamed Hussein

    2015-07-01

    Full Text Available Alzheimer’s disease is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly Q peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

  6. Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders.

    Science.gov (United States)

    Goldberg, Caren S; Pilliod, David S; Arkle, Robert S; Waits, Lisette P

    2011-01-01

    Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

  7. Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders.

    Directory of Open Access Journals (Sweden)

    Caren S Goldberg

    Full Text Available Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus. We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

  8. Performance of Popular XC-Functionals for the Description of Excitation Energies in GFP-Like Chromophore Models

    DEFF Research Database (Denmark)

    List, Nanna Holmgaard; Olsen, Jógvan Magnus Haugaard; Rocha-Rinza, Tomás

    2012-01-01

    this task. We present an evaluation of the performance of commonly used XC-functionals for the prediction of excitation energies of GFP-like chromophores. In particular, we have considered the TD-DFT vertical excitation energies of chromophores displaying different charge states. We compare the quality...

  9. Morphine Preconditioning Downregulates MicroRNA-134 Expression Against Oxygen-Glucose Deprivation Injuries in Cultured Neurons of Mice.

    Science.gov (United States)

    Meng, Fanjun; Li, Yan; Chi, Wenying; Li, Junfa

    2016-07-01

    Brain protection by narcotics such as morphine is clinically relevant due to the extensive use of narcotics in the perioperative period. Morphine preconditioning induces neuroprotection in neurons, but it remains uncertain whether microRNA-134 (miR-134) is involved in morphine preconditioning against oxygen-glucose deprivation-induced injuries in primary cortical neurons of mice. The present study examined this issue. After cortical neurons of mice were cultured in vitro for 6 days, the neurons were transfected by respective virus vector, such as lentiviral vector (LV)-miR-control-GFP, LV-pre-miR-134-GFP, LV-pre-miR-134-inhibitor-GFP for 24 hours; after being normally cultured for 3 days again, morphine preconditioning was performed by incubating the transfected primary neurons with morphine (3 μM) for 1 hour, and then neuronal cells were exposed to oxygen-glucose deprivation (OGD) for 1 hour and oxygen-glucose recovery for 12 hours. The neuronal cells survival rate and the amount of apoptotic neurons were determined by MTT assay or TUNEL staining at designated time; and the expression levels of miR-134 were detected using real-time reverse transcription polymerase chain reaction at the same time. The neuronal cell survival rate was significantly higher, and the amount of apoptotic neurons was significantly decreased in neurons preconditioned with morphine before OGD than that of OGD alone. The neuroprotection induced by morphine preconditioning was partially blocked by upregulating miR-134 expression, and was enhanced by downregulating miR-134 expression. The expression of miR-134 was significantly decreased in morphine-preconditioned neurons alone without transfection. By downregulating miR-134 expression, morphine preconditioning protects primary cortical neurons of mice against injuries induced by OGD.

  10. Co-expression of GAD67 and choline acetyltransferase reveals a novel neuronal phenotype in the mouse medulla oblongata.

    Science.gov (United States)

    Gotts, Jittima; Atkinson, Lucy; Edwards, Ian J; Yanagawa, Yuchio; Deuchars, Susan A; Deuchars, Jim

    2015-12-01

    GABAergic and cholinergic systems play an important part in autonomic pathways. To determine the distribution of the enzymes responsible for the production of GABA and acetylcholine in areas involved in autonomic control in the mouse brainstem, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurones, combined with choline acetyl transferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurones were observed throughout the brainstem. A small number of cells contained both ChAT-IR and GAD67-GFP. Such double labelled cells were observed in the NTS (predominantly in the intermediate and central subnuclei), the area postrema, reticular formation and lateral paragigantocellular nucleus. All ChAT-IR neurones in the area postrema contained GAD67-GFP. Double labelled neurones were not observed in the dorsal vagal motor nucleus, nucleus ambiguus or hypoglossal nucleus. Double labelled ChAT-IR/GAD67-GFP cells in the NTS did not contain neuronal nitric oxide synthase (nNOS) immunoreactivity, whereas those in the reticular formation and lateral paragigantocellular nucleus did. The function of these small populations of double labelled cells is currently unknown, however their location suggests a potential role in integrating signals involved in oromotor behaviours. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton

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    Kaštier Peter

    2017-06-01

    Full Text Available Dodder (Cuscuta species cause severe agricultural damage in many countries throughout the world. To establish strategies for control of its growth and spreading it is important to study its life cycle and survival strategies. For these efforts genetic modification would represent a powerful tool. Here we report on Agrobacteriummediated transformation of dodder using green fluorescent protein (GFP fused to actin-binding protein as a vital marker. Since the shoot of germinating C. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, the shoot apex was used here as explant. The transgene expression was only transient, nevertheless it enabled to detect allocation of actin filaments and studying the cytoskeleton organization in dodder shoot apex. Transient expression of GFP appears to be a suitable method for studying Cuscuta development through cytoskeleton organisation that is presently largely unexplored.

  12. A mammalianized synthetic nitroreductase gene for high-level expression

    International Nuclear Information System (INIS)

    Grohmann, Maik; Paulmann, Nils; Fleischhauer, Sebastian; Vowinckel, Jakob; Priller, Josef; Walther, Diego J

    2009-01-01

    The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans

  13. Systemic colonization of potato plants by a soil-borne, GFP-tagged strain of Dickeya sp. Biovar 3

    NARCIS (Netherlands)

    Czajkowski, R.L.; Boer, de W.; Velvis, H.; Wolf, van der J.M.

    2010-01-01

    Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants

  14. Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

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    Yuki Horiuchi

    2018-01-01

    Full Text Available Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

  15. A review of the biology and conservation of the Cope's giant salamander Dicamptodon copei Nussbaum, 1970 (Amphibia: Caudata: Dicamptodontidae) in the Pacific northwestern region of the USA

    Science.gov (United States)

    Alex D. Foster; Deanna H. Olson; Lawrence L.C. Jones

    2015-01-01

    The Cope’s Giant Salamander Dicamptodon copei is a stream dwelling amphibian reliant on cool streams, native to forested areas primarily west of the crest of the Cascade Range in the Pacific Northwest region, USA. Unlike other members of the genus, adult D. copei are most often found in a paedomorphic form, and rarely transforms to a terrestrial stage. As a result,...

  16. Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

    Science.gov (United States)

    Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2007-12-01

    A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

  17. Transsynaptic transport of wheat germ agglutinin expressed in a subset of type II taste cells of transgenic mice

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    Mosinger Bedrich

    2008-10-01

    Full Text Available Abstract Background Anatomical tracing of neural circuits originating from specific subsets of taste receptor cells may shed light on interactions between taste cells within the taste bud and taste cell-to nerve interactions. It is unclear for example, if activation of type II cells leads to direct activation of the gustatory nerves, or whether the information is relayed through type III cells. To determine how WGA produced in T1r3-expressing taste cells is transported into gustatory neurons, transgenic mice expressing WGA-IRES-GFP driven by the T1r3 promoter were generated. Results Immunohistochemistry showed co-expression of WGA, GFP and endogenous T1r3 in the taste bud cells of transgenic mice: the only taste cells immunoreactive for WGA were the T1r3-expressing cells. The WGA antibody also stained intragemmal nerves. WGA, but not GFP immunoreactivity was found in the geniculate and petrosal ganglia of transgenic mice, indicating that WGA was transported across synapses. WGA immunoreactivity was also found in the trigeminal ganglion, suggesting that T1r3-expressing cells make synapses with trigeminal neurons. In the medulla, WGA was detected in the nucleus of the solitary tract but also in the nucleus ambiguus, the vestibular nucleus, the trigeminal nucleus and in the gigantocellular reticular nucleus. WGA was not detected in the parabrachial nucleus, or the gustatory cortex. Conclusion These results show the usefulness of genetically encoded WGA as a tracer for the first and second order neurons that innervate a subset of taste cells, but not for higher order neurons, and demonstrate that the main route of output from type II taste cells is the gustatory neuron, not the type III cells.