Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

Science.gov (United States)

Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

2017-03-15

Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

Journal of Chemical Sciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Hence, an anticancer agent conjugated to them may render more toxicity in cancer cells due to higher uptake. ... It acts by inducing apoptosis through G2/M phase arrest and encouragingly, is much less toxic to nontumorigenic human embryonic kidney (HEK-293T) and mouse embryonic fibroblast (NIH 3T3) cell lines in vitro ...

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1 Improves Recombinant Protein Expression in the HEK293 Cell Line

Directory of Open Access Journals (Sweden)

Laura Abaandou

2018-06-01

Full Text Available Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA and small interfering RNA (siRNA, to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1 gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293 cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

Polycystin-1 promotes PKCα-mediated NF-κB activation in kidney cells

International Nuclear Information System (INIS)

Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky; Mangolini, Alessandra; Pinton, Paolo; Witzgall, Ralph; Rizzuto, Rosario; Senno, Laura del

2006-01-01

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-κB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293 CTT ), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-κB nuclear levels and NF-κB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-κB promoter activation was mediated by PKCα because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293 CTT cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-κB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCα-mediated NF-κB signalling and cell survival

Biochemical effects of veterinary antibiotics on proliferation and cell cycle arrest of human HEK293 cells.

Science.gov (United States)

Kim, Hyeon Young; Kim, Ki-Tae; Kim, Sang Don

2012-08-01

The purpose of this study was to examine the effects of veterinary antibiotics, including amoxicillin (AMX), chlortetracycline (CTC) and tylosin (TYL), on the biochemical mechanism of human embryonic kidney cells (HEK293). CTC and TYL inhibited HEK293 cell proliferation, in both time- and dose-dependent manners, and changed the cell morphology; whereas, AMX showed no cytotoxic effects. The cell cycle analysis of CTC and TYL revealed G1-arrest in HEK293 cells. Western blot analysis also showed that CTC and TYL affected the activation of DNA damage responsive proteins, as well as cell cycle regulatory proteins, such as p53, p21(Waf1/Cip1) and Rb protein, which are crucial in the G1-S transition. The activation of p21(Waf1/Cip1) was significantly up-regulated over time, but there was no change in the level of CDK2 expression. The results of this study suggest that veterinary antibiotics, even at low level concentrations on continuous exposure, can potentially risk the development of human cells.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

Science.gov (United States)

Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

2015-09-18

BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Data on the construction of a recombinant HEK293 cell line overexpressing hERG potassium channel and examining the presence of hERG mRNA and protein expression

Directory of Open Access Journals (Sweden)

Yi Fan Teah

2017-10-01

Full Text Available The data presented in this article are related to the research article entitled “The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr and human ether-a-go-go-related gene (hERG expression” (Y.F. Teah, M.A. Abduraman, A. Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan [1], which the possible hERG blocking properties of deoxyelephantopin were investigated. This article describes the construction of human embryonic kidney 293 (HEK293 cells overexpressing HERG potassium channel and verification of the presence of hERG mRNA and protein expression in this recombinant cell line.

Agonist-induced desensitization of human β3-adrenoceptors expressed in human embryonic kidney cells

NARCIS (Netherlands)

Michel-Reher, Martina B.; Michel, Martin C.

2013-01-01

β3-Adrenoceptors are resistant to agonist-induced desensitization in some cell types but susceptible in others including transfected human embryonic kidney (HEK) cells. Therefore, we have studied cellular and molecular changes involved in agonist-induced β3-adrenoceptor desensitization in HEK cells.

Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

Science.gov (United States)

2010-01-01

Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is

Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

Directory of Open Access Journals (Sweden)

Münch Jan

2010-05-01

Full Text Available Abstract Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC. Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that

Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

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Jing Li

Full Text Available Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects.

A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

KAUST Repository

Ooi, Amanda Siok Lee

2016-07-19

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

KAUST Repository

Ooi, Amanda Siok Lee; Wong, Aloysius Tze; Esau, Luke; Lemtiri-Chlieh, Fouad; Gehring, Christoph A

2016-01-01

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

DEFF Research Database (Denmark)

Kim, Che Lin; Kwang Ha, Tae; Min Lee, Gyun

2016-01-01

Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (qp)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), Li......Cl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and qp enhancement during TGE (post-treatment) was examined. For the TGE...... of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 m...

Differences in mitochondrial function and morphology during cooling and rewarming between hibernator and non-hibernator derived kidney epithelial cells.

Science.gov (United States)

Hendriks, Koen D W; Lupi, Eleonora; Hardenberg, Maarten C; Hoogstra-Berends, Femke; Deelman, Leo E; Henning, Robert H

2017-11-14

Hibernators show superior resistance to ischemia and hypothermia, also outside the hibernation season. Therefore, hibernation is a promising strategy to decrease cellular damage in a variety of fields, such as organ transplantation. Here, we explored the role of mitochondria herein, by comparing epithelial cell lines from a hibernator (hamster kidney cells, HaK) and a non-hibernator (human embryonic kidney cells, HEK293) during cold preservation at 4 °C and rewarming. Cell survival (Neutral Red), ATP and MDA levels, mitochondrial membrane potential (MMP), mitochondrial morphology (using fluorescent probes) and metabolism (seahorse XF) were assessed. Hypothermia induced dispersion of the tubular mitochondrial network, a loss of MMP, increased oxygen radical (MDA) and decreased ATP production in HEK293. In contrast, HaK maintained MMP and ATP production without an increase in oxygen radicals during cooling and rewarming, resulting in superior cell survival compared to HEK293. Further, normothermic HaK showed a dispersed mitochondrial network and higher respiratory and glycolysis capacity compared to HEK293. Disclosing the mechanisms that hibernators use to counteract cell death in hypothermic and ischemic circumstances may help to eventually improve organ preservation in a variety of fields, including organ transplantation.

Synthesis and characterization of folate-poly(ethylene glycol ...

African Journals Online (AJOL)

Jane

2011-07-04

Jul 4, 2011 ... Cell lines, culture and viability assays. Human embryonic kidney cell line (293T), human colonic cancer cell line (LoVo), human lung adenocarcinoma epithelial cell line. (A549) and human cervical carcinoma cells (Hela) were cultured in. Dulbecco's modified eagle medium (DMEM, Gibco BRL, Paris,.

Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

Science.gov (United States)

Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

2014-01-01

The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

Science.gov (United States)

Long, Lin; He, Jian-Zhong; Chen, Ye; Xu, Xiu-E; Liao, Lian-Di; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

2018-05-07

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with

Identification of a functional nuclear export signal in the green fluorescent protein asFP499

International Nuclear Information System (INIS)

Mustafa, Huseyin; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

2006-01-01

The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified 194 LRMEKLNI 201 as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

Science.gov (United States)

Liste-Calleja, Leticia; Lecina, Martí; Cairó, Jordi Joan

2014-04-01

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian infectious bronchitis coronavirus

Directory of Open Access Journals (Sweden)

Kong Xiangang

2011-03-01

Full Text Available Abstract Background Avian infectious bronchitis (IB is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV. Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection in ovo. In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS. Results 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection. Conclusions To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

Science.gov (United States)

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

International Nuclear Information System (INIS)

Hwang, Young-ha; Tani, Motohiro; Nakagawa, Tetsuto; Okino, Nozomu; Ito, Makoto

2005-01-01

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence

Constitutive Behavior and Processing Map of T2 Pure Copper Deformed from 293 to 1073 K

Science.gov (United States)

Liu, Ying; Xiong, Wei; Yang, Qing; Zeng, Ji-Wei; Zhu, Wen; Sunkulp, Goel

2018-02-01

The deformation behavior of T2 pure copper compressed from 293 to 1073 K with strain rates from 0.01 to 10 s-1 was investigated. The constitutive equations were established by the Arrhenius constitutive model, which can be expressed as a piecewise function of temperature with two sections, in the ranges 293-723 K and 723-1073 K. The processing maps were established according to the dynamic material model for strains of 0.2, 0.4, 0.6, and 0.8, and the optimal processing parameters of T2 copper were determined accordingly. In order to obtain a better understanding of the deformation behavior, the microstructures of the compressed samples were studied by electron back-scattered diffraction. The grains tend to be more refined with decreases in temperature and increases in strain rate.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

Science.gov (United States)

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Partial molar volume of mefenamic acid in alcohol at temperatures between T=293.15 and T=313.15 K

OpenAIRE

Iqbal, Muhammad J.; Siddiquah, Mahrukh

2006-01-01

Apparent molar volume (Vphi), partial molar volume (V), solute-solute interaction parameter (Sv), partial molar expansivity (E(0)2) and isobaric thermal expansion coefficient (alpha2) of mefenamic acid in six different organic solvents namely, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, and 2-butanol, have been calculated from the measured solution densities over a temperature range of T=293.15 and T=313.15±0.1K. The solution densities were measured by an automated vibrating tube de...

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

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Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

HEK293T cell lines defective for O-linked glycosylation.

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James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

Rapid bioreduction of trivalent aurum using banana stem powder and its cytotoxicity against MCF-7 and HEK-293 cell lines

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Arunkumar, Pichaimani [Bharathidasan University, Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences (India); Vedagiri, Hemamalini [Bharathidasan University, Department of Biotechnology (India); Premkumar, Kumpati, E-mail: pkumpati@hotmail.com [Bharathidasan University, Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences (India)

2013-03-15

Bioreduction of metal ions for the synthesis of nanoparticles of well-defined shape and size has been a great challenge in the field of nanotechnology. In this study, we explored the reduction potential of banana stem powder (BSP) for the synthesis of gold nanoparticles (GNP). The kinetics of GNP synthesis was monitored using UV-Vis spectroscopy. The synthesized GNP was characterized using dynamic light scattering (DLS), transmission electron microscopy, and fourier transform infrared spectroscopy. In addition, the cytotoxic potential of the synthesized GNP was investigated using human breast cancer (MCF-7) and normal human embryonic kidney (HEK-293) cell lines, as evaluated by changes in cell morphology, cell viability (MTT), and metabolic activity. BSP exhibited a strong reduction of Au(III) to Au (0) at room temperature within 5 min of reaction time. The synthesized GNP was found to be spherical with an average diameter of 30 nm by DLS analysis. The cytotoxicity analysis reveals a direct dose-response relationship, indicating that the cytotoxicity increases with increasing concentrations of the GNP. Significant cytotoxicity was observed in cancer cells (MCF-7) compared to normal cells (HEK-293). Also the cellular uptake of GNP was more pronounced in MCF-7 cells than HEK-293 cells as evidenced by zeta potential, implicating the possible reason for differential cytotoxicity. Thus the present study demonstrates the importance of these unique, less time-consuming, and stable BSP-mediated GNP as potential drug delivery vehicles in the application of anticancer therapy.

Anti-platelet aggregation of mixtures of betulinic oleanolic and ...

African Journals Online (AJOL)

on human embryonic kidney (HEK293) and hepatocellular carcinoma (HEPG2) cell lines ... exhibited low cytotoxic effect on both HEK293 cells (IC50: 724.43, 269.08 and 407.89 mg/mL ..... Triterpenes from the stem bark of Protorhus longifolia.

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

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Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

2014-10-16

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

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Kuan-Teh Jeang

2012-07-01

Full Text Available A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

Embryonic Stem Cells-loaded Gelatin Microcryogels Slow Progression of Chronic Kidney Disease

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Geng, Xiao-Dong; Zheng, Wei; Wu, Cong-Mei; Wang, Shu-Qiang; Hong, Quan; Cai, Guang-Yan; Chen, Xiang-Mei; Wu, Di

2016-01-01

Background: Chronic kidney disease (CKD) has become a public health problem. New interventions to slow or prevent disease progression are urgently needed. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of embryonic stem cells (ESCs) on the progression of CKD. Methods: Adult male Sprague–Dawley rats were subjected to 5/6 nephrectomy. We used pedicled greater omentum flaps packing ESC-loaded gelatin microcryogels (GMs) on the 5/6 nephrectomized kidney. The viability of ESCs within the GMs was detected using in vitro two-photon fluorescence confocal imaging. Rats were sacrificed after 12 weeks. Renal injury was evaluated using serum creatinine, urea nitrogen, 24 h protein, renal pathology, and tubular injury score results. Structural damage was evaluated by periodic acid-Schiff and Masson trichrome staining. Results: In vitro, ESCs could be automatically loaded into the GMs. Uniform cell distribution, good cell attachment, and viability were achieved from day 1 to 7 in vitro. After 12 weeks, in the pedicled greater omentum flaps packing ESC-loaded GMs on 5/6 nephrectomized rats group, the plasma urea nitrogen levels were 26% lower than in the right nephrectomy group, glomerulosclerosis index was 62% lower and tubular injury index was 40% lower than in the 5/6 nephrectomized rats group without GMs. Conclusions: In a rat model of established CKD, we demonstrated that the pedicled greater omentum flaps packing ESC-loaded GMs on the 5/6 nephrectomized kidney have a long-lasting therapeutic rescue function, as shown by the decreased progression of CKD and reduced glomerular injury. PMID:26879011

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

International Nuclear Information System (INIS)

Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

2010-01-01

Research highlights: → Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. → Adaptor-related protein complex 1 μ1A (AP-1 mu1A) was firstly reported to interact with kAE1. → The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. → AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. → AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl - ) and bicarbonate (HCO 3 - ) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl - /HCO 3 - exchange at the basolateral membrane and failure of proton (H + ) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

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Sawasdee, Nunghathai; Junking, Mutita [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Ngaojanlar, Piengpaga [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Immunology and Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Sukomon, Nattakan; Ungsupravate, Duangporn [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Akkarapatumwong, Varaporn [Institute of Molecular Biosciences, Mahidol University at Salaya Campus, Nakorn Pathom 73170 (Thailand); Noisakran, Sansanee [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

2010-10-08

Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

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Fernández-Del-Río, Lucía; Nag, Anish; Gutiérrez Casado, Elena; Ariza, Julia; Awad, Agape M; Joseph, Akil I; Kwon, Ohyun; Verdin, Eric; de Cabo, Rafael; Schneider, Claus; Torres, Jorge Z; Burón, María I; Clarke, Catherine F; Villalba, José M

2017-09-01

Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q 10 , but due to its highly lipophilic nature, Q 10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13 C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of PTTG on endogenous gene expression in HEK 293 cells

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Panguluri Siva K

2009-12-01

Full Text Available Abstract Background Pituitary tumor transforming gene (PTTG, also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293 cells that do not express PTTG and analyzed the downstream genes using microarray analysis. Results A total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip™ array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad or adenovirus vector expressing GFP (AdGFP. Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of ≤ 0.05 and a fold change of ≥2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family. Conclusion PTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study

PHARMACOLOGICAL IN VITRO MODELS IN PRE-CLINICAL DRUG TESTING - EXAMPLE OF hSERT TRANSFECTED HUMAN EMBRYONIC KIDNEY CELLS

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Mihajlo Jakovljević

2012-06-01

Full Text Available Preclinical drug testing should be considered an important stage during examinations of its efficiency and safety in any likely indication observed. Purpose of the process is acquisition of substantial amount of particular drug-related data before approaching clinical trials in humans. Historical preclinical testing relied on available testing in microbe cultures and animal models. During recent decades laboratory techniques of human cell lines cultivation have been developed and improved. These provide unique possibility of drug acting mechanism testing in a simplified environment lacking basic homeostatic mechanisms. Some examples of these are measuring drug impact to biochemical transport, signaling or anabolic processes. Humane cell lines of embrional kidney 293 are an example of easy-to-grow and disseminate and quite endurable cell line. This methodological article notices some of the details of HEK293 cells cultivation and breading. We took transfection as an example of in vitro model creation for drug testing. Transfection refers to gene introduction into HEK293 cellular genome in order to achieve membrane expression of coded protein. In our case it would be human serotonin transporter. Article contains description of one particular methodological approach in measuring human serotonin transporter expression. The role and importance of serotonin pump in affective disorders genesis was already widely recognized. Aim of the paper was to emphasize feasibility of cell cultivation and its advantages in comparison with alternative traditional methods.

Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.

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Prashanthi Karyala

Full Text Available BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B, β-galactosidase (β-gal and green fluorescent protein (GFP from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

International Nuclear Information System (INIS)

Malkinson, Mertyn; Winocour, Ernest

2005-01-01

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM. AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host

Cytotoxic assessment of silver nanoparticles in embryonic development and kidney tissue in pregnant mice

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Bagher seyedalipour

2015-10-01

Full Text Available Background and Aim: Regarding the widespread use of silver nanoparticles in medecine and lack of a detailed study of toxicity effects of these particles on fetus, this study was carried out to investigate histopathological changes of the kidneys and also embryonic development following exposure to silver nanoparticles. Materials and Methods: In this experimental study, thirty five female NMRI mice were randomly divided into five equal groups i.e. one control group and four experimental groups. The experimental groups intraperitoneally (IP received silver nanoparticles at concentrations of 50, 100, 200 and 400 mg/ kg . .every other day. On the 17th day  of pregnancy, the mice were dissected and  their kidneys and embryos tissues were separated and stained with hematoxylin and eosin for histopathological examinations. .Finally, the obtained data was fed into SPSS software (V:16 using statistical tests including Kolmogrof-Smearnof, one-way variance analysis, Dante, Mann-Whitney and Kruskal-Wallis and P<0.05 was taken as the significant level. Results: Histopathological assessment of kidney tissue following IP administration of silver nanoparticle indicated pathological changes including congestion, necrosis, inflammatory cell infiltration, vacuolar degeneration compared to the control group. Our findings showed that silver nanoparticles during the gestation period affects fetal organogenesis, evolution of neural structure, liver lobulation and fetal growth retardation. Mean number of somites in groups receiving doses of 200 and 400 mg kg, . significantly reduced compared to the control group (P<0.05. Conclusion: The obtained results suggest that  passing of silver nanoparticles through placenta is possible and damage caused by the particles  could lead to the deformity or developmental retardation of the fetus.

Estimation of Total Glomerular Number Using an Integrated Disector Method in Embryonic and Postnatal Kidneys

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Michel G Arsenault

2014-06-01

Full Text Available Congenital Anomalies of the Kidney and Urinary Tract (CAKUT are a polymorphic group of clinical disorders comprising the major cause of renal failure in children. Included within CAKUT is a wide spectrum of developmental malformations ranging from renal agenesis, renal hypoplasia and renal dysplasia (maldifferentiation of renal tissue, each characterized by varying deficits in nephron number. First presented in the Brenner Hypothesis, low congenital nephron endowment is becoming recognized as an antecedent cause of adult-onset hypertension, a leading cause of coronary heart disease, stroke, and renal failure in North America. Genetic mouse models of impaired nephrogenesis and nephron endowment provide a critical framework for understanding the origins of human kidney disease. Current methods to quantitate nephron number include (i acid maceration (ii estimation of nephron number from a small number of tissue sections (iii imaging modalities such as MRI and (iv the gold standard physical disector/fractionator method. Despite its accuracy, the physical disector/fractionator method is rarely employed because it is labour-intensive, time-consuming and costly to perform. Consequently, less rigourous methods of nephron estimation are routinely employed by many laboratories. Here we present an updated, digitized version of the physical disector/fractionator method using free open source Fiji software, which we have termed the integrated disector method. This updated version of the gold standard modality accurately, rapidly and cost-effectively quantitates nephron number in embryonic and post-natal mouse kidneys, and can be easily adapted for stereological measurements in other organ systems.

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

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Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

2010-01-01

AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

Experiment study of tyrosinase gene's expression in HEK293 cell by MR

International Nuclear Information System (INIS)

Yuan Jianpeng; Liang Biling; Zhong Jinglian; Xie Bangkun; Zhang Weidong; Zhang Lin

2004-01-01

Objective: To transfect the tyrosinase gene into HEK293 cell as a reporter gene, and to evaluate the tyrosinase gene's expression by using MRI based on the gene's property of synthesizing large amount of melanin, and to search a way for evaluating the results of gene expression by MR in vitro. Methods: The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin, and MR signals of expressed melanin was observed by scanning the transfected cells with MR sequences of T 1 WI, T 1 WI/SPIR, and T 2 WI. Fontana stain and electric microscopy were used to search for melanin granules in transfected cells, and RT-PCR method was used to search for cDNA of tyrosinase gene. Results: (1) Plasmids of pcDNA3tyr could be transfected into HEK293 cells and could synthesize a large amount of melanin in them. The synthetic melanin in 10 6 cells, which had been transfected with 5 μg, 10 μg, and 20 μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MR and appeared as high signal on MR T 1 WI, T 1 WI/SPIR, and T 2 WI sequences. The more the amounts of transfected plasmids, the higher the signal intensities of MR imaging. On the other hand, 6.25 x 10 4 cells with 20 μg-plasmid of pcDNA3tyr transfection could also be detected by MR; (2) The melanin granules could be found in HEK293 cells in Fontana stain; (3) The melanin granules and their front bodies could be found in intracytoplasm of HEK293 cell by electric microscopy. (4) The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR. Conclusion: The fact that MR could detect the synthetic melanin in HEK293 cells controlled by expression of exogenous gene demonstrated that medical imaging combined with molecular biology technology could evaluate the result of gene expression in vitro, and it also indicated that medical imaging could play an important role in the evaluation of gene therapy following the development

T1-mapping for assessment of ischemia-induced acute kidney injury and prediction of chronic kidney disease in mice

Energy Technology Data Exchange (ETDEWEB)

Hueper, Katja; Gutberlet, Marcel; Wacker, Frank; Hartung, Dagmar [Hannover Medical School, Department of Radiology, Hannover (Germany); Hannover Medical School, REBIRTH Cluster of Excellence, Hannover (Germany); Peperhove, Matti; Tewes, Susanne; Barrmeyer, Amelie [Hannover Medical School, Department of Radiology, Hannover (Germany); Rong, Song [Hannover Medical School, Department of Nephrology, Hannover (Germany); Zunyi Medical College, Laboratory of Organ Transplantation, Zunyi (China); Gerstenberg, Jessica; Haller, Herman; Gueler, Faikah [Hannover Medical School, Department of Nephrology, Hannover (Germany); Mengel, Michael [University of Alberta, Department of Laboratory Medicine and Pathology, Edmonton (Canada); Meier, Martin [Hannover Medical School, REBIRTH Cluster of Excellence, Hannover (Germany); Hannover Medical School, Institute for Animal Science, Hannover (Germany); Chen, Rongjun [Hannover Medical School, Department of Nephrology, Hannover (Germany); Zhejiang University, The Kidney Disease Center of the First Affiliated Hospital, Hangzhou (China)

2014-09-15

To investigate whether T1-mapping allows assessment of acute kidney injury (AKI) and prediction of chronic kidney disease (CKD) in mice. AKI was induced in C57Bl/6N mice by clamping of the right renal pedicle for 35 min (moderate AKI, n = 26) or 45 min (severe AKI, n = 23). Sham animals served as controls (n = 9). Renal histology was assessed in the acute (day 1 + day 7; d1 + d7) and chronic phase (d28) after AKI. Furthermore, longitudinal MRI-examinations (prior to until d28 after surgery) were performed using a 7-Tesla magnet. T1-maps were calculated from a fat-saturated echoplanar inversion recovery sequence, and mean and relative T1-relaxation times were determined. Renal histology showed severe tubular injury at d1 + d7 in both AKI groups, whereas, at d28, only animals with prolonged 45-min ischemia showed persistent signs of AKI. Following both AKI severities T1-values significantly increased and peaked at d7. T1-times in the contralateral kidney without AKI remained stable. At d7 relative T1-values in the outer stripe of the outer medulla were significantly higher after severe than after moderate AKI (138 ± 2 % vs. 121 ± 3 %, p = 0.001). T1-elevation persisted until d28 only after severe AKI. Already at d7 T1 in the outer stripe of the outer medulla correlated with kidney volume loss indicating CKD (r = 0.83). T1-mapping non-invasively detects AKI severity in mice and predicts further outcome. (orig.)

T1-mapping for assessment of ischemia-induced acute kidney injury and prediction of chronic kidney disease in mice

International Nuclear Information System (INIS)

Hueper, Katja; Gutberlet, Marcel; Wacker, Frank; Hartung, Dagmar; Peperhove, Matti; Tewes, Susanne; Barrmeyer, Amelie; Rong, Song; Gerstenberg, Jessica; Haller, Herman; Gueler, Faikah; Mengel, Michael; Meier, Martin; Chen, Rongjun

2014-01-01

To investigate whether T1-mapping allows assessment of acute kidney injury (AKI) and prediction of chronic kidney disease (CKD) in mice. AKI was induced in C57Bl/6N mice by clamping of the right renal pedicle for 35 min (moderate AKI, n = 26) or 45 min (severe AKI, n = 23). Sham animals served as controls (n = 9). Renal histology was assessed in the acute (day 1 + day 7; d1 + d7) and chronic phase (d28) after AKI. Furthermore, longitudinal MRI-examinations (prior to until d28 after surgery) were performed using a 7-Tesla magnet. T1-maps were calculated from a fat-saturated echoplanar inversion recovery sequence, and mean and relative T1-relaxation times were determined. Renal histology showed severe tubular injury at d1 + d7 in both AKI groups, whereas, at d28, only animals with prolonged 45-min ischemia showed persistent signs of AKI. Following both AKI severities T1-values significantly increased and peaked at d7. T1-times in the contralateral kidney without AKI remained stable. At d7 relative T1-values in the outer stripe of the outer medulla were significantly higher after severe than after moderate AKI (138 ± 2 % vs. 121 ± 3 %, p = 0.001). T1-elevation persisted until d28 only after severe AKI. Already at d7 T1 in the outer stripe of the outer medulla correlated with kidney volume loss indicating CKD (r = 0.83). T1-mapping non-invasively detects AKI severity in mice and predicts further outcome. (orig.)

Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study

OpenAIRE

Piroozmand, Ahmad; Yamamoto, Yoshihiko; Khamsri, Boonruang; Fujita, Mikako; Uchiyama, Tsuneo; Adachi, Akio

2007-01-01

We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions u...

Assessment of acute kidney injury with T1 mapping MRI following solid organ transplantation

Energy Technology Data Exchange (ETDEWEB)

Peperhove, Matti; Vo Chieu, Van Dai; Gutberlet, Marcel; Hartung, Dagmar; Tewes, Susanne; Wacker, Frank; Hueper, Katja [Hannover Medical School, Diagnostic and Interventional Radiology, Hannover (Germany); Jang, Mi-Sun; Gwinner, Wilfried; Haller, Hermann; Gueler, Faikah [Nephrology, Hannover Medical School, Hannover (Germany); Warnecke, Gregor; Fegbeutel, Christiane; Haverich, Axel [Hannover Medical School, Cardiothoracic, Transplantation and Vascular Surgery, Hannover (Germany); Lehner, Frank [Hannover Medical School, General, Abdominal and Transplant Surgery, Hannover (Germany); Braesen, Jan Hinrich [Pathology, Hannover Medical School, Hannover (Germany)

2018-01-15

To evaluate T1 mapping as a non-invasive, functional MRI biomarker in patients shortly after solid organ transplantation to detect acute postsurgical kidney damage and to correlate T1 times with renal function. 101 patients within 2 weeks after solid organ transplantation (49 kidney transplantation, 52 lung transplantation) and 14 healthy volunteers were examined by MRI between July 2012 and April 2015 using the modified Look-Locker inversion recovery (MOLLI) sequence. T1 times in renal cortex and medulla and the corticomedullary difference were compared between groups using one-way ANOVA adjusted for multiple comparison with the Tukey test, and T1 times were correlated with renal function using Pearson's correlation. Compared to healthy volunteers T1 times were significantly increased after solid organ transplantation in the renal cortex (healthy volunteers 987 ± 102 ms; kidney transplantation 1299 ± 101 ms, p < 0.001; lung transplantation 1058 ± 96 ms, p < 0.05) and to a lesser extent in the renal medulla. Accordingly, the corticomedullary difference was diminished shortly after solid organ transplantation. T1 changes were more pronounced following kidney compared to lung transplantation, were associated with the stage of renal impairment and significantly correlated with renal function. T1 mapping may be helpful for early non-invasive assessment of acute kidney injury and renal pathology following major surgery such as solid organ transplantation. (orig.)

Assessment of acute kidney injury with T1 mapping MRI following solid organ transplantation

International Nuclear Information System (INIS)

Peperhove, Matti; Vo Chieu, Van Dai; Gutberlet, Marcel; Hartung, Dagmar; Tewes, Susanne; Wacker, Frank; Hueper, Katja; Jang, Mi-Sun; Gwinner, Wilfried; Haller, Hermann; Gueler, Faikah; Warnecke, Gregor; Fegbeutel, Christiane; Haverich, Axel; Lehner, Frank; Braesen, Jan Hinrich

2018-01-01

To evaluate T1 mapping as a non-invasive, functional MRI biomarker in patients shortly after solid organ transplantation to detect acute postsurgical kidney damage and to correlate T1 times with renal function. 101 patients within 2 weeks after solid organ transplantation (49 kidney transplantation, 52 lung transplantation) and 14 healthy volunteers were examined by MRI between July 2012 and April 2015 using the modified Look-Locker inversion recovery (MOLLI) sequence. T1 times in renal cortex and medulla and the corticomedullary difference were compared between groups using one-way ANOVA adjusted for multiple comparison with the Tukey test, and T1 times were correlated with renal function using Pearson's correlation. Compared to healthy volunteers T1 times were significantly increased after solid organ transplantation in the renal cortex (healthy volunteers 987 ± 102 ms; kidney transplantation 1299 ± 101 ms, p < 0.001; lung transplantation 1058 ± 96 ms, p < 0.05) and to a lesser extent in the renal medulla. Accordingly, the corticomedullary difference was diminished shortly after solid organ transplantation. T1 changes were more pronounced following kidney compared to lung transplantation, were associated with the stage of renal impairment and significantly correlated with renal function. T1 mapping may be helpful for early non-invasive assessment of acute kidney injury and renal pathology following major surgery such as solid organ transplantation. (orig.)

Characterisation of the zebrafish serotonin transporter functionally links TM10 to the ligand binding site

DEFF Research Database (Denmark)

Severinsen, Kasper; Müller, Heidi Kaastrup; Wiborg, Ove

2008-01-01

and [(3)H]-escitalopram binding in transiently transfected human embryonic kidney cells; HEK-293-MSR. Residues responsible for altered affinities inhibitors were pinpointed by generating cross-species chimeras and subsequent point mutations by site directed mutagenesis. drSERT has a higher affinity...

Abnormal differentiation, hyperplasia and embryonic/perinatal lethality in BK5-T/t transgenic mice

Science.gov (United States)

Chen, Xin; Schneider-Broussard, Robin; Hollowell, Debra; McArthur, Mark; Jeter, Collene R.; Benavides, Fernando; DiGiovanni, John; Tang, Dean G.

2009-01-01

The cell-of-origin has a great impact on the types of tumors that develop and the stem/progenitor cells have long been considered main targets of malignant transformation. The SV40 large T and small t antigens (T/t), have been targeted to multiple differentiated cellular compartments in transgenic mice. In most of these studies, transgenic animals develop tumors without apparent defects in animal development. In this study, we used the bovine keratin 5 (BK5) promoter to target the T/t antigens to stem/progenitor cell-containing cytokeratin 5 (CK5) cellular compartment. A transgene construct, BK5-T/t, was made and microinjected into the male pronucleus of FVB/N mouse oocytes. After implanting ∼1700 embryos, only 7 transgenics were obtained, including 4 embryos (E9.5, E13, E15, and E20) and 3 postnatal animals, which died at P1, P2, and P18, respectively. Immunohistological analysis revealed aberrant differentiation and prominent hyperplasia in several transgenic CK5 tissues, especially the upper digestive organs (tongue, oral mucosa, esophagus, and forestomach) and epidermis, the latter of which also showed focal dysplasia. Altogether, these results indicate that constitutive expression of the T/t antigens in CK5 cellular compartment results in abnormal epithelial differentiation and leads to embryonic/perinatal animal lethality. PMID:19272531

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner

Directory of Open Access Journals (Sweden)

Zhu Ping

2010-04-01

Full Text Available Abstract Background Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory. Results An anoikis-resistant clone (HEK293ar, derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK inhibitor PD98059. Conclusions Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated

Concise Review: Kidney Generation with Human Pluripotent Stem Cells.

Science.gov (United States)

Morizane, Ryuji; Miyoshi, Tomoya; Bonventre, Joseph V

2017-11-01

Chronic kidney disease (CKD) is a worldwide health care problem, resulting in increased cardiovascular mortality and often leading to end-stage kidney disease, where patients require kidney replacement therapies such as hemodialysis or kidney transplantation. Loss of functional nephrons contributes to the progression of CKD, which can be attenuated but not reversed due to inability to generate new nephrons in human adult kidneys. Human pluripotent stem cells (hPSCs), by virtue of their unlimited self-renewal and ability to differentiate into cells of all three embryonic germ layers, are attractive sources for kidney regenerative therapies. Recent advances in stem cell biology have identified key signals necessary to maintain stemness of human nephron progenitor cells (NPCs) in vitro, and led to establishment of protocols to generate NPCs and nephron epithelial cells from human fetal kidneys and hPSCs. Effective production of large amounts of human NPCs and kidney organoids will facilitate elucidation of developmental and pathobiological pathways, kidney disease modeling and drug screening as well as kidney regenerative therapies. We summarize the recent studies to induce NPCs and kidney cells from hPSCs, studies of NPC expansion from mouse and human embryonic kidneys, and discuss possible approaches in vivo to regenerate kidneys with cell therapies and the development of bioengineered kidneys. Stem Cells 2017;35:2209-2217. © 2017 AlphaMed Press.

Quantitation of two endogenous lactose-inhibitable lectins in embryonic and adult chicken tissues

International Nuclear Information System (INIS)

Beyer, E.C.; Barondes, S.H.

1982-01-01

Two lactose-binding lectins from chicken tissues, chicken-lactose-lectin-I (CLL-I) and chicken-lactose-lectin-II (CLL-II) were quantified with a radioimmunoassay in extracts of a number of developing and adult chicken tissues. Both lectins could be measured in the same extract without separation, because they showed no significant immunological cross- reactivity. Many embryonic and adult tissues, including brain, heart, intestine, kidney, liver, lung, muscle, pancreas, and spleen, contained one or both lectins, although their concentrations differed markedly. For example, embryonic muscle, the richest source of CLL-I contained only traces of CLL-II whereas embryonic kidney, a very rich source of CLL-II contained substantial CLL-I. In both muscle and kidney, lectin levels in adulthood were much lower than in the embryonic state. In contrast, CLL-I in liver and CLL-II in intestine were 10-fold to 30-fold more concentrated in the adult than in the 15-d embryo. CLL-I and CLL-II from several tissues were purified by affinity chromatography and their identity in the various tissues was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping. The results suggest that these lectins might have different functions in the many developing and adult tissues in which they are found

STAT3 Potentiates SIAH-1 Mediated Proteasomal Degradation of β-Catenin in Human Embryonic Kidney Cells.

Science.gov (United States)

Shin, Minkyung; Yi, Eun Hee; Kim, Byung-Hak; Shin, Jae-Cheon; Park, Jung Youl; Cho, Chung-Hyun; Park, Jong-Wan; Choi, Kang-Yell; Ye, Sang-Kyu

2016-11-30

The β-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, β-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of β-catenin. The level of β-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to β-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and β-catenin in HEK293T cells. To our knowledge, this is the first study to report that β-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated β-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active β-catenin via degradation, which stabilized SIAH-1 and increased its interaction with β-catenin. These results suggest that activated STAT3 regulates active β-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of β-catenin in HEK293T cells.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Science.gov (United States)

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

Science.gov (United States)

Ding, Kai; Han, Lei; Zong, Huifang; Chen, Junsheng; Zhang, Baohong; Zhu, Jianwei

2017-03-01

Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

Simvastatin enhances bone morphogenetic protein receptor type II expression

International Nuclear Information System (INIS)

Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

2006-01-01

Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

SLURP1 is a late marker of epidermal differentiation and is absent in Mal de Meleda

DEFF Research Database (Denmark)

Favre, Bertrand; Plantard, Laure; Aeschbach, Lorène

2007-01-01

and MDM skin. SLURP1 was found to be a marker of late differentiation, predominantly expressed in the granular layer of skin, notably the acrosyringium. Moreover, SLURP1 was also identified in several biological fluids such as sweat, saliva, tears, and urine from normal volunteers. In palmoplantar...... sections from MDM patients, as well as in their sweat, mutant SLURP1, including the new variant R71H-SLURP1, was either absent or barely detectable. Transfected human embryonic kidney 293T cells expressed the MDM mutant SLURP1 containing the single amino-acid substitution G86R but did not tolerate the MDM...

Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells

Directory of Open Access Journals (Sweden)

Baek NH

2016-07-01

Full Text Available NamHuk Baek,1,* Ok Won Seo,1,* Jaehwa Lee,1 John Hulme,2 Seong Soo A An2 1Department of Research and Development, NanoEntek Inc., Seoul, 2Department of BioNano Technology, Gachon University, Gyeonggi-do, Korea *These authors contributed equally to this work Abstract: Three-dimensional (3D cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell–cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II or CDDP, on adenosine triphosphate (ATP generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145, testis (F9, embryonic fibroblast (NIH-3T3, muscle (C2C12, embryonic kidney (293T, neuroblastoma (SH-SY5Y, adenocarcinomic alveolar basal epithelial cell (A549, cervical cancer (HeLa, HeLa contaminant (HEp2, pituitary epithelial-like cell (GH3, embryonic cell (PA317, and osteosarcoma (U-2OS cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 µM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be

G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

NARCIS (Netherlands)

Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

International Meeting on Cholinesterases (5th) Held in Madras, India on 24-28 September, 1994.

Science.gov (United States)

1994-09-01

found that hydrolysis of thioesters deviated from simple Michaelis-Menten model. Kinetics was triphasic , displaying complexities of both BuChE and ACHE...of recombinant human acetylcholinesterase (rHuAChE) produced by human embryonic kidney cell line (293) in a fixed-bed reactor (1) was investigated at

Photo-transfection of mammalian cells via femtosecond laser pulses

CSIR Research Space (South Africa)

Mthunzi, P

2009-06-01

Full Text Available on transient photo-transfection of ovary (CHO-Kl), neuroblastoma (NG-I08 & SKN-SH) and embryonic kidney (HEK-293) as well as primary non-differentiated stem cells (EI4g2a) using a tightly focused titanium sapphire laser beam (1.1 urn diameter spot size...

Effects of chlorpyrifos and trichloropyridinol on HEK 293 human embryonic kidney cells

Science.gov (United States)

Chlorpyrifos (CPF) [O, O-diethyl -O-3, 5, 6-trichloro-2-pyridyl phosphorothioate] is an organophosphate insecticide widely used for agricultural and urban pest control. Trichloropyridinol (TCP; 3,5,6-trichloro-2-pyridinol), the primary metabolite of CPF, is often used as a generi...

Phytochemistry, cytotoxicity and apoptosis studies of β-sitosterol-3 ...

African Journals Online (AJOL)

Materials and methods: In this study, compounds from the leaves and bark of this plant were isolated and tested for their cytotoxicity and apoptosis induction in two human cancer cell lines (hepatocellular carcinoma (HepG2) and colorectal carcinoma (Caco-2)) and a non-cancer cell line (embryonic kidney (HEK293)).

Early superoxide scavenging accelerates renal microvascular rarefaction and damage in the stenotic kidney.

Science.gov (United States)

Kelsen, Silvia; He, Xiaochen; Chade, Alejandro R

2012-08-15

Renal artery stenosis (RAS), the main cause of chronic renovascular disease (RVD), is associated with significant oxidative stress. Chronic RVD induces renal injury partly by promoting renal microvascular (MV) damage and blunting MV repair in the stenotic kidney. We tested the hypothesis that superoxide anion plays a pivotal role in MV dysfunction, reduction of MV density, and progression of renal injury in the stenotic kidney. RAS was induced in 14 domestic pigs and observed for 6 wk. Seven RAS pigs were chronically treated with the superoxide dismutase mimetic tempol (RAS+T) to reduce oxidative stress. Single-kidney hemodynamics and function were quantified in vivo using multidetector computer tomography (CT) and renal MV density was quantified ex vivo using micro-CT. Expression of angiogenic, inflammatory, and apoptotic factors was measured in renal tissue, and renal apoptosis and fibrosis were quantified in tissue sections. The degree of RAS and blood pressure were similarly increased in RAS and RAS+T. Renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in the stenotic kidney (280.1 ± 36.8 and 34.2 ± 3.1 ml/min, P < 0.05 vs. control). RAS+T kidneys showed preserved GFR (58.5 ± 6.3 ml/min, P = not significant vs. control) but a similar decreases in RBF (293.6 ± 85.2 ml/min) and further decreases in MV density compared with RAS. These changes were accompanied by blunted angiogenic signaling and increased apoptosis and fibrosis in the stenotic kidney of RAS+T compared with RAS. The current study shows that tempol administration provided limited protection to the stenotic kidney. Despite preserved GFR, renal perfusion was not improved by tempol, and MV density was further reduced compared with untreated RAS, associated with increased renal apoptosis and fibrosis. These results suggest that a tight balance of the renal redox status is necessary for a normal MV repair response to injury, at least at the early stage of RVD, and raise caution

Viability of HEK 293 cells on poly-β-hydroxybutyrate (PHB) biosynthesized from a mutant Azotobacter vinelandii strain. Cast film and electrospun scaffolds.

Science.gov (United States)

Romo-Uribe, Angel; Meneses-Acosta, Angelica; Domínguez-Díaz, Maraolina

2017-12-01

Sterilization, cytotoxicity and cell viability are essential properties defining a material for medical applications and these characteristics were investigated for poly(β-hydroxybutyrate) (PHB) of 230kDa obtained by bacterial synthesis from a mutant strain of Azotobacter vinelandii. Cell viability was investigated for two types of PHB scaffolds, solution cast films and non-woven electrospun fibrous membranes, and the efficiency was compared against a culture dish. The biosynthesized PHB was sterilized by ultraviolet radiation and autoclave, it was found that the thermal properties and intrinsic viscosity remained unchanged indicating that the sterilization methods did not degrade the polymer. Sterilized scaffolds were then seeded with human embryonic kidney 293 (HEK 293) cells to evaluate the cytotoxic response. The cell viability of these cells was evaluated for up to six days, and the results showed that the cell morphology was normal, with no cytotoxic effects. The films and electrospun membranes exhibited over 95% cell viability whereas the viability in culture dishes reached only ca. 90%. The electrospun membrane, however, exhibited significantly higher cell density than the cast film suggesting that the fibrous morphology enables better nutrients transfer. The results indicate that the biosynthesized PHB stands UV and autoclave sterilization methods, it is biocompatible and non-toxic for cell growth of human cell lines. Furthermore, cell culture for up to 18 days showed that 62% and 90% of mass was lost for the film and fibrous electrospun scaffold, respectively. This is a favorable outcome for use in tissue engineering where material degradation, as tissue regenerates, is desirable. Copyright © 2017 Elsevier B.V. All rights reserved.

Fetal Kidney Anomalies: Next Generation Sequencing

DEFF Research Database (Denmark)

Rasmussen, Maria; Sunde, Lone; Nielsen, Marlene Louise

Aim and Introduction Identification of abnormal kidneys in the fetus may lead to termination of the pregnancy and raises questions about the underlying cause and recurrence risk in future pregnancies. In this study, we investigate the effectiveness of targeted next generation sequencing in fetuses...... with prenatally detected kidney anomalies in order to uncover genetic explanations and assess recurrence risk. Also, we aim to study the relation between genetic findings and post mortem kidney histology. Methods The study comprises fetuses diagnosed prenatally with bilateral kidney anomalies that have undergone...... postmortem examination. The approximately 110 genes included in the targeted panel were chosen on the basis of their potential involvement in embryonic kidney development, cystic kidney disease, or the renin-angiotensin system. DNA was extracted from fetal tissue samples or cultured chorion villus cells...

A single nucleotide deletion of 293delT in SEDL gene causing spondyloepiphyseal dysplasia tarda in a four-generation Chinese family

DEFF Research Database (Denmark)

Xiao, Cuiying; Zhang, Sizhong; Wang, Jun

2003-01-01

. The distinctive radiological signs and the X-linked mode of inheritance make it easy to diagnose. Here a four-generation Chinese SEDT family has been analyzed and the disease-causing mutation has been found. After polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis and DNA...... sequencing, a previously unreported deletion of T in exon 5 of SEDL gene (i.e. 293delT) was observed and seven individuals in the family carried the mutation. It results in frameshift and a putative truncated protein with the 97 N-terminal amino acids, and 9 changed amino acids. Therefore, loss of function...

Multiweek Cell Culture Project for Use in Upper-Level Biology Laboratories

Science.gov (United States)

Marion, Rebecca E.; Gardner, Grant E.; Parks, Lisa D.

2012-01-01

This article describes a laboratory protocol for a multiweek project piloted in a new upper-level biology laboratory (BIO 426) using cell culture techniques. Human embryonic kidney-293 cells were used, and several culture media and supplements were identified for students to design their own experiments. Treatments included amino acids, EGF,…

A Simple Model to Study Tau Pathology

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Alexander L. Houck

2016-01-01

Full Text Available Tau proteins play a role in the stabilization of microtubules, but in pathological conditions, tauopathies, tau is modified by phosphorylation and can aggregate into aberrant aggregates. These aggregates could be toxic to cells, and different cell models have been used to test for compounds that might prevent these tau modifications. Here, we have used a cell model involving the overexpression of human tau in human embryonic kidney 293 cells. In human embryonic kidney 293 cells expressing tau in a stable manner, we have been able to replicate the phosphorylation of intracellular tau. This intracellular tau increases its own level of phosphorylation and aggregates, likely due to the regulatory effect of some growth factors on specific tau kinases such as GSK3. In these conditions, a change in secreted tau was observed. Reversal of phosphorylation and aggregation of tau was found by the use of lithium, a GSK3 inhibitor. Thus, we propose this as a simple cell model to study tau pathology in nonneuronal cells due to their viability and ease to work with.

Susceptibility weighted imaging (SWI) of the kidney at 3 T. Initial results

International Nuclear Information System (INIS)

Mie, Moritz B.; Zoellner, Frank G.; Heilmann, Melanie; Schad, Lothar R.; Nissen, Johanna C.; Schoenberg, Stefan O.; Michaely, Henrik J.

2010-01-01

Susceptibility weighted imaging provides diagnostic information in strokes, hemorrhages, and cerebral tumors and has proven to be a valuable tool in imaging venous vessels in the cerebrum. The SWI principle is based on the weighting of T 2 * weighted magnitude images with a phase mask, therewith improving image contrast of veins or neighbouring structures of different susceptibility, in general. T 2 * weighted MRI is already used for assessment of kidney function. In this paper, the feasibility of SWI on kidneys was investigated. Translation of SWI from the brain to the kidneys comes along with two main challenges: (i) organ motion due to breathing and (ii) a higher oxygenation level of renal veins compared to the brain. To handle these problems, the acquisition time has been cut down to allow for breath-hold examinations, and different post-processing methods including a new phase mask were investigated to visualize renal veins. Results showed that by a new post-processing strategy SWI contrast was enhanced on average by a factor of 1.33 compared to the standard phase mask. In summary, initial experiences of SWI on the kidneys demonstrated the feasibility. However, further technical developments have to be performed to make this technology applicable in clinical abdominal MRI. (orig.)

8 CFR 293.1 - Computation of interest.

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2010-01-01

... simple interest table in § 293.3 shall be utilized in the computation of interest under this part. ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Computation of interest. 293.1 Section 293... INTEREST ON CASH RECEIVED TO SECURE IMMIGRATION BONDS § 293.1 Computation of interest. Interest shall be...

Density and viscosity of aqueous solutions of N,N-dimethylethanolamine at p = 0.1 MPa from T = (293.15 to 363.15) K

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Bernal-Garcia, J. Manuel [Instituto Mexicano del Petroleo, Mexico D.F. C.P. 07330 (Mexico); Hall, Kenneth R. [Chemical Engineering Department, Texas A and M University, College Station, TX 77843 (United States); Estrada-Baltazar, Alejandro [Departamento de Ingenieria Quimica, Instituto Tecnologico de Celaya, Celaya, Guanajuato, CP 38010 (Mexico); Iglesias-Silva, Gustavo A. [Departamento de Ingenieria Quimica, Instituto Tecnologico de Celaya, Celaya, Guanajuato, CP 38010 (Mexico)]. E-mail: gais@iqcelaya.itc.mx

2005-08-15

This work presents atmospheric density and viscosity values for (N,N-dimethylethanolamine + water) over the entire composition range from T (293.15 to 363.15) K for density and from T = (313.15 to 353.15) K for viscosity. Density measurements come from a vibrating tube densimeter while we have used three different Cannon-Fenske viscosimeters for the viscosity measurements. Excess molar volumes and viscosity deviations are calculated using a Redlich-Kister type equation. Excess molar volumes present negative deviations from ideality and viscosity deviations are positive at all temperatures and compositions in this work.

Density and viscosity of aqueous solutions of N,N-dimethylethanolamine at p = 0.1 MPa from T = (293.15 to 363.15) K

International Nuclear Information System (INIS)

Bernal-Garcia, J. Manuel; Hall, Kenneth R.; Estrada-Baltazar, Alejandro; Iglesias-Silva, Gustavo A.

2005-01-01

This work presents atmospheric density and viscosity values for (N,N-dimethylethanolamine + water) over the entire composition range from T (293.15 to 363.15) K for density and from T = (313.15 to 353.15) K for viscosity. Density measurements come from a vibrating tube densimeter while we have used three different Cannon-Fenske viscosimeters for the viscosity measurements. Excess molar volumes and viscosity deviations are calculated using a Redlich-Kister type equation. Excess molar volumes present negative deviations from ideality and viscosity deviations are positive at all temperatures and compositions in this work

8 CFR 293.2 - Interest rate.

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2010-01-01

... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Interest rate. 293.2 Section 293.2 Aliens... CASH RECEIVED TO SECURE IMMIGRATION BONDS § 293.2 Interest rate. The Secretary of the Treasury has determined that effective from date of deposit occurring after April 27, 1966, the interest rate shall be 3...

Short-term high dose of quercetin and resveratrol alters aging markers in human kidney cells

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Fatemeh Abharzanjani

2017-01-01

Full Text Available Background: Hyperglycemia-mediated oxidative stress implicates in etiology of kidney cell aging and diabetic nephropathy. We evaluated the effects of different doses of resveratrol and quercetin and their combination therapy on aging marker in human kidney cell culture under hyperglycemia condition. Methods: Human embryonic kidney cell (HEK-293 was cultured in Dulbecco's Modified Eagle Medium (DMEM containing 100 mM (18 mg/L for 24 h. The cells were treated with resveratrol (2.5, 5, 10 μm, quercetin (3, 6, 12 μm, and combination of these (R 2.5 μm, Q 3 μm and (R 5 μm, Q 6 μm and (R 10 μm, Q 12 μm for 48 h, and then, cells were lysed to access RNA and lysate. Results: The analysis of data showed that beta-galactosidase enzyme gene expression as an aging marker in all treatment groups has reduced in a dose-dependent manner. Gene expression of Sirtuin1 and thioredoxin (Trx in all treated groups in comparison to control group increased in a dose-dependent fashion. Trx interacting protein (TXNIP gene expression decreased in a dose-dependent manner in all treated groups, especially in resveratrol and combination therapy. Conclusions: According to the results of this research, quercetin, resveratrol, and especially combination treatments with increased expression levels of antioxidants, can reduce aging markers in HEK cell line in hyperglycemia conditions. These results lead us to use flavonoids such as resveratrol for anti-aging potential.

Characterization of glycolipid galactosyltransferases from embryonic chicken brain

International Nuclear Information System (INIS)

Kyle, J.W.

1985-01-01

Glycolipid galactosyltransferases (GalT-3 and GalT-4) were solubilized from a membrane fraction isolated from embryonic chicken brain. The profiles of specific activity and total units per brain of GalT-3 and GalT-4 varied with embryonic age. GalT-4 had the highest specific activity at 9 days of embryonic development and showed a steady decrease until hatching. GalT-3 showed a gradual increase in specific activity. Both GalT3 and GalT-4 showed a steady increase in total units per brain throughout embryonic development. The solubilized enzymes could be separated using gel filtration, ion exchange chromatography or affinity chromatography on α-lactalbumin-agarose. Data obtained in the study imply that GalT-4 is involved in both glycoprotein and glycolipid biosynthesis. Glycosphingolipid products from GalT-3 and GalT-4 catalyzed reactions labeled with [ 14 C]galactose comigrated with authentic GMI and nLcOse 4 Cer, when examined by thin layer chromatography and autoradiography. Studies with galactosidases revealed that all of the enzyme products formed by GalT-3 and GalT-4 contained a [ 14 C]-galactose in a β anomeric linkage. Periodate oxidation studies of Gal-[ 14 C]GlcNAc, formed by purified GalT-4 using [ 14 C]GlcNAc as the acceptor, demonstrated that approximately 70% of the linkage formed was Galβ1-4GlcNAc and 30% was Galβ1-3GlcNAc. Studies on the susceptibility of [ 14 C]Gal-GlcNAc to base catalyzed β-elimination also suggested the presence of approximately 30% Galβ1-3GlcNAc

Volumetric and viscometric studies of amino acids in L-ascorbic acid aqueous solutions at T = (293.15 to 323.15) K

International Nuclear Information System (INIS)

Jiang, Xiaofeng; Zhu, Chunying; Ma, Youguang

2014-01-01

Graphical abstract: The densities and viscosities of L-alanine in L-ascorbic acid aqueous solutions at T = 293.15 K. Highlights: • Densities and viscosities of five amino acids in L-ascorbic acid aqueous solutions were measured. • Based on the experimental data, a series of volumetric and viscometric parameters were calculated. • The group additivity analysis has been applied to analyze the V φ 0 and B-coefficients. -- Abstract: Densities and viscosities of glycine, L-alanine, L-valine, L-threonine and L-arginine in aqueous solutions of (0.0, 0.1, 0.2, 0.3 and 0.4) mol · kg −1 L-ascorbic acid have been measured at T = (293.15, 303.15, 313.15 and 323.15) K under atmospheric pressure. The apparent molar volumes (V φ ), limiting partial molar volumes (V φ 0 ), limiting partial molar volumes of transfer (Δ tr V φ 0 ) and limiting partial molar expansibilities (E 2 0 ) were computed by densities. The extended Jones–Dole equation was used to correlate the viscosities in order to obtain viscosity B-coefficients and the free energies of activation per mole of solvent (Δμ 1 0≠ ) and solute (Δμ 2 0≠ ) were also calculated. The contributions of zwitterionic end group (NH 3 + , COO − ), CH 2 group, OH group and CNHNHNH 2 group to V φ 0 and viscosity B-coefficients were obtained through the group additivity analysis

8 CFR 293.3 - Simple interest table.

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2010-01-01

... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Simple interest table. 293.3 Section 293.3 Aliens and Nationality DEPARTMENT OF HOMELAND SECURITY IMMIGRATION REGULATIONS DEPOSIT OF AND INTEREST ON CASH RECEIVED TO SECURE IMMIGRATION BONDS § 293.3 Simple interest table. Following is a simple interest...

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

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Lu Wang

2014-07-01

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

Pretransplantation recipient regulatory T cell suppressive function predicts delayed and slow graft function after kidney transplantation.

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Nguyen, Minh-Tri J P; Fryml, Elise; Sahakian, Sossy K; Liu, Shuqing; Michel, Rene P; Lipman, Mark L; Mucsi, Istvan; Cantarovich, Marcelo; Tchervenkov, Jean I; Paraskevas, Steven

2014-10-15

Delayed graft function (DGF) and slow graft function (SGF) are a continuous spectrum of ischemia-reperfusion-related acute kidney injury (AKI) that increases the risk for acute rejection and graft loss after kidney transplantation. Regulatory T cells (Tregs) are critical in transplant tolerance and attenuate murine AKI. In this prospective observational cohort study, we evaluated whether pretransplantation peripheral blood recipient Treg frequency and suppressive function are predictors of DGF and SGF after kidney transplantation. Deceased donor kidney transplant recipients (n=53) were divided into AKI (n=37; DGF, n=10; SGF, n=27) and immediate graft function (n=16) groups. Pretransplantation peripheral blood CD4CD25FoxP3 Treg frequency was quantified by flow cytometry. Regulatory T-cell suppressive function was measured by suppression of autologous effector T-cell proliferation by Treg in co-culture. Pretransplantation Treg suppressive function, but not frequency, was decreased in AKI recipients (Paccounting for the effects of cold ischemic time and donor age, Treg suppressive function discriminated DGF from immediate graft function recipients in multinomial logistic regression (odds ratio, 0.77; Pfunction is a potential independent pretransplantation predictor of DGF and SGF.

T2' imaging of native kidneys and renal allografts. A feasibility study

International Nuclear Information System (INIS)

Mathys, C.; Blondin, D.; Wittsack, H.J.; Miese, F.R.; Rybacki, K.; Walther, C.; Holstein, A.; Lanzman, R.S.

2011-01-01

Purpose: To evaluate the feasibility of T2' mapping in native kidneys and renal allografts. Materials and Methods: Following approval of the local ethics committee, 24 renal allograft recipients and 10 control subjects (healthy volunteers) were included in this study. Multi-echo T2 and T2 * imaging was performed on a 1.5 Tesla scanner. Allograft recipients were assigned to two groups: group (a), 8 patients with good (glomerular filtration rate of more than 40 ml/min) allograft function and no evidence of transplant rejection, transplant renal artery stenosis or ureteral obstruction; group (b), 16 patients with deterioration of renal graft function (glomerular filtration rate (GFR) of 40 ml/min or less). Two different imaging protocols were tested. Results: The mean T2' relaxation parameters were 108.33 msec ± 13.34, 100.00 msec ± 18.89 and 124.57 msec ± 6.51 for groups (a), (b) and for control subjects, respectively. The reduction of T2' values in patient group (b) was not statistically significant. However, significant correlations could be demonstrated between T2' values and the glomerular filtration rate (GFR) of renal allograft function. The reproducibility was tested and the coefficients of variation of T2' values in the cortex of transplanted kidneys were 11.1 % within subjects and 11.3 % between subjects. Conclusion: Our results indicate that T2' imaging is a promising non-enhanced technique, which seems to reveal information on transplant function. Further studies are required to determine the clinical value of T2' mapping for monitoring renal allograft recipients. (orig.)

Direct and Indirect Effects of Cytomegalovirus-induced gamma-delta T Cells after Kidney Transplantation

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Lionel eCouzi

2015-01-01

Full Text Available Despite effective anti-viral therapies, cytomegalovirus (CMV is still associated with direct (CMV disease and indirect effects (rejection and poor graft survival in kidney transplant recipients. Recently, an unconventional T cell population (collectively designated as Vδ2neg γδ T cells has been characterized during the anti-CMV immune response in all solid-organ and bone-marrow transplant recipients, neonates, and healthy people. These CMV-induced γδ T cells undergo a dramatic and stable expansion after CMV infection, in a conventional ‘adaptive’ manner. Similarly as CMV-specific CD8+ αβ T cells, they exhibit an effector/memory TEMRA phenotype and cytotoxic effector functions. Activation of Vd2neg gd T cells by CMV-infected cells involves the TCR and still ill-defined co-stimulatory molecules such LFA-1. A multiple of Vd2neg gd TCR ligands are apparently recognized on CMV-infected cells, the first one identified being the MHC-related molecule endothelial protein C receptor (EPCR. A singularity of CMV-induced Vd2neg gd T cells is to acquire CD16 expression and to exert an antibody-dependent cell-mediated inhibition on CMV replication, which is controlled by a specific cytokine microenvironment. Beyond the well-demonstrated direct anti-CMV effect of Vδ2neg γδ T cells, unexpected indirect effects of these cells have been also observed in the context of kidney transplantation. CMV-induced Vδ2neg γδ T cells have been involved in surveillance of malignancy subsequent to long term immunosuppression. Moreover, CMV-induced CD16+ γδ T cells are cell effectors of antibody-mediated rejection of kidney transplants, and represent a new physiopathological contribution to the well-known association between CMV infection and poor graft survival. All these basic and clinical studies paved the road to the development of a future γδ T cell-based immunotherapy. In the meantime, γδ T cell monitoring should prove a valuable immunological

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

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Kaminski, Michael M; Tosic, Jelena; Pichler, Roman; Arnold, Sebastian J; Lienkamp, Soeren S

2017-07-01

Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

Mutant tamm-horsfall glycoprotein accumulation in endoplasmic reticulum induces apoptosis reversed by colchicine and sodium 4-phenylbutyrate.

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Choi, Sung Won; Ryu, Ok Hee; Choi, Sun Jin; Song, In Sun; Bleyer, Anthony J; Hart, Thomas C

2005-10-01

As a consequence of uromodulin gene mutations, individuals develop precocious hyperuricemia, gout, and progressive renal failure. In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfall glycoprotein (THP) occurs in the endoplasmic reticulum (ER), but the pathophysiology of renal damage is unclear. It was hypothesized that programmed cell death triggered by accumulation of misfolded THP in the ER causes progressive renal disease. Stably transfected human embryonic kidney 293 cells and immortalized thick ascending limb of Henle's loop cells with wild-type and mutated uromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry, ELISA, and deglycosylation studies indicated that accumulation of mutant THP occurred in the ER. FACS analyses showed a significant increase in early apoptosis signal in human embryonic kidney 293 and thick ascending limb of Henle's loop cells that were transfected with mutant uromodulin constructs. Colchicine and sodium 4-phenylbutyrate treatment increased secretion of THP from the ER to the cell membrane and into the culture media and significantly improved cell viability. These findings indicate that intracellular accumulation of THP facilitates apoptosis and that this may provide the pathologic mechanism responsible for the progressive renal damage associated with uromodulin gene mutations. Colchicine and sodium 4-phenylbutyrate reverse these processes and could potentially be beneficial in ameliorating the progressive renal damage in uromodulin-associated kidney diseases.

Pre-transplant donor-specific T-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy.

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Elena Crespo

Full Text Available Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90, to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67. We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction

Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

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Baek N

2016-11-01

Full Text Available NamHuk Baek,1,* Ok Won Seo,1,* MinSung Kim,1 John Hulme,2 Seong Soo A An2 1Department of R & D, NanoEntek Inc., Seoul, Republic of Korea; 2Department of BioNano Technology Gachon University, Gyeonggi-do, Republic of Korea *These authors contributed equally to this work Abstract: Recently, increasing numbers of cell culture experiments with 3D spheroids presented better correlating results in vivo than traditional 2D cell culture systems. 3D spheroids could offer a simple and highly reproducible model that would exhibit many characteristics of natural tissue, such as the production of extracellular matrix. In this paper numerous cell lines were screened and selected depending on their ability to form and maintain a spherical shape. The effects of increasing concentrations of doxorubicin (DXR on the integrity and viability of the selected spheroids were then measured at regular intervals and in real-time. In total 12 cell lines, adenocarcinomic alveolar basal epithelial (A549, muscle (C2C12, prostate (DU145, testis (F9, pituitary epithelial-like (GH3, cervical cancer (HeLa, HeLa contaminant (HEp2, embryo (NIH3T3, embryo (PA317, neuroblastoma (SH-SY5Y, osteosarcoma U2OS, and embryonic kidney cells (293T, were screened. Out of the 12, 8 cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U2OS formed regular spheroids and the effects of DXR on these structures were measured at regular intervals. Finally, 5 cell lines, A549, HeLa, SH-SY5Y, U2OS, and 293T, were selected for real-time monitoring and the effects of DXR treatment on their behavior were continuously recorded for 5 days. A potential correlation regarding the effects of DXR on spheroid viability and ATP production was measured on days 1, 3, and 5. Cytotoxicity of DXR seemed to occur after endocytosis, since the cellular activities and ATP productions were still viable after 1 day of the treatment in all spheroids, except SH-SY5Y. Both cellular activity and ATP production were

Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec.

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Lister, James A; Lane, Brandon M; Nguyen, Anhthu; Lunney, Katherine

2011-11-01

The MiT family comprises four genes in mammals: Mitf, Tfe3, Tfeb, and Tfec, which encode transcription factors of the basic-helix-loop-helix/leucine zipper class. Mitf is well-known for its essential role in the development of melanocytes, however the functions of the other members of this family, and of interactions between them, are less well understood. We have now characterized the complete set of MiT genes from zebrafish, which totals six instead of four. The zebrafish genome contain two mitf (mitfa and mitfb), two tfe3 (tfe3a and tfe3b), and single tfeb and tfec genes; this distribution is shared with other teleosts. We present here the sequence and embryonic expression patterns for the zebrafish tfe3b, tfeb, and tfec genes, and identify a new isoform of tfe3a. These findings will assist in elucidating the roles of the MiT gene family over the course of vertebrate evolution. Copyright © 2011 Wiley-Liss, Inc.

Oncogenic roles of TOPK and MELK, and effective growth suppression by small molecular inhibitors in kidney cancer cells.

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Kato, Taigo; Inoue, Hiroyuki; Imoto, Seiya; Tamada, Yoshinori; Miyamoto, Takashi; Matsuo, Yo; Nakamura, Yusuke; Park, Jae-Hyun

2016-04-05

T-lymphokine-activated killer cell-originated protein kinase (TOPK) and maternal embryonic leucine zipper kinase (MELK) have been reported to play critical roles in cancer cell proliferation and maintenance of stemness. In this study, we investigated possible roles of TOPK and MELK in kidney cancer cells and found their growth promotive effect as well as some feedback mechanism between these two molecules. Interestingly, the blockade of either of these two kinases effectively caused downregulation of forkhead box protein M1 (FOXM1) activity which is known as an oncogenic transcriptional factor in various types of cancer cells. Small molecular compound inhibitors against TOPK (OTS514) and MELK (OTS167) effectively suppressed the kidney cancer cell growth, and the combination of these two compounds additively worked and showed the very strong growth suppressive effect on kidney cancer cells. Collectively, our results suggest that both TOPK and MELK are promising molecular targets for kidney cancer treatment and that dual blockade of OTS514 and OTS167 may bring additive anti-tumor effects with low risk of side effects.

IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

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Karthikeyan, Chandrabose; Lee, Crystal; Moore, Joshua; Mittal, Roopali; Suswam, Esther A; Abbott, Kodye L; Pondugula, Satyanarayana R; Manne, Upender; Narayanan, Narayanan K; Trivedi, Piyush; Tiwari, Amit K

2015-02-01

Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

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Hardman, P.; Klement, B. J.; Spooner, B. S.

1993-01-01

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.

MRI diagnosis of embryonal tumors in the spinal canal

International Nuclear Information System (INIS)

Sun Jilin; Zhang Xinchuan; Zhang Huaning; Liu Lianxiang; Wu Yujin

1997-01-01

To evaluate MRI diagnostic value of the embryonal tumors in the spinal canal. Materials and methods: The MRI appearances of 15 cases of histologically confirmed embryonal tumors in the spinal canal were analyzed. (1) Lipoma (3 cases) had characteristic MRI appearance, demonstrating high signal intensity on T 1 WI, and moderately high signal on T 2 WI. High signal intensity of the lipoma was turned into low signal intensity by fat suppression technique. (2) Dermoids (2 cases) and epidermoid (7 cases) exhibiting low or iso-low signal on T 1 WI and high or iso-high signal on T 2 WI. All had an iso-intense capsule on T 1 WI. However, the two tumors could not be distinguished from each other. (3) Teratoma (3 cases) appeared as a mass of inhomo-generous signals in the spinal canal including soft tissue, fatty tissue and calcification within the same tumor. The diagnosis of embryonal tumors in the spinal canal mainly depend on their MRI appearances, specific tumor location and patient's age

36 CFR 293.11 - Water rights.

Science.gov (United States)

2010-07-01

... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Water rights. 293.11 Section...-PRIMITIVE AREAS § 293.11 Water rights. Nothing in the regulations in this part constitutes an expressed or implied claim or denial on the part of the Department of Agriculture as to exemption from State water laws. ...

Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development

OpenAIRE

Parent, Audrey V.; Russ, Holger A.; Khan, Imran S.; LaFlam, Taylor N.; Metzger, Todd C.; Anderson, Mark S.; Hebrok, Matthias

2013-01-01

Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into th...

36 CFR 293.10 - Jurisdiction over wildlife and fish.

Science.gov (United States)

2010-07-01

... and fish. 293.10 Section 293.10 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE WILDERNESS-PRIMITIVE AREAS § 293.10 Jurisdiction over wildlife and fish. Nothing in the... States with respect to wildlife and fish in the National Forests. ...

Kidney Cancer

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... kind of kidney cancer called Wilms' tumor. The incidence of kidney cancer seems to be increasing. One ... doesn't go away Loss of appetite Unexplained weight loss Tiredness Fever, which usually comes and goes ( ...

Kidney Failure

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Healthy kidneys clean your blood by removing excess fluid, minerals, and wastes. They also make hormones that keep your ... strong and your blood healthy. But if the kidneys are damaged, they don't work properly. Harmful ...

Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

DEFF Research Database (Denmark)

Molina, Henrik; Horn, David M; Tang, Ning

2007-01-01

Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total...... of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 ( approximately 80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more...... fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single...

PTBP1 is required for embryonic development before gastrulation.

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Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

2011-02-17

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

36 CFR 293.7 - Grazing of livestock.

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2010-07-01

... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Grazing of livestock. 293.7...-PRIMITIVE AREAS § 293.7 Grazing of livestock. (a) The grazing of livestock, where such use was established..., shall be permitted to continue under the general regulations covering grazing of livestock on the...

Alterations of the intracellular peptidome in response to the proteasome inhibitor bortezomib.

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Julia S Gelman

Full Text Available Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours, and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.

Effects of the β1 auxiliary subunit on modification of Rat Na{sub v}1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin

Energy Technology Data Exchange (ETDEWEB)

He, Bingjun [College of Life Sciences, Nankai University, Tianjin 300071 (China); Soderlund, David M., E-mail: dms6@cornell.edu [Department of Entomology, Cornell University, Geneva, NY 14456 (United States)

2016-01-15

We expressed rat Na{sub v}1.6 sodium channels with or without the rat β1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Na{sub v}1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~ 18 mV for tefluthrin and ~ 24 mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~ 10–14 mV in the voltage dependence of steady-state inactivation and increased in the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Na{sub v}1.6 with the β1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the β1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons. - Highlights: • We expressed Na{sub v}1.6 sodium channels with or without β1 subunits in HEK293 cells. • Tefluthrin and deltamethrin

Interkinetic nuclear migration in the mouse embryonic ureteric epithelium: Possible implication for congenital anomalies of the kidney and urinary tract.

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Motoya, Tomoyuki; Ogawa, Noriko; Nitta, Tetsuya; Rafiq, Ashiq Mahmood; Jahan, Esrat; Furuya, Motohide; Matsumoto, Akihiro; Udagawa, Jun; Otani, Hiroki

2016-05-01

Interkinetic nuclear migration (INM) is a phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium, which is characterized by the presence of apical primary cilia, in synchrony with the cell cycle in a manner of apical mitosis. INM is suggested to regulate not only stem/progenitor cell proliferation/differentiation but also organ size and shape. INM has been reported in epithelia of both ectoderm and endoderm origin. We examined whether INM exists in the mesoderm-derived ureteric epithelium. At embryonic day (E) 11.5, E12.5 and E13.5, C57BL/6J mouse dams were injected with 5-bromo-2'-deoxyuridine (BrdU) and embryos were killed 1, 2, 4, 6, 8, 10 and 12 h later. We immunostained transverse sections of the ureter for BrdU, and measured the position of BrdU (+) nuclei in the ureteric epithelia along the apico-basal axis at each time point. We analyzed the distribution patterns of BrdU (+) nuclei in histograms using the multidimensional scaling. Changes in the nucleus distribution patterns suggested nucleus movement characteristic of INM in the ureteric epithelia, and the mode of INM varied throughout the ureter development. While apical primary cilia are related with INM by providing a centrosome for the apical mitosis, congenital anomalies of the kidney and urinary tract (CAKUT) include syndromes linked to primary ciliary dysfunction affecting epithelial tubular organs such as kidney, ureter, and brain. The present study showed that INM exists in the ureteric epithelium and suggests that INM may be related with the CAKUT etiology via primary ciliary protein function. © 2015 Japanese Teratology Society.

Magnetic resonance imaging of the transplanted kidneys

International Nuclear Information System (INIS)

Matsui, Suguru; Lee, Chol-Joo; Hamashima, Takashi

1987-01-01

Magnetic resonance imaging (MRI) is a new noninvasive means for evaluating pathological changes of kidney transplants. Thirty kidney transplants were examined by MRI study, comparing with 12 donor kidneys as control. Imaging of well functioning grafts using inversion recovery (IR) method displayed a clear figure of corticomedullary differentiation (CMD). Kidneys under acute rejection, chronic rejection, and ciclosporin nephrotoxicity displayed poor CMD. CMD of Kidneys under ATN was poor on IR imaging, but clear on T 1 weightened imaging. T 1 values of kidney grafts were obtained as the mean value of T 1 relaxation time of three areas including upper pole, lower pole, and the middle of the cortex. T 1 value of the grafts under chronic rejection was similar to that of well functioning grafts. The value increased in case of acute rejection, ATN, and ciclosporin nephrotoxicity and decreased as the graft function was getting better. Imaging and the estimation of T 1 value of kidney transplants of MRI were effective for evaluating graft function but of no use for differentiation of causes of graft deterioration. (author)

Localization of Mg2+-sensing shark kidney calcium receptor SKCaR in kidney of spiny dogfish, Squalus acanthias.

Science.gov (United States)

Hentschel, Hartmut; Nearing, Jacqueline; Harris, H William; Betka, Marlies; Baum, Michelle; Hebert, Steven C; Elger, Marlies

2003-09-01

We recently cloned a homologue of the bovine parathyroid calcium receptor from the kidney of a spiny dogfish (Squalus acanthias) and termed this new protein SKCaR. SKCaR senses alterations in extracellular Mg2+ after its expression in human embryonic kidney cells (Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M, Bai M, Chattopadyhay N, Brown E, Hebert S, and Harris HW. Proc Natl Acad. Sci USA 99: 9231-9236, 2002). In this report, we used light and electron microscopic immunocytochemical techniques to study the distribution of SKCaR in dogfish kidney. SKCaR antiserum bound to the apical membranes of shark kidney epithelial cells in the following tubular segments: proximal tubules (PIa and PIIb), late distal tubule, and collecting tubule/collecting duct as well as diffusely labeled cells of early distal tubule. The highly specific distribution of SKCaR in mesial tissue as well as lateral countercurrent bundles of dogfish kidney is compatible with a role for SKCaR to sense local tubular Mg2+ concentrations. This highly specific distribution of SKCaR protein in dogfish kidney could possibly work in concert with the powerful Mg2+ secretory system present in the PIIa segment of elasmobranch fish kidney to affect recycling of Mg2+ from putative Mg2+-sensing/Mg2+-reabsorbing segments. These data provide support for the possible existence of Mg2+ cycling in elasmobranch kidney in a manner analogous to that described for mammals.

Characterization of the Antioxidant Effects of γ-Oryzanol: Involvement of the Nrf2 Pathway

OpenAIRE

W. Rungratanawanich; G. Abate; M. M. Serafini; M. Guarienti; M. Catanzaro; M. Marziano; M. Memo; C. Lanni; D. Uberti

2018-01-01

γ-Oryzanol (ORY) is well known for its antioxidant potential. However, the mechanism by which ORY exerts its antioxidant effect is still unclear. In this paper, the antioxidant properties of ORY were investigated for its potential effects as a reactive oxygen and nitrogen species (ROS/RNS) scavenger and in activating antioxidant-promoting intracellular pathways utilizing the human embryonic kidney cells (HEK-293). The 24 h ORY exposure significantly prevented hydrogen peroxide- (H2O2-) induce...

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

Science.gov (United States)

Brast, Sabine; Grabner, Alexander; Sucic, Sonja; Sitte, Harald H; Hermann, Edwin; Pavenstädt, Hermann; Schlatter, Eberhard; Ciarimboli, Giuliano

2012-03-01

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.

Spontaneous focal activation of invariant natural killer T (iNKT cells in mouse liver and kidney

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Zeng Jia

2010-11-01

Full Text Available Abstract Background Invariant natural killer T (iNKT cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported. Results We used an interferon (IFN-γ-inducible cytoplasmic protein, Irga6, as a histological marker for local IFN-γ production. Irga6 was intensely expressed in small foci of liver parenchymal cells and kidney tubular epithelium. Focal Irga6 expression was unaffected by germ-free status or loss of TLR signalling and was totally dependent on IFN-γ secreted by T cells in the centres of expression foci. These were shown to be iNKT cells by diagnostic T cell receptor usage and their activity was lost in both CD1 d and Jα-deficient mice. Conclusions This is the first report that supplies direct evidence for explicit activation events of NKT cells in vivo and raises issues about the triggering mechanism and consequences for immune functions in liver and kidney.

Viscosities of binary mixtures of some n-ethoxyethanols with ethyl tert-butyl ether at T = (293.15, 298.15, and 303.15) K

International Nuclear Information System (INIS)

Cwiklinska, Aneta; Dzikowski, Tomasz; Szychowski, Dariusz; Kinart, Wojciech J.; Kinart, Cezary M.

2007-01-01

Viscosities at T = (293.15, 298.15, and 303.15) K in the binary mixtures of ethyl tert-butyl ether with 2-ethoxyethanol, 2-(2-ethoxyethoxy)ethanol, and 2-[2-(2-ethoxyethoxy)ethoxy]ethanol have been measured over the entire range of mixture compositions. From the experimental data, deviations in the viscosity (Δln η) and excess energies of activation for viscous flow (ΔG *E ) have been calculated. The viscosity data were correlated with equations of Hind et al., Grunberg and Nissan, Auslaender, and McAllister. The results for Δln η and ΔG *E are discussed in terms of intermolecular interactions and structure of studied binary mixtures

Integrated Kidney Exosome Analysis for the Detection of Kidney Transplant Rejection.

Science.gov (United States)

Park, Jongmin; Lin, Hsing-Ying; Assaker, Jean Pierre; Jeong, Sangmoo; Huang, Chen-Han; Kurdi, A; Lee, Kyungheon; Fraser, Kyle; Min, Changwook; Eskandari, Siawosh; Routray, Sujit; Tannous, Bakhos; Abdi, Reza; Riella, Leonardo; Chandraker, Anil; Castro, Cesar M; Weissleder, Ralph; Lee, Hakho; Azzi, Jamil R

2017-11-28

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.

Hedgehog Signalling in the Embryonic Mouse Thymus

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Alessandro Barbarulo

2016-07-01

Full Text Available T cells develop in the thymus, which provides an essential environment for T cell fate specification, and for the differentiation of multipotent progenitor cells into major histocompatibility complex (MHC-restricted, non-autoreactive T cells. Here we review the role of the Hedgehog signalling pathway in T cell development, thymic epithelial cell (TEC development, and thymocyte–TEC cross-talk in the embryonic mouse thymus during the last week of gestation.

Screening Fabry's disease in chronic kidney disease patients not on dialysis: a multicenter study.

Science.gov (United States)

Yeniçerioğlu, Yavuz; Akdam, Hakan; Dursun, Belda; Alp, Alper; Sağlam Eyiler, Funda; Akın, Davut; Gün, Yelda; Hüddam, Bülent; Batmazoğlu, Mehmet; Gibyeli Genek, Dilek; Pirinççi, Serhat; Ersoy, İsmail Rıfkı; Üzüm, Atilla; Soypaçacı, Zeki; Tanrısev, Mehmet; Çolak, Hülya; Demiral Sezer, Sibel; Bozkurt, Gökay; Akyıldız, Utku Oğan; Akyüz Ünsal, Ayşe İpek; Ünübol, Mustafa; Uslu, Meltem; Eryılmaz, Ufuk; Günel, Ceren; Meteoğlu, İbrahim; Yavaşoğlu, İrfan; Ünsal, Alparslan; Akar, Harun; Okyay, Pınar

2017-11-01

Fabry's disease is an X-linked inherited, rare, progressive, lysosomal storage disorder, affecting multiple organs due to the deficient activity of α-galactosidase A (α-Gal A) enzyme. The prevalence has been reported to be 0.15-1% in hemodialysis patients; however, the information on the prevalence in chronic kidney disease not on dialysis is lacking. This study aimed to determine the prevalence of Fabry's disease in chronic kidney disease. The patients older than 18 years, enclosing KDIGO 2012 chronic kidney disease definitions, not on dialysis, were enrolled. Dried blood spots on Guthrie papers were used to analyze α-Gal A enzyme and genetic analysis was performed in individuals with enzyme activity ≤1.2 μmol/L/h. A total of 1453 chronic kidney disease patients not on dialysis from seven clinics in Turkey were screened. The mean age of the study population was 59.3 ± 15.9 years. 45.6% of patients were female. The creatinine clearance of 77.3% of patients was below 60 mL/min/1.73 m 2 , 8.4% had proteinuria, and 2.5% had isolated microscopic hematuria. The mean value of patients' α-Gal A enzyme was detected as 2.93 ± 1.92 μmol/L/h. 152 patients had low levels of α-Gal A enzyme activity (≤1.2 μmol/L/h). In mutation analysis, A143T and D313Y variants were disclosed in three male patients. The prevalence of Fabry's disease in chronic kidney disease not on dialysis was found to be 0.2% (0.4% in male, 0.0% in female). Fabry's disease should be considered in the differential diagnosis of chronic kidney disease with unknown etiology even in the absence of symptoms and signs suggestive of Fabry's disease.

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

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The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

Thermophysical and sonochemical behaviour of binary mixtures of decan-1-ol with halohydrocarbons at (T = 293.15 and 313.15) K

International Nuclear Information System (INIS)

Bhatia, Subhash C.; Bhatia, Rachna; Dubey, Gyan P.

2010-01-01

Densities and ultrasonic velocities of binary mixtures of decan-1-ol with 1,2-dichloroethane, 1,2-dibromoethane, and 1,1,2,2-tetrachloroethene have been measured over the entire range of composition at T = (293.15 and 313.15) K and at atmospheric pressure. From these results, the excess molar volumes, molar free volumes, excess molar isentropic compressibilities, limiting excess partial molar volumes, and isentropic compressibilities, intermolecular free lengths, and available volumes by three methods, thermal expansion coefficients, parameters related to space-filling ability, intermolecular free lengths, and molecular radii have been calculated. The experimental ultrasonic velocities have been analyzed in terms of the ideal mixture relations of Nomoto and Van Dael, Jacobson's free length, Schaaff's collision factor, Flory's statistical, and Prigogine-Flory-Patterson theories and thermoacoustical parameters.

Rhabdomyosarcoma of the kidney

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Alaa Samkari

2018-05-01

Full Text Available Rhabdomyosarcoma is considered the most common soft tissue sarcoma arising in patients younger than 15 years old, accounting for 5%–10% of childhood solid tumors. Sarcoma of the kidney represents 1% of all primary renal malignancies. Primary renal rhabdomyosarcoma is a very rare entity with limited number of cases reported in the literature. In this paper we present two cases of primary renal rhabdomyosarcoma in pediatric patients. The two tumors involved the renal parenchyma and occurred in 2-year-old girl and 6-year-old boy, respectively. Histopathology examination and immunohistochemistry studies confirm the diagnosis of embryonal rhabdomyosarcoma with pleomorphic component, and pleomorphic rhabdomyosarcoma, respectively. Both cases are treated with chemotherapy and show a good response with no evidence of recurrence or metastasis. The aim of this paper is to expand the differential diagnosis of primary mesenchymal kidney tumors in pediatric age group. Keywords: Rhabdomyosarcoma, Renal neoplasm, Pediatric, Oncology

Multi-modality imaging review of congenital abnormalities of kidney and upper urinary tract.

Science.gov (United States)

Ramanathan, Subramaniyan; Kumar, Devendra; Khanna, Maneesh; Al Heidous, Mahmoud; Sheikh, Adnan; Virmani, Vivek; Palaniappan, Yegu

2016-02-28

Congenital abnormalities of the kidney and urinary tract (CAKUT) include a wide range of abnormalities ranging from asymptomatic ectopic kidneys to life threatening renal agenesis (bilateral). Many of them are detected in the antenatal or immediate postnatal with a significant proportion identified in the adult population with varying degree of severity. CAKUT can be classified on embryological basis in to abnormalities in the renal parenchymal development, aberrant embryonic migration and abnormalities of the collecting system. Renal parenchymal abnormalities include multi cystic dysplastic kidneys, renal hypoplasia, number (agenesis or supernumerary), shape and cystic renal diseases. Aberrant embryonic migration encompasses abnormal location and fusion anomalies. Collecting system abnormalities include duplex kidneys and Pelvi ureteric junction obstruction. Ultrasonography (US) is typically the first imaging performed as it is easily available, non-invasive and radiation free used both antenatally and postnatally. Computed tomography (CT) and magnetic resonance imaging (MRI) are useful to confirm the ultrasound detected abnormality, detection of complex malformations, demonstration of collecting system and vascular anatomy and more importantly for early detection of complications like renal calculi, infection and malignancies. As CAKUT are one of the leading causes of end stage renal disease, it is important for the radiologists to be familiar with the varying imaging appearances of CAKUT on US, CT and MRI, thereby helping in prompt diagnosis and optimal management.

NFAT5 is activated by hypoxia: role in ischemia and reperfusion in the rat kidney.

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Sandra Villanueva

Full Text Available The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10-18 mmHg; however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO(2 on NFAT5 activity. We found that 1 Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2 Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3 The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O(2. 4 At 2.5% of O(2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5 siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6 siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7 NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla from rats subjected to an experimental model of ischemia and reperfusion (I/R. 7 Experimental I/R increased the NFAT5-target gene aldose reductase (AR. 8 NFAT5 activators (ATM and PI3K were induced in vitro (HEK293 cells and in vivo (I/R kidneys with the same timing of NFAT5. 8 Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage.

Action of bicyclic isoxazole GABA analogues on GABA transporters and its relation to anticonvulsant activity

DEFF Research Database (Denmark)

Bolvig, T; Larsson, O M; Pickering, D S

1999-01-01

The inhibitory action of bicyclic isoxazole gamma-aminobutyric acid (GABA) analogues and their 4,4-diphenyl-3-butenyl (DPB) substituted derivatives has been investigated in cortical neurones and astrocytes as well as in human embryonic kidney (HEK 293) cells transiently expressing either mouse GA...... anticonvulsant activity, lack of proconvulsant activity and the ability of THPO to increase extracellular GABA concentration, indicate that these bicyclic isoxazole GABA analogues and their DPB derivatives may be useful lead structures in future search for new antiepileptic drugs....

Fatigue-induced dislocation structure of titanium alloy VT5-1ct at temperatures of 293-11 K

Energy Technology Data Exchange (ETDEWEB)

Grinberg, N.M. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine)); Aleksenko, E.N. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine)); Moskalenko, V.A. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine)); Smirnov, A.R.N. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine)); Yakovenko, L.F. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine)); Mozhaev, A.V. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine)); Arinushkin, I.A. (Inst. for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Kharkov (Ukraine))

1993-07-05

The dislocation structure formed during the final stage of fatigue at high- and low-amplitude stresses at T=293 K in air and T=293, 93 and 11 K in high vacuum is studied on the Ti alloy VT5-1ct which has been prepared by two processing methods. The [sigma]-N curves are plotted for corresponding experimental conditions. It is shown that slip alone is responsible for the plastic deformation. The characteristic features of the dislocation structure formed are reported. The morphology of the a phase does not influence the character of the dislocation structure. At lower temperatures, the substructure remains practically unaltered, although the likelihood of uniformly distributed dislocations is lower. The lifetime is essentially dependent on the environment, temperature and the alloy microstructure, the latter being especially important at low temperatures in the high-amplitude region. (orig.)

Pharmacological characterization of emerging synthetic cannabinoids in HEK293T cells and hippocampal neurons.

Science.gov (United States)

Costain, Willard J; Tauskela, Joseph S; Rasquinha, Ingrid; Comas, Tanya; Hewitt, Melissa; Marleau, Vincent; Soo, Evelyn C

2016-09-05

There has been a worldwide proliferation of synthetic cannabinoids that have become marketed as legal alternatives to cannabis (marijuana). Unfortunately, there is a dearth of information about the pharmacological effects of many of these emerging synthetic cannabinoids (ESCs), which presents a challenge for regulatory authorities that need to take such scientific evidence into consideration in order to regulate ECSs as controlled substances. We aimed to characterize the pharmacological properties of ten ESCs using two cell based assays that enabled the determination of potency and efficacy relative to a panel of well-characterized cannabinoids. Agonist-mediated inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live HEK293T cells transfected with human cannabinoid receptor 1 gene (CNR1) and pGloSensor-22F. Pharmacological analysis of this data indicated that all of the ESCs tested were full agonists, with the following rank order of potency: Win 55212-2≈5F-PB-22≈AB-PINACA≈EAM-2201≈MAM-2201>JWH-250≈ PB-22>AKB48 N-(5FP)>AKB-48≈STS-135>XLR-11. Assessment of agonist-stimulated depression of Ca(2+) transients was also used to confirm the efficacy of five ESCs (XLR-11, JWH-250, AB-PINACA, 5F-PB-22, and MAM-2201) in cultured primary hippocampal neurons. This work aims to help inform decisions made by regulatory agencies concerned with the profusion of these poorly characterized recreational drugs. Copyright © 2016. Published by Elsevier B.V.

Screening of plant extracts for human tyrosinase inhibiting effects.

Science.gov (United States)

Kim, M; Park, J; Song, K; Kim, H G; Koh, J-S; Boo, Y C

2012-04-01

Screening for tyrosinase (TYR) inhibitors potentially useful for control of skin pigmentation has been hampered by the limited availability of human TYR. To overcome this hurdle, we have established human embryonic kidney (HEK293)-TYR cells that constitutively express human TYR. In the current study, we assayed human TYR inhibition activities of 50 plant extracts using the lysates of transformed HEK293-TYR cells. The strongest inhibition of human TYR was shown by the extract of Vaccinium bracteatum Thunberg, followed by the extract of Morus bombycis Koidzumi. The former extract did not inhibit mushroom TYR activity whereas significant inhibition was observed with the latter extract, demonstrating the importance of using human TYR in the screening for human TYR inhibitors. Upon liquid-liquid partitioning of the extract from V. bracteatum, the active constituents were enriched in the ethyl acetate fraction, and the subsequent preparatory thin-layer chromatography identified p-coumaric acid (PCA) as the main active constituent. The hypo-pigmentation of PCA was verified in the MelanoDerm™ Skin Model. This study demonstrates that transformed HEK293-TYR cells could expedite the discovery of human TYR-specific inhibitors from natural sources which might be useful in the control of skin pigmentation. © 2012 The Authors. ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

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Duangtum, Natapol [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Junking, Mutita; Sawasdee, Nunghathai [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Cheunsuchon, Boonyarit [Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai, E-mail: limjindaporn@yahoo.com [Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

2011-09-16

Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

International Nuclear Information System (INIS)

Duangtum, Natapol; Junking, Mutita; Sawasdee, Nunghathai; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2011-01-01

Highlights: → Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). → The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. → The co-localization between kAE and KIF3B was detected in human kidney tissues. → A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. → KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl - /HCO 3 - exchange and the failure of proton (H + ) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.

Cell Penetrating Polymers Containing Guanidinium Trigger Apoptosis in Human Hepatocellular Carcinoma Cells unless Conjugated to a Targeting N-Acetyl-Galactosamine Block.

Science.gov (United States)

Tan, Zhe; Dhande, Yogesh K; Reineke, Theresa M

2017-12-20

A series of 3-guanidinopropyl methacrylamide (GPMA)-based polymeric gene delivery vehicles were developed via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers have been evaluated for their cellular internalization ability, transfection efficiency, and cytotoxicity. Two homopolymers: P(GPMA 20 ), P(GPMA 34 ), were synthesized to study the effect of guanidium polymer length on delivery efficiency and toxicity. In addition, an N-acetyl-d-galactosamine (GalNAc)-based hydrophilic block was incorporated to produce diblock polymers, which provides a neutral hydrophilic block that sterically protects plasmid-polymer complexes (polyplexes) from colloidal aggregation and aids polyplex targeting to hepatocytes via binding to asialoglycoprotein receptors (ASGPRs). Polyplexes formed with P(GPMA x ) (x = 20, 34) homopolymers were shown to be internalized via both energy-dependent and independent pathways, whereas polyplexes formed with block polymers were internalized through endocytosis. Notably, P(GPMA x ) polyplexes enter cells very efficiently but are also very toxic to human hepatocellular carcinoma (HepG2) cells and triggered cell apoptosis. In comparison, the presence of a carbohydrate block in the polymer structures reduced the cytotoxicity of the polyplex formulations and increased gene delivery efficiency with HepG2 cells. Transfection efficiency and toxicity studies were also carried out with HEK 293T (human embryonic kidney) cells for comparison. Results showed that polyplexes formed with the P(GPMA x ) homopolymers exhibit much higher transfection efficiency and lower toxicity with HEK 293T cells. The presence of the carbohydrate block did not further increase transfection efficiency in comparison to the homopolymers with HEK 293T cells, likely due to the lack of ASGPRs on the HEK 293T cell line. This study revealed that although guanidinium-based polymers have high membrane permeability, their application as plasmid

In vitro regeneration of kidney from pluripotent stem cells

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Osafune, Kenji, E-mail: osafu@cira.kyoto-u.ac.jp [Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507 (Japan); PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); JST Yamanaka iPS Cell Special Project, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan)

2010-10-01

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.

In vitro regeneration of kidney from pluripotent stem cells

International Nuclear Information System (INIS)

Osafune, Kenji

2010-01-01

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.

Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3: Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

Directory of Open Access Journals (Sweden)

Mathis Kopp

Full Text Available Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE. Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1 and lysosomes (shown by the marker Lamp1. There, it was still intact and functional (i.e. properly folded as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic effect of a protein inside a cell.

Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

Science.gov (United States)

Kopp, Mathis; Rotan, Olga; Papadopoulos, Chrisovalantis; Schulze, Nina; Meyer, Hemmo; Epple, Matthias

2017-01-01

Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

Modeling Kidney Disease with iPS Cells

Science.gov (United States)

Freedman, Benjamin S.

2015-01-01

Induced pluripotent stem cells (iPSCs) are somatic cells that have been transcriptionally reprogrammed to an embryonic stem cell (ESC)-like state. iPSCs are a renewable source of diverse somatic cell types and tissues matching the original patient, including nephron-like kidney organoids. iPSCs have been derived representing several kidney disorders, such as ADPKD, ARPKD, Alport syndrome, and lupus nephritis, with the goals of generating replacement tissue and ‘disease in a dish’ laboratory models. Cellular defects in iPSCs and derived kidney organoids provide functional, personalized biomarkers, which can be correlated with genetic and clinical information. In proof of principle, disease-specific phenotypes have been described in iPSCs and ESCs with mutations linked to polycystic kidney disease or focal segmental glomerulosclerosis. In addition, these cells can be used to model nephrotoxic chemical injury. Recent advances in directed differentiation and CRISPR genome editing enable more specific iPSC models and present new possibilities for diagnostics, disease modeling, therapeutic screens, and tissue regeneration using human cells. This review outlines growth opportunities and design strategies for this rapidly expanding and evolving field. PMID:26740740

INTEGRAL reports renewed activity from KS 1741-293

DEFF Research Database (Denmark)

Chenevez, Jérôme; Kuulkers, E.; Alfonso-Garzón, J.

2010-01-01

The low-mass X-ray binary and burster source KS 1741-293 has been detected during recent INTEGRAL Galactic bulge (see ATel #438) observations by the JEM-X instrument. On February 25, 2010, between UTC 13:04 and 14:08, KS 1741-293 was detected at a 3-10 keV flux of 9 +/- 4 mCrab, and an upper limi...

Transgenic Xenopus laevis Line for In Vivo Labeling of Nephrons within the Kidney

Directory of Open Access Journals (Sweden)

Mark E. Corkins

2018-04-01

Full Text Available Xenopus laevis embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between Xenopus and humans, allowing for the study of human disease-causing genes. Xenopus embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this cdh17:eGFP line, showing green fluorescent protein (GFP expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of cdh17:eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes.

Rapid Fabrication of Cell-Laden Alginate Hydrogel 3D Structures by Micro Dip-Coating.

Science.gov (United States)

Ghanizadeh Tabriz, Atabak; Mills, Christopher G; Mullins, John J; Davies, Jamie A; Shu, Wenmiao

2017-01-01

Development of a simple, straightforward 3D fabrication method to culture cells in 3D, without relying on any complex fabrication methods, remains a challenge. In this paper, we describe a new technique that allows fabrication of scalable 3D cell-laden hydrogel structures easily, without complex machinery: the technique can be done using only apparatus already available in a typical cell biology laboratory. The fabrication method involves micro dip-coating of cell-laden hydrogels covering the surface of a metal bar, into the cross-linking reagents calcium chloride or barium chloride to form hollow tubular structures. This method can be used to form single layers with thickness ranging from 126 to 220 µm or multilayered tubular structures. This fabrication method uses alginate hydrogel as the primary biomaterial and a secondary biomaterial can be added depending on the desired application. We demonstrate the feasibility of this method, with survival rate over 75% immediately after fabrication and normal responsiveness of cells within these tubular structures using mouse dermal embryonic fibroblast cells and human embryonic kidney 293 cells containing a tetracycline-responsive, red fluorescent protein (tHEK cells).

Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293.

Science.gov (United States)

Kanhayuwa, Lakkhana; Coutts, Robert H A

2016-01-01

Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4-14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140-493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3'-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50-65% and 60-75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259-343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.

Antiproliferative Effects of Bacillus coagulans Unique IS2 in Colon Cancer Cells.

Science.gov (United States)

Madempudi, Ratna Sudha; Kalle, Arunasree M

2017-10-01

In the present study, the in vitro anticancer (antiproliferative) effects of Bacillus coagulans Unique IS2 were evaluated on human colon cancer (COLO 205), cervical cancer (HeLa), and chronic myeloid leukemia (K562) cell lines with a human embryonic kidney cell line (HEK 293T) as noncancerous control cells. The Cytotoxicity assay (MTT) clearly demonstrated a 22%, 31.7%, and 19.5% decrease in cell proliferation of COLO 205, HeLa, and K562 cells, respectively, when compared to the noncancerous HEK 293T cells. Normal phase-contrast microscopic images clearly suggested that the mechanism of cell death is by apoptosis. To further confirm the induction of apoptosis by Unique IS2, the sub-G0-G1 peak of the cell cycle was quantified using a flow cytometer and the data indicated 40% of the apoptotic cells in Unique IS2-treated COLO cells when compared with their untreated control cells. The Western blot analysis showed an increase in pro-apoptotic protein BAX, decrease in antiapoptotic protein, Bcl2, decrease in mitochondrial membrane potential, increase in cytochrome c release, increase in Caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. The present study suggests that the heat-killed culture supernatant of B. coagulans can be more effective in inducing apoptosis of colon cancer cells and that can be considered for adjuvant therapy in the treatment of colon carcinoma.

CD4 T cell knockout does not protect against kidney injury and worsens cancer.

Science.gov (United States)

Ravichandran, Kameswaran; Wang, Qian; Ozkok, Abdullah; Jani, Alkesh; Li, Howard; He, Zhibin; Ljubanovic, Danica; Weiser-Evans, Mary C; Nemenoff, Raphael A; Edelstein, Charles L

2016-04-01

Most previous studies of cisplatin-induced acute kidney injury (AKI) have been in models of acute, high-dose cisplatin administration that leads to mortality in non-tumor-bearing mice. The aim of the study was to determine whether CD4 T cell knockout protects against AKI and cancer in a clinically relevant model of low-dose cisplatin-induced AKI in mice with cancer. Kidney function, serum neutrophil gelatinase-associated lipocalin (NGAL), acute tubular necrosis (ATN), and tubular apoptosis score were the same in wild-type and CD4 -/- mice with AKI. The lack of protection against AKI in CD4 -/- mice was associated with an increase in extracellular signal-regulated kinase (ERK), p38, CXCL1, and TNF-α, mediators of AKI and fibrosis, in both cisplatin-treated CD4 -/- mice and wild-type mice. The lack of protection was independent of the presence of cancer or not. Tumor size was double, and cisplatin had an impaired therapeutic effect on the tumors in CD4 -/- vs. wild-type mice. Mice depleted of CD4 T cells using the GK1.5 antibody were not protected against AKI and had larger tumors and lesser response to cisplatin. In summary, in a clinically relevant model of cisplatin-induced AKI in mice with cancer, (1) CD4 -/- mice were not protected against AKI; (2) ERK, p38, CXCL1, and TNF-α, known mediators of AKI, and interstitial fibrosis were increased in CD4 -/- kidneys; and (3) CD4 -/- mice had faster tumor growth and an impaired therapeutic effect of cisplatin on the tumors. The data warns against the use of CD4 T cell inhibition to attenuate cisplatin-induced AKI in patients with cancer. A clinically relevant low-dose cisplatin model of AKI in mice with cancer was used. CD4 -/- mice were not functionally or histologically protected against AKI. CD4 -/- mice had faster tumor growth. CD4 -/- mice had an impaired therapeutic effect of cisplatin on the tumors. Mice depleted of CD4 T cells were not protected against AKI and had larger tumors.

ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

Science.gov (United States)

Jonsson, Frida; Westin, Ida Maria; Österman, Lennart; Sandgren, Ola; Burstedt, Marie; Holmberg, Monica; Golovleva, Irina

2018-02-20

Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable. © 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Regenerative Medicine, Disease Modelling, and Drug Discovery in Human Pluripotent Stem Cell-Derived Kidney Tissue

Directory of Open Access Journals (Sweden)

Navin Gupta

2017-08-01

Full Text Available The multitude of research clarifying critical factors in embryonic organ development has been instrumental in human stem cell research. Mammalian organogenesis serves as the archetype for directed differentiation protocols, subdividing the process into a series of distinct intermediate stages that can be chemically induced and monitored for the expression of stage-specific markers. Significant advances over the past few years include established directed differentiation protocols of human embryonic stem cells and human induced pluripotent stem cells (hiPSC into human kidney organoids in vitro. Human kidney tissue in vitro simulates the in vivo response when subjected to nephrotoxins, providing a novel screening platform during drug discovery to facilitate identification of lead candidates, reduce developmental expenditures, and reduce future rates of drug-induced acute kidney injury. Patient-derived hiPSC, which bear naturally occurring DNA mutations, may allow for modelling of human genetic diseases to enable determination of pathological mechanisms and screening for novel therapeutics. In addition, recent advances in genome editing with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 enable the generation of specific mutations to study genetic disease, with non-mutated lines serving as an ideal isogenic control. The growing population of patients with end-stage kidney disease is a worldwide healthcare problem, with high morbidity and mortality rates, that warrants the discovery of novel forms of renal replacement therapy. Coupling the outlined advances in hiPSC research with innovative bioengineering techniques, such as decellularised kidney and three-dimensional printed scaffolds, may contribute to the development of bioengineered transplantable human kidney tissue as a means of renal replacement therapy.

Conserved and Divergent Features of Human and Mouse Kidney Organogenesis.

Science.gov (United States)

Lindström, Nils O; McMahon, Jill A; Guo, Jinjin; Tran, Tracy; Guo, Qiuyu; Rutledge, Elisabeth; Parvez, Riana K; Saribekyan, Gohar; Schuler, Robert E; Liao, Christopher; Kim, Albert D; Abdelhalim, Ahmed; Ruffins, Seth W; Thornton, Matthew E; Basking, Laurence; Grubbs, Brendan; Kesselman, Carl; McMahon, Andrew P

2018-03-01

Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species. Copyright © 2018 by the American Society of Nephrology.

Independent Activation of Hepatitis B Virus Biosynthesis by Retinoids, Peroxisome Proliferators, and Bile Acids

Science.gov (United States)

Reese, Vanessa C.; Oropeza, Claudia E.

2013-01-01

In the human hepatoma cell line HepG2, retinoic acid, clofibric acid, and bile acid treatment can only modestly increase hepatitis B virus (HBV) biosynthesis. Utilizing the human embryonic kidney cell line 293T, it was possible to demonstrate that the retinoid X receptor α (RXRα) plus its ligand can support viral biosynthesis independently of additional nuclear receptors. In addition, RXRα/peroxisome proliferator-activated receptor α (PPARα) and RXRα/farnesoid X receptor α (FXRα) heterodimeric nuclear receptors can also mediate ligand-dependent HBV transcription and replication when activated by clofibric acid and bile acid, respectively, independently of a requirement for the ligand-dependent activation of RXRα. These observations indicate that there are at least three possible modes of ligand-mediated activation of HBV transcription and replication existing within hepatocytes, suggesting that multiple independent mechanisms control viral production in the livers of infected individuals. PMID:23135717

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

DEFF Research Database (Denmark)

Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

2015-01-01

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses....... Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

In vivo sodium ({sup 23}Na) imaging of the human kidneys at 7 T: Preliminary results

Energy Technology Data Exchange (ETDEWEB)

Haneder, Stefan [University Medical Center Mannheim, Heidelberg University, Institute of Clinical Radiology and Nuclear Medicine, Mannheim (Germany); Medical University of Vienna/Vienna General Hospital, Department of Biomedical Imaging and Image-guided Therapy, Department of Radiology, Vienna (Austria); Juras, Vladimir; Trattnig, Siegfried; Zbyn, Stefan [Medical University of Vienna/Vienna General Hospital, Department of Biomedical Imaging and Image-guided Therapy, Department of Radiology, Vienna (Austria); Michaely, Henrik J.; Schoenberg, Stefan O. [University Medical Center Mannheim, Heidelberg University, Institute of Clinical Radiology and Nuclear Medicine, Mannheim (Germany); Deligianni, Xeni; Bieri, Oliver [University of Basel Hospital, Department of Radiology, Division of Radiological Physics, Basel (Switzerland)

2014-02-15

To evaluate the feasibility of in vivo {sup 23}Na imaging of the corticomedullary {sup 23}Na gradient and to measure {sup 23}Na transverse relaxation times (T2*) in human kidneys. In this prospective, IRB-approved study, eight healthy volunteers (4 female, 4 male; mean age 29.4 ± 3.6 years) were examined on a 7-T whole-body MR system using a {sup 23}Na-only spine-array coil. For morphological {sup 23}Na-MRI, a 3D gradient echo (GRE) sequence with a variable echo time scheme (vTE) was used. T2* times were calculated using a multiecho 3D vTE-GRE approach. {sup 23}Na signal-to-noise ratios (SNR) were given on a pixel-by-pixel basis for a 20-mm section from the cortex in the direction of the medulla. T2* maps were calculated by fitting the {sup 23}Na signal decay monoexponentially on a pixel-by-pixel basis, using least squares fit. Mean corticomedullary {sup 23}Na-SNR increased from the cortex (32.2 ± 5.6) towards the medulla (85.7 ± 16.0). The SNR increase ranged interindividually from 57.2 % to 66.3 %. Mean {sup 23}Na-T2* relaxation times differed statistically significantly (P < 0.001) between the cortex (17.9 ± 0.8 ms) and medulla (20.6 ± 1.0 ms). The aim of this study was to evaluate the feasibility of in vivo {sup 23}Na MRI of the corticomedullary {sup 23}Na gradient and to measure the {sup 23}Na T2* relaxation times of human kidneys at 7 T. (orig.)

Fabrication of Si-based planar type patch clamp biosensor using silicon on insulator substrate

International Nuclear Information System (INIS)

Zhang, Z.L.; Asano, T.; Uno, H.; Tero, R.; Suzui, M.; Nakao, S.; Kaito, T.; Shibasaki, K.; Tominaga, M.; Utsumi, Y.; Gao, Y.L.; Urisu, T.

2008-01-01

The aim of this paper is to fabricate the planar type patch clamp ion-channel biosensor, which is suitable for the high throughput screening, using silicon-on-insulator (SOI) substrate. The micropore with 1.2 μm diameter is formed through the top Si layer and the SiO 2 box layer of the SOI substrate by focused ion beam (FIB). Then the substrate is assembled into the microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with transient receptor potential vanilloid type 1 (TRPV1) is positioned on the micropore and the whole-cell configuration is formed by the suction. Capsaicin is added to the extracellular solution as a ligand molecule, and the channel current showing the desensitization unique to TRPV1 is measured successfully

Fabrication of Si-based planar type patch clamp biosensor using silicon on insulator substrate

Energy Technology Data Exchange (ETDEWEB)

Zhang, Z.L.; Asano, T. [Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585 (Japan); Uno, H. [Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan); Tero, R. [Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585 (Japan); Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan); Suzui, M.; Nakao, S. [Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan); Kaito, T. [SII NanoTechnology Inc., 36-1, Takenoshita, Oyama-cho, Sunto-gun, Shizuoka, 410-1393 (Japan); Shibasaki, K.; Tominaga, M. [Okazaki Institute for Integrative Bioscience, 5-1, Higashiyama, Myodaiji, Okazaki, 444-8787 (Japan); Utsumi, Y. [Laboratory of Advanced Science and Technology for Industry, University of Hyogo, 3-1-2, Koto, Kamigori, Ako-gun, Hyogo, 678-1205 (Japan); Gao, Y.L. [Department of Physics and Astronomy, Rochester University, Rochester, New York 14627 (United States); Urisu, T. [Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585 (Japan); Institute for Molecular Science, Myodaiji, Okazaki, 444-8585 (Japan)], E-mail: urisu@ims.ac.jp

2008-03-03

The aim of this paper is to fabricate the planar type patch clamp ion-channel biosensor, which is suitable for the high throughput screening, using silicon-on-insulator (SOI) substrate. The micropore with 1.2 {mu}m diameter is formed through the top Si layer and the SiO{sub 2} box layer of the SOI substrate by focused ion beam (FIB). Then the substrate is assembled into the microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with transient receptor potential vanilloid type 1 (TRPV1) is positioned on the micropore and the whole-cell configuration is formed by the suction. Capsaicin is added to the extracellular solution as a ligand molecule, and the channel current showing the desensitization unique to TRPV1 is measured successfully.

ABO-incompatible kidney transplantation

DEFF Research Database (Denmark)

Schousboe, Karoline; Titlestad, Kjell; Baudier, Francois

2010-01-01

INTRODUCTION: Kidney transplantation is the optimal treatment for many patients with end-stage renal disease (ESRD). Due to shortage of donor kidneys in Denmark, there is a need to expand the possibilities for donation. At the Odense University Hospital (OUH), we have introduced ABO......-incompatible kidney transplantation. We used antigenspecific immunoadsorptions to remove blood group antibodies and anti-CD20 antibody (rituximab) to inhibit the antibody production. The aim of introducing the ABO-incompatible kidney transplantation at the OUH was to increase the rate of living donor kidney...... transplantation without increasing rejection or mortality rates. MATERIAL AND METHODS: Retrospective evaluation. Eleven patients received ABO-incompatible kidney transplantation. The patients were followed for 3-26 months. RESULTS: One patient had an antibody-mediated rejection, one patient suffered T...

The herpes simplex virus 1-encoded envelope glycoprotein B activates NF-κB through the Toll-like receptor 2 and MyD88/TRAF6-dependent signaling pathway.

Directory of Open Access Journals (Sweden)

Mingsheng Cai

Full Text Available The innate immune response plays a critical role in the host defense against invading pathogens, and TLR2, a member of the Toll-like receptor (TLR family, has been implicated in the immune response and initiation of inflammatory cytokine secretion against several human viruses. Previous studies have demonstrated that infectious and ultraviolet-inactivated herpes simplex virus 1 (HSV-1 virions lead to the activation of nuclear factor kappa B (NF-κB and secretion of proinflammatory cytokines via TLR2. However, except for the envelope glycoprotein gH and gL, whether there are other determinants of HSV-1 responsible for TLR2 mediated biological effects is not known yet. Here, we demonstrated that the HSV-1-encoded envelope glycoprotein gB displays as molecular target recognized by TLR2. gB coimmunoprecipitated with TLR2, TLR1 and TLR6 in transfected and infected human embryonic kidney (HEK 293T cells. Treatment of TLR2-transfected HEK293T (HEK293T-TLR2 cells with purified gB results in the activation of NF-κB reporter, and this activation requires the recruitment of the adaptor molecules myeloid differentiation primary-response protein 88 (MyD88 and tumor necrosis factor receptor-associated factor 6 (TRAF6 but not CD14. Furthermore, activation of NF-κB was abrogated by anti-gB and anti-TLR2 blocking antibodies. In addition, the expression of interleukin-8 induced by gB was abrogated by the treatment of the human monocytic cell line THP-1 with anti-TLR2 blocking antibody or by the incubation of gB with anti-gB antibody. Taken together, these results indicate the importance and potency of HSV-1 gB as one of pathogen-associated molecular patterns (PAMPs molecule recognized by TLR2 with immediate kinetics.

NIR fluorescent chitosan-based nanoparticles for tracking and delivery of cancer therapeutic molecule in living systems

Science.gov (United States)

Suarato, Giulia; Chin, Amanda; Meng, Yizhi

2013-03-01

Tumor metastasis is associated with the epithelial-to-mesenchymal transition (EMT), in which cells lose their polarized phenotype to acquire the asymmetry and motility of mesenchymal cells. Among the many molecular determinants for EMT is bone morphogenetic protein-7 (BMP-7), a critical regulator of skeletal tissue formation and kidney development. Current treatments for metastatic cancer primarily involve surgery and chemotherapy, both with considerable side effects. Therefore the goal of our research is to evaluate the ability of BMP-7 to reverse EMT using a delivery system based on glycol chitosan nanoparticles (GCNP), naturally biodegradable. The GCNP are labeled with Cy5.5, a near infrared (NIR) excitable dye that enables non-invasive imaging in living systems. The chitosan shell provides affinity for the cell surface and protection from intracellular enzymes during transport. Preliminary data show that Cy5.5-GCNP vehicles were successfully delivered to murine preosteoblast (MC3T3-E1), rat osteosarcoma (ROS) 17/2.8 and human embryonic kidney (HEK293) cells. Release kinetics using a model protein (BSA) and BMP-7, and the stability of the protein nano-cargo are currently being evaluated. Cell morphology will be examined with immunofluorescence microscopy.

The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

Science.gov (United States)

Xue, Qiao

2017-01-01

Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects domestic and wild cloven-hoofed animals. The structural protein VP1 plays an important role in FMDV pathogenesis. However, the interacting partners of VP1 in host cells and the effects of these interactions in FMDV replication remain incompletely elucidated. Here, we identified a porcine cell protein, serine/threonine kinase 3 (STK3), which interacts with FMDV VP1 using the yeast two-hybrid system. The VP1-STK3 interaction was further confirmed by coimmunoprecipitation experiments in human embryonic kidney 293T and porcine kidney 15 (PK-15) cells. The carboxyl-terminal region (amino acids 180–214) of VP1 was essential for its interaction with STK3. The effects of overexpression and underexpressing of STK3 in PK-15 cells were assessed, and the results indicated that STK3 significantly inhibited FMDV replication. Our data expand the role of STK3 during viral infection, provide new information regarding the host cell kinases that are involved in viral replication, and identify potential targets for future antiviral strategies. PMID:29226127

Embryonic exposure of medaka (Oryzias latipes) to propylparaben: Effects on early development and post-hatching growth

International Nuclear Information System (INIS)

González-Doncel, Miguel; García-Mauriño, José Enrique; San Segundo, Laura; Beltrán, Eulalia M.; Sastre, Salvador; Fernández Torija, Carlos

2014-01-01

Here we proposed a battery of non-invasive biomarkers and a histological survey to examine physiological/anatomical features in embryos, eleutheroembryos (13 days post-fertilization, dpf), and larvae (28–42 dpf) of medaka to investigate the effects of embryonic exposure to propylparaben (PrP). Concentrations <1000 μg PrP/L didn't exert early or late toxic effects. However, survivorship was affected at 4000 μg/L in eleutheroembryos and at ≥1000 μg/L in larvae. Histological alterations were found in 37.5% of eleutheroembryos exposed to 4000 μg PrP/L. Morphometric analysis of the gallbladder revealed significant dilation at ≥400 μg/L throughout embryo development. Ethoxyresorufin-O-deethylase (EROD), as indicator of cytochrome P4501A activity, didn't reveal induction/inhibition although its combination with a P4501A agonist (i.e. β-naphthoflavone) resulted in a synergic EROD response. Results suggest a low toxicity of PrP for fish and support the use of fish embryos and eleutheroembryos as alternatives of in vivo biomarkers indicative of exposure/toxicity. -- Highlights: • Addressing pre- and post-hatch effects from medaka embryo exposure to propylparaben. • Macroscopical effects (length, mortality) seen primarily after hatch at ≥400 μg/L. • Synergic EROD embryonic response when propylparaben combined with a CYP1A agonist. • Significant gallbladder dilation seen at ≥400 μg PrP/L and as soon as discernible. • Histological harm to eleutheroembryos in peritoneal cavity, liver, kidney and brain. -- PrP resulted in low toxicity based on non-invasive biomarkers and histological tools to analyze pre- and post-hatch effects after medaka embryo exposure

47 CFR 80.293 - Check bearings by authorized ship personnel.

Science.gov (United States)

2010-10-01

....293 Section 80.293 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL... comparison of simultaneous visual and radio direction finder bearings. At least one comparison bearing must be taken in each quadrant, within plus or minus 20 degrees from the following bearings relative to...

Asymmetric BMP4 signalling improves the realism of kidney organoids.

Science.gov (United States)

Mills, Christopher G; Lawrence, Melanie L; Munro, David A D; Elhendawi, Mona; Mullins, John J; Davies, Jamie A

2017-11-01

We present a strategy for increasing the anatomical realism of organoids by applying asymmetric cues to mimic spatial information that is present in natural embryonic development, and demonstrate it using mouse kidney organoids. Existing methods for making kidney organoids in mice yield developing nephrons arranged around a symmetrical collecting duct tree that has no ureter. We use transplant experiments to demonstrate plasticity in the fate choice between collecting duct and ureter, and show that an environment rich in BMP4 promotes differentiation of early collecting ducts into uroplakin-positive, unbranched, ureter-like epithelial tubules. Further, we show that application of BMP4-releasing beads in one place in an organoid can break the symmetry of the system, causing a nearby collecting duct to develop into a uroplakin-positive, broad, unbranched, ureter-like 'trunk' from one end of which true collecting duct branches radiate and induce nephron development in an arrangement similar to natural kidneys. The idea of using local symmetry-breaking cues to improve the realism of organoids may have applications to organoid systems other than the kidney.

Short Interspersed Nuclear Element (SINE Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293.

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Lakkhana Kanhayuwa

Full Text Available Novel families of short interspersed nuclear element (SINE sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4-14 bp flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140-493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3'-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50-65% and 60-75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259-343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.

SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

Energy Technology Data Exchange (ETDEWEB)

Almeida, Luciana O.; Goto, Renata N. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Neto, Marinaldo P.C. [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Sousa, Lucas O. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Curti, Carlos [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Leopoldino, Andréia M., E-mail: andreiaml@usp.br [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil)

2015-03-06

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.

Apparent diffusion coefficient measurements of bilateral kidneys at 3 T MRI: Effects of age, gender, and laterality in healthy adults

International Nuclear Information System (INIS)

Suo, S.-T.; Cao, M.-Q.; Ding, Y.-Z.; Yao, Q.-Y.; Wu, G.-Y.; Xu, J.-R.

2014-01-01

Aim: To investigate the effects of age and gender on apparent diffusion coefficient (ADC) measurements of bilateral kidneys at 3 T MRI, and compare the ADC values of left and right kidneys. Materials and methods: In all, 137 healthy participants (mean age 42.8 ± 14.7 years; age range 16–75 years) comprising 68 male and 69 female participants were enrolled. Three Tesla echo-planar diffusion-weighted imaging (DWI) of bilateral kidneys was performed and ADC values were measured in the cortex, medulla, and whole parenchyma. Pearson correlation analysis and linear regression were performed to determine the associations between the ADC values in each region and age. Effects of age and gender on ADC values were analysed using two-factor analysis of variance (ANOVA). The paired-samples t-test was established to compare the ADC values between left and right kidneys. Results: ADC values were significantly higher in the young group (≤50 years) than in the old group (>50 years), and correlated inversely with the age in all regions. Male participants had higher ADC values than female participants in all regions except left medulla. Two-factor ANOVA of age × gender showed no significant interactions between the variables age and gender were found. No significant differences in ADC values between left and right kidneys were observed. Conclusion: Renal ADC values are age- and gender-dependent, and show no significant difference between left and right kidneys. Age- and gender-related effects should be taken into consideration in future renal DWI studies when using normal ADC values from health controls. - Highlights: • Renal apparent diffusion coefficient (ADC) values decrease with ageing. • Men tend to have higher renal ADC values than women. • Bilateral kidneys seem to have no significantly different ADC values

Horseshoe kidney with growth retardation: Don't forget Turner syndrome.

Science.gov (United States)

Arslansoyu-Çamlar, Seçil; Soylu, Alper; Abacı, Ayhan; Türkmen, Mehmet Atilla; Ülgenalp, Ayfer; Kavukçu, Salih

2016-01-01

Horseshoe kidney is the most frequent renal fusion anomaly that is usually asymptomatic and isolated malformation. However it can be seen with various syndromes and chromosomal anomalies. It was reported that 15-35% of Turner syndrome cases (TS) also display horseshoe kidney condition. TS is a chromosomal anomaly that had been characterized by delayed puberty, short body height and gonadal dysgenesis. In this report a five-year-old girl with horseshoe kidney, which has growth retardation during follow-up as only symptom of Turner syndrome.

Blockade of T-lymphocyte KCa3.1 and Kv1.3 channels as novel immunosuppression strategy to prevent kidney allograft rejection

DEFF Research Database (Denmark)

Grgic, I; Wulff, H; Eichler, I

2009-01-01

. Kidney sections were stained with periodic acid-Schiff or hematoxylin-eosin and histochemically for markers of macrophages (CD68), T-lymphocytes (CD43), or cytotoxic T-cells (CD8). Our results showed that treatment with TRAM-34 and ShK reduced total interstitial mononuclear cell infiltration (-42...

Activation of ERK mitogen-activated protein kinase in human cells by the mycotoxin patulin

International Nuclear Information System (INIS)

Wu, T.-S.; Yu, F.-Y.; Su, C.-C.; Kan, J.-C.; Chung, C.-P.; Liu, B.-H.

2005-01-01

Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 μM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 μM of PAT. Treatment of human PBMCs for 30 min with 30 μM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 μM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression

Do You Have Symptoms of a Kidney Stone?

Science.gov (United States)

... Or, the stone will be removed with treatment. Dogs, Cats, and Kidney Stones Humans aren't the only ones affected by kidney and bladder stones. Dogs, cats, and other animals can also have kidney ...

The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

DEFF Research Database (Denmark)

Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

2010-01-01

Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells

International Nuclear Information System (INIS)

Qin Zhaoling; Zhao Ping; Zhang Xiaolian; Yu Jianguo; Cao Mingmei; Zhao Lanjuan; Luan Jie; Qi Zhongtian

2004-01-01

Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV

Thermodynamic and physicochemical properties of binary mixtures of nitromethane with {2-methoxyethanol + 2-butoxyethanol} systems at T = (293.15, 298.15, 303.15, 308.15, and 313.15) K

International Nuclear Information System (INIS)

Cwiklinska, Aneta; Kinart, Cezary M.

2011-01-01

The density, relative permittivity, viscosity and speed of sound at T = (293.15, 298.15, 303.15, 308.15, and 313.15) K in the binary mixtures of nitromethane with 2-methoxyethanol and 2-butoxyethanol have been measured as a function of composition. From the experimental results, the excess molar volumes V E , excess Gibbs free energy of activation for viscous flow (ΔG *E ), excess isentropic compressibility (κ s E ) and the deviations in the relative permittivity, viscosity, and speed of sound from a mole fraction average have been calculated. The viscosity data, at T = 298.15 K, were correlated with equations of Hind et al., Grunberg and Nissan, Frenkel, and McAllister. The results are discussed in terms of intermolecular interactions and structure of studied binary mixtures.

Endocrine control of embryonic diapause in the Australian sharpnose shark Rhizoprionodon taylori.

Directory of Open Access Journals (Sweden)

Daniela Waltrick

Full Text Available The reproductive cycle of the Australian sharpnose shark, Rhizoprionodon taylori, includes a temporary suspension of development at the commencement of embryogenesis termed embryonic diapause. This study investigated levels of 17β-estradiol (E2, testosterone (T and progesterone (P4 in plasma samples of mature wild female R. taylori captured throughout the reproductive cycle and correlated them with internal morphological changes. Levels of T were elevated through most of the embryonic diapause period, suggesting a role of this hormone in the maintenance of this condition. Increasing plasma T concentrations from late diapause to early active development were associated with a possible role of androgens in the termination of embryonic diapause. As in other elasmobranchs, a concomitant increase of E2 with ovarian follicle size indicated a direct role of this hormone in regulating vitellogenesis, while a peak in P4 suggested this hormone is associated with preovulation and ovulation. Additionally, significant correlations between photoperiod or water temperature and maximum follicular diameter and hepatosomatic index suggest that these abiotic factors may also play a role triggering and regulating the synchrony and timing of reproductive events.

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

Directory of Open Access Journals (Sweden)

Elisa Fanunza

2018-02-01

Full Text Available The interferon (IFN system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24 is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE. Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

36 CFR 293.15 - Gathering information about resources other than minerals.

Science.gov (United States)

2010-07-01

... resources other than minerals. 293.15 Section 293.15 Parks, Forests, and Public Property FOREST SERVICE... than minerals. (a) The Chief, Forest Service, shall allow any activity, for the purposes of gathering information about resources, other than minerals, in National Forest Wilderness, except that any such activity...

Regenerative medicine in kidney disease: where we stand and where to go.

Science.gov (United States)

Borges, Fernanda T; Schor, Nestor

2017-07-22

The kidney is a complex organ with more than 20 types of specialized cells that play an important role in maintaining the body's homeostasis. The epithelial tubular cell is formed during embryonic development and has little proliferative capacity under physiological conditions, but after acute injury the kidney does have regenerative capacity. However, after repetitive or severe lesions, it may undergo a maladaptation process that predisposes it to chronic kidney injury. Regenerative medicine includes various repair and regeneration techniques, and these have gained increasing attention in the scientific literature. In the future, not only will these techniques contribute to the repair and regeneration of the human kidney, but probably also to the construction of an entire organ. New mechanisms studied for kidney regeneration and repair include circulating stem cells as mesenchymal stromal/stem cells and their paracrine mechanisms of action; renal progenitor stem cells; the leading role of tubular epithelial cells in the tubular repair process; the study of zebrafish larvae to understand the process of nephron development, kidney scaffold and its repopulation; and, finally, the development of organoids. This review elucidates where we are in terms of current scientific knowledge regarding these mechanisms and the promises of future scientific perspectives.

5 CFR 293.504 - Composition of, and access to, the Employee Medical File System.

Science.gov (United States)

2010-01-01

... Employee Medical File System. 293.504 Section 293.504 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PERSONNEL RECORDS Employee Medical File System Records § 293.504 Composition of, and access to, the Employee Medical File System. (a) All employee occupational medical records...

Development and Evaluation of a Semi-automated Segmentation Tool and a Modified Ellipsoid Formula for Volumetric Analysis of the Kidney in Non-contrast T2-Weighted MR Images.

Science.gov (United States)

Seuss, Hannes; Janka, Rolf; Prümmer, Marcus; Cavallaro, Alexander; Hammon, Rebecca; Theis, Ragnar; Sandmair, Martin; Amann, Kerstin; Bäuerle, Tobias; Uder, Michael; Hammon, Matthias

2017-04-01

Volumetric analysis of the kidney parenchyma provides additional information for the detection and monitoring of various renal diseases. Therefore the purposes of the study were to develop and evaluate a semi-automated segmentation tool and a modified ellipsoid formula for volumetric analysis of the kidney in non-contrast T2-weighted magnetic resonance (MR)-images. Three readers performed semi-automated segmentation of the total kidney volume (TKV) in axial, non-contrast-enhanced T2-weighted MR-images of 24 healthy volunteers (48 kidneys) twice. A semi-automated threshold-based segmentation tool was developed to segment the kidney parenchyma. Furthermore, the three readers measured renal dimensions (length, width, depth) and applied different formulas to calculate the TKV. Manual segmentation served as a reference volume. Volumes of the different methods were compared and time required was recorded. There was no significant difference between the semi-automatically and manually segmented TKV (p = 0.31). The difference in mean volumes was 0.3 ml (95% confidence interval (CI), -10.1 to 10.7 ml). Semi-automated segmentation was significantly faster than manual segmentation, with a mean difference = 188 s (220 vs. 408 s); p T2-weighted MR data delivers accurate and reproducible results and was significantly faster than manual segmentation. Applying a modified ellipsoid formula quickly provides an accurate kidney volume.

Healthy Kidneys (A Cup of Health with CDC)

Centers for Disease Control (CDC) Podcasts

Kidneys that function properly are critical for maintaining good health, however, more than one in seven American adults have kidney disease and most aren't aware of their condition. In this podcast, Nilka Rios Burrows discusses the importance of maintaining healthy kidneys.

Adaptive regulation of taurine and beta-alanine uptake in a human kidney cell line from the proximal tubule

DEFF Research Database (Denmark)

Jessen, H; Jacobsen, Christian

1997-01-01

1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine...

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Science.gov (United States)

Miura, Jiro; Sakai, Manabu; Uchida, Hitoshi; Nakamura, Wataru; Nohara, Kanji; Maruyama, Yusuke; Hattori, Atsuhiko; Sakai, Takayoshi

2015-01-01

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development. PMID:25876057

Aberrant expression of interleukin-22 and its targeting microRNAs in oral lichen planus: a preliminary study.

Science.gov (United States)

Shen, Zhengyu; Du, Guanhuan; Zhou, Zengtong; Liu, Wei; Shi, Linjun; Xu, Hui

2016-08-01

Oral lichen planus (OLP) is a T cell-mediated autoimmune disease involving oral mucosa. Interleukin-22 (IL-22) as the signature cytokine of T helper 22 cells is increasingly recognized as a key regulator in various autoimmune diseases. Our previous study reported that IL-22 immunoexpression in OLP was significantly increased compared with the normal controls. The objective of this preliminary study was to compare the IL-22 expression levels in oral biopsies from patients with OLP (n = 50) against normal oral mucosa (n = 19) using RT-qPCR and Western blot, identify the potential targeting miRNAs of IL-22, and examine the miRNA expression levels in OLP. Interleukin-22 expression level in OLP was significantly increased compared with the normal controls. The Dual-Luciferase reporter assay system in human embryonic kidney 293 (HEK293) cells demonstrated that miR-562 and miR-203 were the target miRNAs of IL-22, which was consistent with predictions from bioinformatics software analyses. Interestingly, miR-562 expression in OLP was significantly decreased, but miR-203 expression in OLP was significantly increased compared with the normal controls. This preliminary study for the first time reported that aberrant expression levels of miR-562 and miR-203 were associated with high expression of IL-22 and demonstrated the target relationship between miRNAs and IL-22 in HEK293 cells. Our data indicated that IL-22 and its targeting miRNAs contribute to the pathogenesis of OLP. Further studies are required to investigate the regulatory pathways of IL-22 and miR-562 and miR-203 in OLP. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Circulating CD4+CD28null T Cells May Increase the Risk of an Atherosclerotic Vascular Event Shortly after Kidney Transplantation

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Michiel G. H. Betjes

2013-01-01

Full Text Available Proinflammatory CD4+ T cells without the costimulatory molecule CD28 (CD4+CD28null T cells are expanded in patients with end-stage renal disease (ESRD and associated with atherosclerotic vascular events (AVE. In a prospective study, the number of circulating CD4+CD28null T cells was established in 295 ESRD patients prior to receiving a kidney allograft. Within the first year after transplantation, an AVE occurred in 20 patients. Univariate analysis showed that besides a history of cardiovascular disease (CVDpos, HR 8.1, , age (HR 1.04, , dyslipidaemia (HR 8.8, , and the % of CD4+CD28null T cells (HR 1.04 per % increase, 95% CI 1.00–1.09, were significantly associated with the occurrence of a posttransplantation AVE. In a multivariate analysis, only CVDpos remained a significant risk factor with a significant and positive interaction between the terms CVDpos and the % of CD4+CD28null T cells (HR 1.05, 95% CI 1.03–1.11, . Within the CVDpos group, the incidence of an AVE was 13% in the lowest tertile compared to 25% in the highest tertile of % of CD4+CD28null T cells. In conclusion, the presence of circulating CD4+CD28null T cells is associated with an increased risk for a cardiovascular event shortly after kidney transplantation.

APOPTOSIS DURING HUMAN FETAL KIDNEY DEVELOPMENT

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Rade Čukuranović

2005-01-01

Full Text Available Kidney morphogenesis is a complex and stepwise process. The formation of mature kidney in mammals is preceded by two primitive embryonic kidneys known as pronephros and mesonephros. Metanephros develops as a result of reciprocal inductive interactions between two primordial mesodermal derivates: ureteric bud, an epithelial outgrowth of the Wolffian duct, and metanephric blastema, a group of mesenchymal cells. The ureteric bud induces the metanephric mesenchyme to differentiate and form nephrons, whilst the metanephric mesenchyme induces the ureteric bud to grow and branch to form collecting ducts. The nephron goes through four developmental stages, which are described as: 1 vesicle, 2 comma-shaped and S-shaped stages, 3 developing capillary loop, and finally 4 maturing glomerulus. Apoptosis (programmed cell death is a predominant form of physiological cell death, by which organism eliminate unwanted or damaged cells. It is the major component of normal development and disease. Apoptosis is the result of series of biochemical processes happening in certain order in a dying cell, among which the most important is activation of enzyme families called caspases which influence different cell components. Apoptosis is characterized by membrane blebbing, shrinkage of the cell, nuclear fragmentation and chromatin condensation. Organelles are preserved almost intact. Cell surface molecules change. A variety of physiological and pathological stimuli can initiate apoptosis. They act via receptor mechanisms, through biochemical agents, or cause DNA and cell membrane damage. Apoptosis is an important component of fetal development. It is thought that apoptosis is the one of the main regulatory events involved in kidney morphogenesis, considering that among great number of developed cells, only a few of them are involved in the developing program by escaping apoptosis. In any period during kidney development about 3 to 5%of cells are apoptotic. Thorough

Evaluation of Porcine Pancreatic Islets Transplanted in the Kidney Capsules of Diabetic Mice Using a Clinically Approved Superparamagnetic Iron Oxide (SPIO) and a 1.5T MR Scanner

International Nuclear Information System (INIS)

Kim, Hoe Suk; Kim, Hyoung Su; Park, Kyong Soo; Moon, Woo Kyung

2010-01-01

To evaluate transplanted porcine pancreatic islets in the kidney capsules of diabetic mice using a clinically approved superparamagnetic iron oxide (SPIO) and a 1.5T MR scanner. Various numbers of porcine pancreatic islets labeled with Resovist, a carboxydextran-coated SPIO, were transplanted into the kidney capsules of normal mice and imaged with a 3D FIESTA sequence using a 1.5T clinical MR scanner. Labeled (n = 3) and unlabeled (n = 2) islets were transplanted into the kidney capsules of streptozotocin-induced diabetic mice. Blood glucose levels and MR signal intensities were monitored for 30 days post-transplantation. There were no significant differences in viability or insulin secretion between labeled and unlabeled islets. A strong correlation (γ 2 > 0.94) was evident between the number of transplanted islets and T 2 relaxation times quantified by MRI. Transplantation with labeled or unlabeled islets helped restore normal sustained glucose levels in diabetic mice, and nephrectomies induced the recurrence of diabetes. The MR signal intensity of labeled pancreatic islets decreased by 80% over 30 days. The transplantation of SPIO-labeled porcine islets into the kidney capsule of diabetic mice allows to restore normal glucose levels, and these islets can be visualized and quantified using a 1.5T clinical MR scanner

Polycystic kidney disease

Science.gov (United States)

... don't have other diseases may be good candidates for a kidney transplant. Possible Complications Health problems ... www.urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. ...

Healthy Kidneys (A Cup of Health with CDC)

Centers for Disease Control (CDC) Podcasts

Kidneys serve as the body’s filtering system, removing waste and excess water from the blood. If your kidneys are damaged or don’t function properly, you can have severe health problems. In this podcast, Nilka Rios Burrows discusses the dangers of kidney disease.

UV laser radiation alters the embryonic protein profile of adrenal-kidney-gonadal complex and gonadal differentiation in the lizard, Calotes Versicolor.

Science.gov (United States)

Khodnapur, Bharati S; Inamdar, Laxmi S; Nindi, Robertraj S; Math, Shivkumar A; Mulimani, B G; Inamdar, Sanjeev R

2015-02-01

To examine the impact of ultraviolet (UV) laser radiation on the embryos of Calotes versicolor in terms of its effects on the protein profile of the adrenal-kidney-gonadal complex (AKG), sex determination and differentiation, embryonic development and hatching synchrony. The eggs of C. versicolor, during thermo-sensitive period (TSP), were exposed to third harmonic laser pulses at 355 nm from a Q-switched Nd:YAG laser for 180 sec. Subsequent to the exposure they were incubated at the male-producing temperature (MPT) of 25.5 ± 0.5°C. The AKG of hatchlings was subjected to protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and to histology. The UV laser radiation altered the expression of the protein banding pattern in the AKG complex of hatchlings and it also affected the gonadal sex differentiation. SDS-PAGE of AKG of one-day-old hatchlings revealed a total of nine protein bands in the control group whereas UV laser irradiated hatchlings expressed a total of seven protein bands only one of which had the same Rf as a control band. The UV laser treated hatchlings have an ovotestes kind of gonad exhibiting a tendency towards femaleness instead of the typical testes. It is inferred that 355 nm UV laser radiation during TSP induces changes in the expression of proteins as well as their secretions. UV laser radiation had an impact on the gonadal differentiation pathway but no morphological anomalies were noticed.

Diffusion-weighted MR imaging of kidneys in patients with chronic kidney disease: initial study

Energy Technology Data Exchange (ETDEWEB)

Xu, Xueqin; Fang, Wenqiang; Ling, Huawei; Chai, Weimin; Chen, Kemin [Ruijin Hospital Shanghai, Jiaotong University School of Medicine, Department of Radiology, Shanghai (China)

2010-04-15

To prospectively evaluate the feasibility of diffusion-weighted (DW) magnetic resonance (MR) imaging in the assessment of renal function in patients with chronic kidney disease (CKD). Seventy-two healthy volunteers and 43 patients underwent coronal echo-planar DW MR imaging of the kidneys with a single breath-hold time of 16 s. The patients were grouped according to five stages as indicated by the K/DOQI CKD (kidney disease outcome quality initiative). The apparent diffusion coefficient (ADC) value of the kidneys was calculated with high b values (b = 500 s/mm{sup 2}). The ADC values were compared between patients and healthy volunteers, and among different stages. For statistical analysis, Student's t tests, ANOVA, Pearson's correlation tests, and Spearman's correlation tests were used. No difference between the cortex and medulla could be observed on DW images of all volunteers. Patients with CKD had significantly lower renal ADC (t = -4.383, P = 0.000) than volunteers. The ADC values of kidneys were significantly lower than normal at most stages of CKD, except CKD1. There was a negative correlation between the ADCs and serum creatinine (sCr) level (P = 0.000) amongst the patients. Diffusion-weighted MR imaging is feasible in the assessment of renal function, especially in the detection of early stage renal failure of CKD. (orig.)

Diffusion-weighted MR imaging of kidneys in patients with chronic kidney disease: initial study

International Nuclear Information System (INIS)

Xu, Xueqin; Fang, Wenqiang; Ling, Huawei; Chai, Weimin; Chen, Kemin

2010-01-01

To prospectively evaluate the feasibility of diffusion-weighted (DW) magnetic resonance (MR) imaging in the assessment of renal function in patients with chronic kidney disease (CKD). Seventy-two healthy volunteers and 43 patients underwent coronal echo-planar DW MR imaging of the kidneys with a single breath-hold time of 16 s. The patients were grouped according to five stages as indicated by the K/DOQI CKD (kidney disease outcome quality initiative). The apparent diffusion coefficient (ADC) value of the kidneys was calculated with high b values (b = 500 s/mm 2 ). The ADC values were compared between patients and healthy volunteers, and among different stages. For statistical analysis, Student's t tests, ANOVA, Pearson's correlation tests, and Spearman's correlation tests were used. No difference between the cortex and medulla could be observed on DW images of all volunteers. Patients with CKD had significantly lower renal ADC (t = -4.383, P = 0.000) than volunteers. The ADC values of kidneys were significantly lower than normal at most stages of CKD, except CKD1. There was a negative correlation between the ADCs and serum creatinine (sCr) level (P = 0.000) amongst the patients. Diffusion-weighted MR imaging is feasible in the assessment of renal function, especially in the detection of early stage renal failure of CKD. (orig.)

Excess molar volumes and viscosities of (1,1,2,2-tetrabromoethane + 1-alkanols) at T = (293.15 and 303.15) K

International Nuclear Information System (INIS)

Al-Hayan, M.N.M.; Abdul-latif, Abdul-Haq M.

2006-01-01

Density and viscosity measurements for binary mixtures of (1,1,2,2-tetrabromoethane + 1-pentanol, or + 1-hexanol, or + 1-heptanol, or + 1-octanol, or + 1-decanol) at T = (293.15 and 303.15) K, have been conducted at atmospheric pressure. The excess molar volumes V E , have been calculated from the experimental measurements, and the results were fitted to Redlich-Kister equation. The viscosity data were correlated with the model of Grunberg and Nissan, and McAllister four-body model. The excess molar volumes of (1,1,2,2-tetrabromoethane + 1-pentanol, or + 1-haxanol, or + 1-heptanol, or + 1-octanol) had a sigmoidal shape and the values varied from negative to positive with the increase in the molar fraction of 1,1,2,2-tetrabromoethane. The remaining binary mixture of (1,1,2,2-tetrabromoethane + 1-decanol) was positive over the entire composition range. The effects of the 1-alkanol chain length as well as the temperature on the excess molar volume have been studied. The results have been qualitatively used to explain the molecular interaction between the components of these mixtures

Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease.

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Arianne van Koppen

Full Text Available Chronic kidney disease (CKD is a major health care problem, affecting more than 35% of the elderly population worldwide. New interventions to slow or prevent disease progression are urgently needed. Beneficial effects of mesenchymal stem cells (MSC have been described, however it is unclear whether the MSCs themselves or their secretome is required. We hypothesized that MSC-derived conditioned medium (CM reduces progression of CKD and studied functional and structural effects in a rat model of established CKD. CKD was induced by 5/6 nephrectomy (SNX combined with L-NNA and 6% NaCl diet in Lewis rats. Six weeks after SNX, CKD rats received either 50 µg CM or 50 µg non-CM (NCM twice daily intravenously for four consecutive days. Six weeks after treatment CM administration was functionally effective: glomerular filtration rate (inulin clearance and effective renal plasma flow (PAH clearance were significantly higher in CM vs. NCM-treatment. Systolic blood pressure was lower in CM compared to NCM. Proteinuria tended to be lower after CM. Tubular and glomerular damage were reduced and more glomerular endothelial cells were found after CM. DNA damage repair was increased after CM. MSC-CM derived exosomes, tested in the same experimental setting, showed no protective effect on the kidney. In a rat model of established CKD, we demonstrated that administration of MSC-CM has a long-lasting therapeutic rescue function shown by decreased progression of CKD and reduced hypertension and glomerular injury.

Multidrug Resistance Protein-4 Influences Aspirin Toxicity in Human Cell Line

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Isabella Massimi

2015-01-01

Full Text Available Overexpression of efflux transporters, in human cells, is a mechanism of resistance to drug and also to chemotherapy. We found that multidrug resistance protein-4 (MRP4 overexpression has a role in reducing aspirin action in patients after bypass surgery and, very recently, we found that aspirin enhances platelet MRP4 levels through peroxisome proliferator activated receptor-α (PPARα. In the present paper, we verified whether exposure of human embryonic kidney-293 cells (Hek-293 to aspirin modifies MRP4 gene expression and its correlation with drug elimination and cell toxicity. We first investigated the effect of high-dose aspirin in Hek-293 and we showed that aspirin is able to increase cell toxicity dose-dependently. Furthermore, aspirin effects, induced at low dose, already enhance MRP4 gene expression. Based on these findings, we compared cell viability in Hek-293, after high-dose aspirin treatment, in MRP4 overexpressing cells, either after aspirin pretreatment or in MRP4 transfected cells; in both cases, a decrease of selective aspirin cell growth inhibition was observed, in comparison with the control cultures. Altogether, these data suggest that exposing cells to low nontoxic aspirin dosages can induce gene expression alterations that may lead to the efflux transporter protein overexpression, thus increasing cellular detoxification of aspirin.

Cloning and expression of sheep renal K-CI cotransporter-1.

Science.gov (United States)

Zhang, Jin J; Misri, Sandeep; Adragna, Norma C; Gagnon, Kenneth B E; Fyffe, Robert E W; Lauf, Peter K

2005-01-01

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide. Copyright (c) 2005 S. Karger AG, Basel.

Tissue distribution in mice of BPP 10c, a potent proline-rich anti-hypertensive peptide of Bothrops jararaca.

Science.gov (United States)

Silva, Carlos A; Portaro, Fernanda C V; Fernandes, Beatriz L; Ianzer, Danielle A; Guerreiro, Juliano R; Gomes, Claudiana L; Konno, Katsuhiro; Serrano, Solange M T; Nascimento, Nanci; Camargo, Antonio C M

2008-03-15

The snake venom proline-rich peptide BPP 10c is an active somatic angiotensin-converting enzyme (sACE) inhibitors. Recently we demonstrated that the anti-hypertensive effect of BPP 10c is not related to the inhibition of sACE alone, thus suggesting that this enzyme is not its only target for blood pressure reduction. In the present work, a biodistribution study in Swiss mice of [(125)I]-BPP 10c in the absence or in the presence of a saturating concentration of captopril, a selective active-site inhibitor of sACE, demonstrated that: (1) [(125)I]-BPP 10c was present in several organs and the renal absorption was significantly high; (2) [(125)I]-BPP 10c showed a clear preference for the kidney, maintaining a high concentration in this organ in the presence of captopril for at least 3h; (3) The residual amount of [(125)I]-BPP 10c in the kidney of animals simultaneously treated with captopril suggest that the peptide can interact with other targets different from sACE in this organ. We also showed that Cy3-labeled BPP 10c was internalized by human embryonic kidney cells (HEK-293T). Taken together, these results suggest that sACE inhibition by captopril affects the tissue distribution of [(125)I]-BPP 10c and that the anti-hypertensive effects of BPP 10c are not only dependent on sACE inhibition.

Expression of Nek1 during kidney development and cyst formation in multiple nephron segments in the Nek1-deficient kat2J mouse model of polycystic kidney disease.

Science.gov (United States)

Chen, Yumay; Chiang, Huai-Chin; Litchfield, Patricia; Pena, Michelle; Juang, Charity; Riley, Daniel J

2014-07-17

Neks, mammalian orthologs of the fungal protein kinase never-in-mitosis A, have been implicated in the pathogenesis of polycystic kidney disease. Among them, Nek1 is the primary protein inactivated in kat2J mouse models of PKD. We report the expression pattern of Nek1 and characterize the renal cysts that develop in kat2J mice. Nek1 is detectable in all murine tissues but its expression in wild type and kat2J heterozygous kidneys decrease as the kidneys mature, especially in tubular epithelial cells. In the embryonic kidney, Nek1 expression is most prominent in cells that will become podocytes and proximal tubules. Kidney development in kat2J homozygous mice is aberrant early, before the appearance of gross cysts: developing cortical zones are thin, populated by immature glomeruli, and characterized by excessive apoptosis of several cell types. Cysts in kat2J homozygous mice form postnatally in Bowman's space as well as different tubular subtypes. Late in life, kat2J heterozygous mice form renal cysts and the cells lining these cysts lack staining for Nek1. The primary cilia of cells lining cysts in kat2J homozygous mice are morphologically diverse: in some cells they are unusually long and in others there are multiple cilia of varying lengths. Our studies indicate that Nek1 deficiency leads to disordered kidney maturation, and cysts throughout the nephron.

Cationic Polyamidoamine Dendrimers as Modulators of EGFR Signaling In Vitro and In Vivo.

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Saghir Akhtar

Full Text Available Cationic polyamidoamine (PAMAM dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented generation (G 6 PAMAM dendrimer, Superfect (SF, stimulated epidermal growth factor receptor (EGFR tyrosine kinase signaling-an important signaling cascade that regulates cell growth, survival and apoptosis- in cultured human embryonic kidney (HEK 293 cells. Here, we firstly studied the in vitro effects of Polyfect (PF, a non-activated (intact G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2 and p38 mitogen-activated protein kinase (MAPK in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM

A novel Golgi retention signal RPWS for tumor suppressor UBIAD1.

Directory of Open Access Journals (Sweden)

Xian Wang

Full Text Available UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder's corneal dystrophy, Parkinson's disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER, but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.

Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection.

Science.gov (United States)

Yun, Kangsun; Perantoni, Alan O

The Wnt5a null mouse is a complex developmental model which, among its several posterior-localized axis defects, exhibits multiple kidney phenotypes, including duplex kidney and loss of the medullary zone. We previously reported that ablation of Wnt5a in nascent mesoderm causes duplex kidney formation as a result of aberrant development of the nephric duct and abnormal extension of intermediate mesoderm. However, these mice also display a loss of the medullary region late in gestation. We have now genetically isolated duplex kidney formation from the medullary defect by specifically targeting the progenitors for both the ureteric bud and metanephric mesenchyme. The conditional mutants fail to form a normal renal medulla but no longer exhibit duplex kidney formation. Approximately 1/3 of the mutants develop hydronephrosis in the kidneys either uni- or bilaterally when using Dll1Cre. The abnormal kidney phenotype becomes prominent at E16.5, which approximates the time when urine production begins in the mouse embryonic kidney, and is associated with a dramatic increase in apoptosis only in mutant kidneys with hydronephrosis. Methylene blue dye injection and histologic examination reveal that aberrant cell death likely results from urine toxicity due to an abnormal ureter-bladder connection. This study shows that Wnt5a is not required for development of the renal medulla and that loss of the renal medullary region in the Wnt5a-deleted kidney is caused by an abnormal ureter-bladder connection. Published by Elsevier B.V.

Healthy Kidneys (A Cup of Health with CDC)

Centers for Disease Control (CDC) Podcasts

2017-03-02

Kidneys that function properly are critical for maintaining good health, however, more than one in seven American adults have kidney disease and most aren’t aware of their condition. In this podcast, Nilka Rios Burrows discusses the importance of maintaining healthy kidneys.  Created: 3/2/2017 by MMWR.   Date Released: 3/2/2017.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Science.gov (United States)

Rota, Paola; Cirillo, Federica; Piccoli, Marco; Gregorio, Antonio; Tettamanti, Guido; Allevi, Pietro; Anastasia, Luigi

2015-10-05

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Vivo Clonal Analysis Reveals Lineage-Restricted Progenitor Characteristics in Mammalian Kidney Development, Maintenance, and Regeneration

Directory of Open Access Journals (Sweden)

Yuval Rinkevich

2014-05-01

Full Text Available The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

On the nanotoxicity of PAMAM dendrimers: Superfect® stimulates the EGFR-ERK1/2 signal transduction pathway via an oxidative stress-dependent mechanism in HEK 293 cells.

Science.gov (United States)

Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Yousif, Mariam H M; Benter, Ibrahim F

2013-05-01

Polyamidoamine (PAMAM) dendrimers are cationic branch-like macromolecules that may serve as drug delivery systems for gene-based therapies such as RNA interference. For their safe use in the clinic, they should ideally only enhance drug delivery to target tissues and exhibit no adverse effects. However, little is known about their toxicological profiles in terms of their interactions with cellular signal transduction pathways such as the epidermal growth factor receptor (EGFR). The EGFR is an important signaling cascade that regulates cell growth, differentiation, migration, survival and apoptosis. Here, we investigated the impact of naked, unmodified Superfect (SF), a commercially available generation 6 PAMAM dendrimer, on the epidermal growth factor receptor (EGFR) tyrosine kinase-extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway in human embryonic kidney (HEK 293) cells. At concentrations routinely used for transfection, SF exhibited time and dose-dependent stimulation of EGFR and ERK1/2 phosphorylation whereas AG1478, a selective EGFR tyrosine kinase antagonist, inhibited EGFR-ERK1/2 signaling. SF-induced phosphorylation of EGFR for 1h was partly reversible upon removal of the dendrimer and examination of cells 24 later. Co-treatment of SF with epidermal growth factor (EGF) ligand resulted in greater EGFR stimulation than either agent alone implying that the stimulatory effects of SF and the ligand are synergistic. Dendrimer-induced stimulation of EGFR-ERK1/2 signaling could be attenuated by the antioxidants apocynin, catalase and tempol implying that an oxidative stress dependent mechanism was involved. These results show for the first time that PAMAM dendrimers, aside from their ability to improve drug delivery, can modulate the important EGFR-ERK1/2 cellular signal transduction pathway - a novel finding that may have a bearing on their safe application as drug delivery systems. Copyright © 2013 Elsevier B.V. All rights reserved.

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway

International Nuclear Information System (INIS)

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-01-01

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis.

A spatially-averaged mathematical model of kidney branching morphogenesis

KAUST Repository

Zubkov, V.S.

2015-08-01

© 2015 Published by Elsevier Ltd. Kidney development is initiated by the outgrowth of an epithelial ureteric bud into a population of mesenchymal cells. Reciprocal morphogenetic responses between these two populations generate a highly branched epithelial ureteric tree with the mesenchyme differentiating into nephrons, the functional units of the kidney. While we understand some of the mechanisms involved, current knowledge fails to explain the variability of organ sizes and nephron endowment in mice and humans. Here we present a spatially-averaged mathematical model of kidney morphogenesis in which the growth of the two key populations is described by a system of time-dependant ordinary differential equations. We assume that branching is symmetric and is invoked when the number of epithelial cells per tip reaches a threshold value. This process continues until the number of mesenchymal cells falls below a critical value that triggers cessation of branching. The mathematical model and its predictions are validated against experimentally quantified C57Bl6 mouse embryonic kidneys. Numerical simulations are performed to determine how the final number of branches changes as key system parameters are varied (such as the growth rate of tip cells, mesenchyme cells, or component cell population exit rate). Our results predict that the developing kidney responds differently to loss of cap and tip cells. They also indicate that the final number of kidney branches is less sensitive to changes in the growth rate of the ureteric tip cells than to changes in the growth rate of the mesenchymal cells. By inference, increasing the growth rate of mesenchymal cells should maximise branch number. Our model also provides a framework for predicting the branching outcome when ureteric tip or mesenchyme cells change behaviour in response to different genetic or environmental developmental stresses.

A spatially-averaged mathematical model of kidney branching morphogenesis

KAUST Repository

Zubkov, V.S.; Combes, A.N.; Short, K.M.; Lefevre, J.; Hamilton, N.A.; Smyth, I.M.; Little, M.H.; Byrne, H.M.

2015-01-01

© 2015 Published by Elsevier Ltd. Kidney development is initiated by the outgrowth of an epithelial ureteric bud into a population of mesenchymal cells. Reciprocal morphogenetic responses between these two populations generate a highly branched epithelial ureteric tree with the mesenchyme differentiating into nephrons, the functional units of the kidney. While we understand some of the mechanisms involved, current knowledge fails to explain the variability of organ sizes and nephron endowment in mice and humans. Here we present a spatially-averaged mathematical model of kidney morphogenesis in which the growth of the two key populations is described by a system of time-dependant ordinary differential equations. We assume that branching is symmetric and is invoked when the number of epithelial cells per tip reaches a threshold value. This process continues until the number of mesenchymal cells falls below a critical value that triggers cessation of branching. The mathematical model and its predictions are validated against experimentally quantified C57Bl6 mouse embryonic kidneys. Numerical simulations are performed to determine how the final number of branches changes as key system parameters are varied (such as the growth rate of tip cells, mesenchyme cells, or component cell population exit rate). Our results predict that the developing kidney responds differently to loss of cap and tip cells. They also indicate that the final number of kidney branches is less sensitive to changes in the growth rate of the ureteric tip cells than to changes in the growth rate of the mesenchymal cells. By inference, increasing the growth rate of mesenchymal cells should maximise branch number. Our model also provides a framework for predicting the branching outcome when ureteric tip or mesenchyme cells change behaviour in response to different genetic or environmental developmental stresses.

Chronic kidney disease: pathological and functional assessment with diffusion tensor imaging at 3T MR

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhiling; Zhang, Jie; Cai, Shifeng; Yuan, Xianshun; Liu, Qingwei [Shandong University, Department of Radiology, Provincial Hospital Affiliated to Shandong University, Jinan (China); Xu, Ying; Wang, Rong [Shandong University, Department of Nephrology, Provincial Hospital Affiliated to Shandong University, Jinan (China); Zhen, Junhui [Shandong University, Department of Pathology, Qilu Hospital of Shandong University, Jinan (China)

2014-10-11

Our objective was to evaluate pathological and functional changes in chronic kidney disease (CKD) using diffusion tensor imaging (DTI) at 3 T. There were fifty-one patients with CKD who required biopsy and 19 healthy volunteers who were examined using DTI at 3 T. The mean values of fractional anisotropy (FA) and the apparent diffusion coefficient (ADC) were obtained from the renal parenchyma (cortex and medulla). Correlations between imaging results and the estimated glomerular filtration rate (eGFR), as well as pathological damage (glomerular lesion and tubulointerstitial injury), were evaluated. The renal cortical FA was significantly lower than the medullary in both normal and affected kidneys (p < 0.001). The parenchymal FA was significantly lower in patients than healthy controls, regardless of whether eGFR was reduced. There were positive correlations between eGFR and FA (cortex, r = 0.689, p = 0.000; and medulla, r = 0.696, p = 0.000), and between eGFR and ADC (cortex, r = 0.310, p = 0.017; and medulla, r = 0.356, p = 0.010). Negative correlations were found between FA and the glomerular lesion (cortex, r = -0.499, p = 0.000; and medulla, r = -0.530, p = 0.000), and between FA and tubulointerstitial injury (cortex, r = -0.631, p = 0.000; and medulla, r = -0.724, p = 0.000). DTI is valuable for noninvasive assessment of renal function and pathology in patients with CKD. A decrease in FA could identify the glomerular lesions, tubulointerstitial injuries, and eGFR. (orig.)

Chronic kidney disease: pathological and functional assessment with diffusion tensor imaging at 3T MR

International Nuclear Information System (INIS)

Liu, Zhiling; Zhang, Jie; Cai, Shifeng; Yuan, Xianshun; Liu, Qingwei; Xu, Ying; Wang, Rong; Zhen, Junhui

2015-01-01

Our objective was to evaluate pathological and functional changes in chronic kidney disease (CKD) using diffusion tensor imaging (DTI) at 3 T. There were fifty-one patients with CKD who required biopsy and 19 healthy volunteers who were examined using DTI at 3 T. The mean values of fractional anisotropy (FA) and the apparent diffusion coefficient (ADC) were obtained from the renal parenchyma (cortex and medulla). Correlations between imaging results and the estimated glomerular filtration rate (eGFR), as well as pathological damage (glomerular lesion and tubulointerstitial injury), were evaluated. The renal cortical FA was significantly lower than the medullary in both normal and affected kidneys (p < 0.001). The parenchymal FA was significantly lower in patients than healthy controls, regardless of whether eGFR was reduced. There were positive correlations between eGFR and FA (cortex, r = 0.689, p = 0.000; and medulla, r = 0.696, p = 0.000), and between eGFR and ADC (cortex, r = 0.310, p = 0.017; and medulla, r = 0.356, p = 0.010). Negative correlations were found between FA and the glomerular lesion (cortex, r = -0.499, p = 0.000; and medulla, r = -0.530, p = 0.000), and between FA and tubulointerstitial injury (cortex, r = -0.631, p = 0.000; and medulla, r = -0.724, p = 0.000). DTI is valuable for noninvasive assessment of renal function and pathology in patients with CKD. A decrease in FA could identify the glomerular lesions, tubulointerstitial injuries, and eGFR. (orig.)

Healthy Kidneys (A Cup of Health with CDC)

Centers for Disease Control (CDC) Podcasts

2014-03-13

Kidneys serve as the body’s filtering system, removing waste and excess water from the blood. If your kidneys are damaged or don’t function properly, you can have severe health problems. In this podcast, Nilka Rios Burrows discusses the dangers of kidney disease.  Created: 3/13/2014 by MMWR.   Date Released: 3/13/2014.

A higher risk of acute rejection of human kidney allografts can be predicted from the level of CD45RC expressed by the recipients' CD8 T cells.

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Laurence Ordonez

Full Text Available Although transplantation is the common treatment for end-stage renal failure, allograft rejection and marked morbidity from the use of immunosuppressive drugs remain important limitations. A major challenge in the field is to identify easy, reliable and noninvasive biomarkers allowing the prediction of deleterious alloreactive immune responses and the tailoring of immunosuppressive therapy in individuals according to the rejection risk. In this study, we first established that the expression of the RC isoform of the CD45 molecule (CD45RC on CD4 and CD8 T cells from healthy individuals identifies functionally distinct alloreactive T cell subsets that behave differently in terms of proliferation and cytokine secretion. We then investigated whether the frequency of the recipients CD45RC T cell subsets before transplantation would predict acute graft rejection in a cohort of 89 patients who had undergone their first kidney transplantation. We showed that patients exhibiting more than 54.7% of CD8 CD45RC(high T cells before transplantation had a 6 fold increased risk of acute kidney graft rejection. In contrast, the proportions of CD4 CD45RC T cells were not predictive. Thus, a higher risk of acute rejection of human kidney allografts can be predicted from the level of CD45RC expressed by the recipients' CD8 T cells.

EMBRYOLOGICAL ASPECTS OF СONGENITAL ANOMALIES OF THE KIDNEY AND URINARY TRACT (CAKUT: REVIEW

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A. O. Vasilyev

2015-01-01

Full Text Available In clinical practice, urologists and nephrologists abnormalities called structural and / or functional abnormalities of the urinary and reproductive systems, caused by disturbance of embryonic development. A significant increase in the number of birth defects may be due to the fact that in embryogenesis kidney is the target organ for exposure to various damaging factors in nature, among which a special place is occupied by medication and physical status of the mother. Violation of prenatal development of the kidneys can often be combined with defects of the lower urinary tract. This condition is often called CAKUT in the development of the role played by the combination of gene mutations. In this article, we describe the majority of congenital anomalies of the kidneys and urinary tract. Significant improvement in antenatal diagnosis of malformations also contributed to the increase in this indicator. Understanding the embryology urinary organs allows to diagnose disorders in the mother-placenta-fetus system.

Agglomeration, sedimentation, and cellular toxicity of alumina nanoparticles in cell culture medium

International Nuclear Information System (INIS)

Yoon, Dokyung; Woo, Daekwang; Kim, Jung Heon; Kim, Moon Ki; Kim, Taesung; Hwang, Eung-Soo; Baik, Seunghyun

2011-01-01

The cytotoxicity of alumina nanoparticles (NPs) was investigated for a wide range of concentration (25–200 μg/mL) and incubation time (0–72 h) using floating cells (THP-1) and adherent cells (J774A.1, A549, and 293). Alumina NPs were gradually agglomerated over time although a significant portion of sedimentation occurred at the early stage within 6 h. A decrease of the viability was found in floating (THP-1) and adherent (J774A.1 and A549) cells in a dose-dependent manner. However, the time-dependent decrease in cell viability was observed only in adherent cells (J774A.1 and A549), which is predominantly related with the sedimentation of alumina NPs in cell culture medium. The uptake of alumina NPs in macrophages and an increased cell-to-cell adhesion in adherent cells were observed. There was no significant change in the viability of 293 cells. This in vitro test suggests that the agglomeration and sedimentation of alumina NPs affected cellular viability depending on cell types such as monocytes (THP-1), macrophages (J774A.1), lung carcinoma cells (A549), and embryonic kidney cells (293).

Agglomeration, sedimentation, and cellular toxicity of alumina nanoparticles in cell culture medium

Science.gov (United States)

Yoon, Dokyung; Woo, Daekwang; Kim, Jung Heon; Kim, Moon Ki; Kim, Taesung; Hwang, Eung-Soo; Baik, Seunghyun

2011-06-01

The cytotoxicity of alumina nanoparticles (NPs) was investigated for a wide range of concentration (25-200 μg/mL) and incubation time (0-72 h) using floating cells (THP-1) and adherent cells (J774A.1, A549, and 293). Alumina NPs were gradually agglomerated over time although a significant portion of sedimentation occurred at the early stage within 6 h. A decrease of the viability was found in floating (THP-1) and adherent (J774A.1 and A549) cells in a dose-dependent manner. However, the time-dependent decrease in cell viability was observed only in adherent cells (J774A.1 and A549), which is predominantly related with the sedimentation of alumina NPs in cell culture medium. The uptake of alumina NPs in macrophages and an increased cell-to-cell adhesion in adherent cells were observed. There was no significant change in the viability of 293 cells. This in vitro test suggests that the agglomeration and sedimentation of alumina NPs affected cellular viability depending on cell types such as monocytes (THP-1), macrophages (J774A.1), lung carcinoma cells (A549), and embryonic kidney cells (293).

Effects of bisphenol A treatment during pregnancy on kidney development in mice: a stereological and histopathological study.

Science.gov (United States)

Nuñez, P; Fernandez, T; García-Arévalo, M; Alonso-Magdalena, P; Nadal, A; Perillan, C; Arguelles, J

2018-04-01

Bisphenol A (BPA) is a chemical found in plastics that resembles oestrogen in organisms. Developmental exposure to endocrine-disrupting chemicals, such as BPA, increases the susceptibility to type 2 diabetes (T2DM) and cardiovascular diseases. Animal studies have reported a nephron deficit in offspring exposed to maternal diabetes. The aim of this study was to investigate the prenatal BPA exposure effects on nephrogenesis in a mouse model that was predisposed to T2DM. This study quantitatively evaluated the renal structural changes using stereology and histomorphometry methods. The OF1 pregnant mice were treated with a vehicle or BPA (10 or 100 μg/kg/day) during days 9-16 of gestation (early nephrogenesis). The 30-day-old offspring were sacrificed, and tissue samples were collected and prepared for histopathological and stereology studies. Glomerular abnormalities and reduced glomerular formation were observed in the BPA offspring. The kidneys of the BPA10 and BPA100 female offspring had a significantly lower glomerular number and density than those of the CONTROL female offspring. The glomerular histomorphometry revealed a significant difference between the female and male CONTROL offspring for the analysed glomerular parameters that disappeared in the BPA10 and BPA100 offspring. In addition, the kidney histopathological examination showed typical male cuboidal epithelial cells of the Bowman capsule in the female BPA offspring. Exposure to environmentally relevant doses of BPA during embryonic development altered nephrogenesis. These structural changes could be associated with an increased risk of developing cardiometabolic diseases later in life.

The sustained-release behavior and in vitro and in vivo transfection of pEGFP-loaded core-shell-structured chitosan-based composite particles

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Wang, Yun; Lin, Fu-xing; Zhao, Yu; Wang, Mo-zhen; Ge, Xue-wu; Gong, Zheng-xing; Bao, Dan-dan; Gu, Yu-fang

2014-01-01

Novel submicron core-shell-structured chitosan-based composite particles encapsulated with enhanced green fluorescent protein plasmids (pEGFP) were prepared by complex coacervation method. The core was pEGFP-loaded thiolated N-alkylated chitosan (TACS) and the shell was pH- and temperature-responsive hydroxybutyl chitosan (HBC). pEGFP-loaded TACS-HBC composite particles were spherical, and had a mean diameter of approximately 120 nm, as measured by transmission electron microscopy and particle size analyzer. pEGFP showed sustained release in vitro for >15 days. Furthermore, in vitro transfection in human embryonic kidney 293T and human cervix epithelial cells, and in vivo transfection in mice skeletal muscle of loaded pEGFP, were investigated. Results showed that the expression of loaded pEGFP, both in vitro and in vivo, was slow but could be sustained over a long period. pEGFP expression in mice skeletal muscle was sustained for >60 days. This work indicates that these submicron core-shell-structured chitosan-based composite particles could potentially be used as a gene vector for in vivo controlled gene transfection. PMID:25364253

X-ray fluorescence microscopy artefacts in elemental maps of topologically complex samples: Analytical observations, simulation and a map correction method

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Billè, Fulvio; Kourousias, George; Luchinat, Enrico; Kiskinova, Maya; Gianoncelli, Alessandra

2016-08-01

XRF spectroscopy is among the most widely used non-destructive techniques for elemental analysis. Despite the known angular dependence of X-ray fluorescence (XRF), topological artefacts remain an unresolved issue when using X-ray micro- or nano-probes. In this work we investigate the origin of the artefacts in XRF imaging of topologically complex samples, which are unresolved problems in studies of organic matter due to the limited travel distances of low energy XRF emission from the light elements. In particular we mapped Human Embryonic Kidney (HEK293T) cells. The exemplary results with biological samples, obtained with a soft X-ray scanning microscope installed at a synchrotron facility were used for testing a mathematical model based on detector response simulations, and for proposing an artefact correction method based on directional derivatives. Despite the peculiar and specific application, the methodology can be easily extended to hard X-rays and to set-ups with multi-array detector systems when the dimensions of surface reliefs are in the order of the probing beam size.

Mass spectroscopic phosphoprotein mapping of Ral binding protein 1 (RalBP1/Rip1/RLIP76)

International Nuclear Information System (INIS)

Herlevsen, Mikael C.; Theodorescu, Dan

2007-01-01

RalBP1, a multifunctional protein implicated in cancer cell proliferation, radiation and chemoresistance, and ligand dependent receptor internalization, is upregulated in bladder cancer and is a downstream effector of RalB, a GTPase associated with metastasis. RalBP1 can be regulated by phosphorylation by protein kinase C (PKC). No studies have comprehensively mapped RalBP1 phosphorylation sites or whether RalB affects these. We identified 14 phosphorylation sites of RalBP1 in human bladder carcinoma UMUC-3 and embryonic kidney derived 293T cells. The phosphorylated residues are concentrated at the N-terminus. Ten of the first 100 amino acids of the primary structure were phosphorylated. Nine were serine residues, and one a threonine. We evaluated the effect of RalB overexpression on RalBP1 phosphorylation and found the largest change in phosphorylation status at S463 and S645. Further characterization of these sites will provide novel insights on RalBP1 biology, its functional relationship to RalB and possible avenues for therapeutic intervention

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

International Nuclear Information System (INIS)

Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

2008-01-01

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

A rapid and sustained improvement of calcification propensity score (serum T50 ) after successful kidney transplantation: Reanalysis of a randomized controlled trial of ibandronate.

Science.gov (United States)

Smerud, Knut T; Åsberg, Anders; Kile, Håkon; Pasch, Andreas; Dahle, Dag O; Bollerslev, Jens; Godang, Kristin; Hartmann, Anders

2017-12-01

A serum test called T 50 assesses the overall propensity for calcification of the blood and is associated with cardiovascular outcomes. We aimed to examine T 50 over time in kidney transplant recipients and also address any effects of ibandronate. Serum samples taken from kidney transplant patients included in a prospective, randomized placebo controlled study of ibandronate were analyzed in retrospect. Adequate analyses were performed at baseline (approximately 3 weeks after transplantation) in 129 patients, at 10 weeks in 127 patients and at 1 year in 123 patients. There were no statistical differences between ibandronate and placebo treatment in terms of T 50 at 10 weeks (P = .094) or at 1 year (P = .116). Baseline T 50 was a significant covariate (P < .0001) for T 50 scores at 10 weeks and 1 year. In the total cohort, there was a highly significant (P < .0001) increase in T 50 of 26.6% after 10 weeks and T 50 remained stable after 1 year. T 50 change was inversely correlated to phosphate of -0.515 (P < .0001) and to change in serum albumin (P < .03). We found that T 50 increased from baseline to 10 weeks after transplantation with no further change after 1 year. Ibandronate had no effect on T 50 . © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons

International Nuclear Information System (INIS)

Kyriakis, J.M.; Hausman, R.E.; Peterson, S.W.

1987-01-01

The effect of insulin on the appearance of the enzyme choline acetyltransferase in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the 125 I-insulin concentration in the medium during culture, cell-surface insulin receptors decreased by ≅ 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. The findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina

25-Hydroxyvitamin D3 1-Alpha-Hydroxylase-Dependent Stimulation of Renal Klotho Expression by Spironolactone

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Ioana Alesutan

2013-11-01

Full Text Available Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1 by FGF23. Conversely, 1,25(OH2D3 stimulates, by activating the vitamin D3 receptor (Vdr, the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293 or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR or of mineralocorticoid receptor (NR3C2. Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours and mice (8 hours or 5 days. Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours. Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of

10 CFR 431.293 - Materials incorporated by reference.

Science.gov (United States)

2010-01-01

....293 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL.../code_of_federal_regulations/ibr_locations.html. This material is also available for inspection at U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Building Technologies Program, 6th...

Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway.

Science.gov (United States)

Chen, Chunhai; Zhou, Zhou; Zhong, Min; Li, Maoquan; Yang, Xuesen; Zhang, Yanwen; Wang, Yuan; Wei, Aimin; Qu, Mingyue; Zhang, Lei; Xu, Shangcheng; Chen, Shude; Yu, Zhengping

2011-07-01

Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

Metabolomic Profiling in Individuals with a Failing Kidney Allograft.

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Roberto Bassi

Full Text Available Alteration of certain metabolites may play a role in the pathophysiology of renal allograft disease.To explore metabolomic abnormalities in individuals with a failing kidney allograft, we analyzed by liquid chromatography-mass spectrometry (LC-MS/MS; for ex vivo profiling of serum and urine and two dimensional correlated spectroscopy (2D COSY; for in vivo study of the kidney graft 40 subjects with varying degrees of chronic allograft dysfunction stratified by tertiles of glomerular filtration rate (GFR; T1, T2, T3. Ten healthy non-allograft individuals were chosen as controls.LC-MS/MS analysis revealed a dose-response association between GFR and serum concentration of tryptophan, glutamine, dimethylarginine isomers (asymmetric [A]DMA and symmetric [S]DMA and short-chain acylcarnitines (C4 and C12, (test for trend: T1-T3 = p<0.05; p = 0.01; p<0.001; p = 0.01; p = 0.01; p<0.05, respectively. The same association was found between GFR and urinary levels of histidine, DOPA, dopamine, carnosine, SDMA and ADMA (test for trend: T1-T3 = p<0.05; p<0.01; p = 0.001; p<0.05; p = 0.001; p<0.001; p<0.01, respectively. In vivo 2D COSY of the kidney allograft revealed significant reduction in the parenchymal content of choline, creatine, taurine and threonine (all: p<0.05 in individuals with lower GFR levels.We report an association between renal function and altered metabolomic profile in renal transplant individuals with different degrees of kidney graft function.

PLK-1 Silencing in Bladder Cancer by siRNA Delivered With Exosomes.

Science.gov (United States)

Greco, Kristin A; Franzen, Carrie A; Foreman, Kimberly E; Flanigan, Robert C; Kuo, Paul C; Gupta, Gopal N

2016-05-01

To use exosomes as a vector to deliver small interfering ribonucleic acid (siRNA) to silence the polo-like kinase 1 (PLK-1) gene in bladder cancer cells. Exosomes were isolated from both human embryonic kidney 293 (HEK293) cell and mesenchymal stem cell (MSC) conditioned media. Fluorescently labeled exosomes were co-cultured with bladder cancer and normal epithelial cells and uptake was quantified by image cytometry. PLK-1 siRNA and negative control siRNA were loaded into HEK293 and MSC exosomes using electroporation. An invasive bladder cancer cell line (UMUC3) was co-cultured with the electroporated exosomes. Quantitative reverse transcriptase polymerase chain reaction was performed. Protein analysis was performed by Western blot. Annexin V staining and MTT assays were used to investigate effects on apoptosis and viability. Bladder cancer cell lines internalize an increased percentage of HEK293 exosomes when compared to normal bladder epithelial cells. Treatment of UMUC3 cells with exosomes electroporated with PLK-1 siRNA achieved successful knockdown of PLK-1 mRNA and protein when compared to cells treated with negative control exosomes. HEK293 and MSC exosomes were effectively used as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in selective gene silencing of PLK-1. The use of exosomes as a delivery vector for potential intravesical therapy is attractive. Copyright © 2016 Elsevier Inc. All rights reserved.

Microencapsulation of Lefty-secreting engineered cells for pulmonary fibrosis therapy in mice.

Science.gov (United States)

Ma, Hongge; Qiao, Shupei; Wang, Zeli; Geng, Shuai; Zhao, Yufang; Hou, Xiaolu; Tian, Weiming; Chen, Xiongbiao; Yao, Lifen

2017-05-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that causes unremitting deposition of extracellular matrix proteins, thus resulting in distortion of the pulmonary architecture and impaired gas exchange. Associated with high morbidity and mortality, IPF is generally refractory to current pharmacological therapies. Lefty A, a potent inhibitor of transforming growth factor-β signaling, has been shown to have promising antifibrotic ability in vitro for the treatment of renal fibrosis and other potential organ fibroses. Here, we determined whether Lefty A can attenuate bleomycin (BLM)-induced pulmonary fibrosis in vivo based on a novel therapeutic strategy where human embryonic kidney 293 (HEK293) cells are genetically engineered with the Lefty A-associated GFP gene. The engineered HEK293 cells were encapsulated in alginate microcapsules and then subcutaneously implanted in ICR mice that had 1 wk earlier been intratracheally administered BLM to induce pulmonary fibrosis. The severity of fibrosis in lung tissue was assessed using pathological morphology and collagen expression to examine the effect of Lefty A released from the microencapsulated cells. The engineered HEK293 cells with Lefty A significantly reduced the expression of connective tissue growth factor and collagen type I mRNA, lessened the morphological fibrotic effects induced by BLM, and increased the expression of matrix metalloproteinase-9. This illustrates that engineered HEK293 cells with Lefty A can attenuate pulmonary fibrosis in vivo, thus providing a novel method to treat human pulmonary fibrotic disease and other organ fibroses. Copyright © 2017 the American Physiological Society.

Functional expression of the Na-K-2Cl cotransporter NKCC2 in mammalian cells fails to confirm the dominant-negative effect of the AF splice variant.

Science.gov (United States)

Hannemann, Anke; Christie, Jenny K; Flatman, Peter W

2009-12-18

The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive (86)Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

Science.gov (United States)

Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

Conditional ablation of glycogen synthase kinase 3β in postnatal mouse kidney.

Science.gov (United States)

Ge, Yan; Si, Jin; Tian, Li; Zhuang, Shougang; Dworkin, Lance D; Gong, Rujun

2011-01-01

Glycogen synthase kinase (GSK)3 is a ubiquitously expressed serine/threonine kinase existing in two isoforms, namely GSK3α and GSK3β. Aside from the long-recognized role in insulin signal transduction and glycogen biosynthesis, GSK3β has been recently coined as a master control molecule in nuclear factor-κB activation and inflammatory kidney injury. Nevertheless, previous studies are less conclusive because they relied greatly on small molecule inhibitors, which lack selectivity and barely distinguish between the GSK3 isoforms. In addition, early embryonic lethality after global knockout of GSK3β precludes interrogation of the biological role of GSK3β in the adult kidney. To circumvent these issues, the Cre/loxP system was used to generate a conditional knockout mouse model in which the GSK3β gene was specifically deleted in kidney cortical tubules at postnatal mature stage. Kidney-specific ablation of GSK3β resulted in a phenotype no different from control littermates. Knockout mice (KO) were viable and exhibited normal development and normal kidney physiology in terms of kidney function, urine albumin excretion, and urine-concentrating ability. It is noteworthy that apart from normal glomerular and tubulointerstitial morphology, the kidneys from KO demonstrated more glycogen accumulation in the renal cortical tubules as assessed by both periodic acid-Schiff staining for light microscopy and direct biochemical assay, consistent with an elevated glycogen synthetic activity as evidenced by diminished inhibitory phosphorylation of glycogen synthase that occurred subsequent to GSK3β ablation. This finding was further validated by electron microscopic observations of increased deposition of glycogen particles in the renal tubules of KO, suggesting that GSK3α could not fully compensate for the loss of GSK3β in regulating glycogen metabolism in the kidney. Collectively, our study suggests that kidney-specific ablation of GSK3β barely affects kidney function

Physicochemical properties of green solvent 1-ethyl-3-methylimidazolium tetrafluoroborate with aniline from T = (293.15 to 323.15) K at atmospheric pressure

International Nuclear Information System (INIS)

Srinivasa Rao, V.; Vijaya Krishna, T.; Madhu Mohan, T.; Madhusudana Rao, P.

2017-01-01

Highlights: • Nature of interactions in the binary mixture of [Emim][BF 4 ] + aniline are studied. • Excess properties are calculated and correlated using Redlich–Kister equation. • Temperature dependence of the calculated thermophysical propertiesis discussed. - Abstract: Density and speed of sound values are measured for the binary mixture of 1-ethyl-3-methylimidazolium tetrafluoroborate and aniline over the entire range of mole fraction at temperatures from T = (293.15 to 323.15) K under atmospheric pressure. Using the basic experimental results for the molar volume, isentropic compressibility, molar isentropic compressibility, inter molecular free length, excess molar volume, excess isentropic compressibility, excess molar isentropic compressibility and excess intermolecular free length, these values are calculated. The partial molar volumes and partial molar isentropic compressibilities at infinite dilutions have also been calculated. The trends of variation of the properties have been interpreted in light of the solute–solvent interactions occurring in the system. The excess values are fitted to Redlich–Kister polynomial equation to estimate the binary coefficients and standard deviation between the experimental and calculated values. Further, the molecular interactions in the binary mixture system are analysed using the experimental FT-IR spectrum recorded at room temperature.

Density, dynamic viscosity, and derived properties of binary mixtures of methanol or ethanol with water, ethyl acetate, and methyl acetate at T (293.15, 298.15, and 303.15) K

Energy Technology Data Exchange (ETDEWEB)

Gonzalez, Begona; Calvar, Noelia; Gomez, Elena [Chemical Engineering Department, University of Vigo, 36200 Vigo (Spain); Dominguez, Angeles [Chemical Engineering Department, University of Vigo, 36200 Vigo (Spain)], E-mail: admguez@uvigo.es

2007-12-15

Densities and dynamic viscosities for methanol or ethanol with water, ethyl acetate, and methyl acetate at several temperatures T = (293.15, 298.15, and 303.15) K have been measured over the whole composition range and 0.1 MPa, along with the properties of the pure components. Excess molar volumes, viscosity deviations, and excess free energy of activation for the binary systems at the above-mentioned temperatures, were calculated and fitted to the Redlich-Kister equation to determine the fitting parameters and the root-mean-square deviations. UNIQUAC equation was used to correlate the experimental viscosity data. The UNIFAC-VISCO method and ASOG-VISCO method, based on contribution groups, were used to predict the dynamic viscosities of the binary mixtures.

Density, dynamic viscosity, and derived properties of binary mixtures of methanol or ethanol with water, ethyl acetate, and methyl acetate at T (293.15, 298.15, and 303.15) K

International Nuclear Information System (INIS)

Gonzalez, Begona; Calvar, Noelia; Gomez, Elena; Dominguez, Angeles

2007-01-01

Densities and dynamic viscosities for methanol or ethanol with water, ethyl acetate, and methyl acetate at several temperatures T = (293.15, 298.15, and 303.15) K have been measured over the whole composition range and 0.1 MPa, along with the properties of the pure components. Excess molar volumes, viscosity deviations, and excess free energy of activation for the binary systems at the above-mentioned temperatures, were calculated and fitted to the Redlich-Kister equation to determine the fitting parameters and the root-mean-square deviations. UNIQUAC equation was used to correlate the experimental viscosity data. The UNIFAC-VISCO method and ASOG-VISCO method, based on contribution groups, were used to predict the dynamic viscosities of the binary mixtures

Mechanobiology of embryonic limb development.

Science.gov (United States)

Nowlan, Niamh C; Murphy, Paula; Prendergast, Patrick J

2007-04-01

Considerable evidence exists to support the hypothesis that mechanical forces have an essential role in healthy embryonic skeletal development. Clinical observations and experimental data indicate the importance of muscle contractions for limb development. However, the influence of these forces is seldom referred to in biological descriptions of bone development, and perhaps this is due to the fact that the hypothesis that mechanical forces are essential for normal embryonic skeletal development is difficult to test and elaborate experimentally in vivo, particularly in humans. Computational modeling has the potential to address this issue by simulating embryonic growth under a range of loading conditions but the potential of such models has yet to be fully exploited. In this article, we review the literature on mechanobiology of limb development in three main sections: (a) experimental alteration of the mechanical environment, (b) mechanical properties of embryonic tissues, and (c) the use of computational models. Then we analyze the main issues, and suggest how experimental and computational fields could work closer together to enhance our understanding of mechanobiology of the embryonic skeleton.

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

Science.gov (United States)

Ji, Li; Zhu, Huayuan; Chen, Hong; Fan, Wenyong; Chen, Junjie; Chen, Jing; Zhu, Guoqing; Wang, Juejin

2015-12-01

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

NARCIS (Netherlands)

Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

2008-01-01

Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

The volumetric properties of (1,2-propanediol carbonate+benzene, or toluene, or styrene) binary mixtures at temperatures from T=293.15 K to T=353.15 K

International Nuclear Information System (INIS)

Wang Haijun; Wu Yonghua; Huang Jihou

2006-01-01

The densities and excess molar volumes V m E for binary liquid mixtures of (1,2-propanediol carbonate+benzene, or toluene, or ethylbenzene, or styrene) have been measured as a function of compositions using a vibrating-tube densimeter in the temperature range of (293.15 to 353.15) K and at atmospheric pressure. The V m E results were correlated using the fourth-order Redlich-Kister equation. It was found that the V m E in these systems studied increases with rising temperature

Cytoprotective effect of glutaraldehyde erythropoietin on HEK293 kidney cells after silver nanoparticle exposure

Directory of Open Access Journals (Sweden)

Sooklert K

2016-02-01

Full Text Available Kanidta Sooklert,1,2 Supreecha Chattong,3 Krissanapong Manotham,3 Chawikan Boonwong,1 I-yanut Klaharn,1 Depicha Jindatip,4 Amornpun Sereemaspun1,4 1Nanobiomedicine Laboratory, Department of Anatomy, Faculty of Medicine, 2Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, 3Renal Unit, Department of Medicine, Lerdsin General Hospital, 4Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, ThailandAbstract: The toxic effects from exposure to silver nanoparticles (AgNPs, which are broadly present in many consumer products, have long raised concerns. Many studies have focused on the mechanisms of nanosilver, which cause toxicity in human cells, but little is known about prevention of this type of injury. This study investigated the in vitro effects of glutaraldehyde erythropoietin (GEPO, a cytoprotective compound derived from erythropoietin, in terms of cell protection against AgNP-induced injury. HEK293 cells were pretreated with or without GEPO before administration of AgNPs. The protective effects of GEPO in this cell line were assessed by the percentage of viable cells, alterations of cell morphology, and the proliferative capability of the cells. In addition, we assessed the role of GEPO in lowering cellular oxidative stress and regulating expression of the anti-apoptotic protein Bcl2. The results showed rescue effects on the percentage of viable and proliferative cells among GEPO pretreated cells. Pretreatment with GEPO maintained the normal cell shape and ultrastructural morphology. Moreover, GEPO reduced the generation of reactive oxygen species in cells and activated expression of Bcl2, which are the major mechanisms in protection against cellular toxicity induced by AgNPs. In conclusion, our study showed that the cytotoxic effects from exposure to AgNPs can be prevented by GEPO. Keywords: glutaraldehyde erythropoietin, silver nanoparticles, cytoprotection

The developmental programme for genesis of the entire kidney is recapitulated in Wilms tumour

Science.gov (United States)

Anaka, Matthew R.; Morison, Ian M.; Reeve, Anthony E.

2017-01-01

Wilms tumour (WT) is an embryonal tumour that recapitulates kidney development. The normal kidney is formed from two distinct embryological origins: the metanephric mesenchyme (MM) and the ureteric bud (UB). It is generally accepted that WT arises from precursor cells in the MM; however whether UB-equivalent structures participate in tumorigenesis is uncertain. To address the question of the involvement of UB, we assessed 55 Wilms tumours for the molecular features of MM and UB using gene expression profiling, immunohistochemsitry and immunofluorescence. Expression profiling primarily based on the Genitourinary Molecular Anatomy Project data identified molecular signatures of the UB and collecting duct as well as those of the proximal and distal tubules in the triphasic histology group. We performed immunolabeling for fetal kidneys and WTs. We focused on a central epithelial blastema pattern which is the characteristic of triphasic histology characterized by UB-like epithelial structures surrounded by MM and MM-derived epithelial structures, evoking the induction/aggregation phase of the developing kidney. The UB-like epithelial structures and surrounding MM and epithelial structures resembling early glomerular epithelium, proximal and distal tubules showed similar expression patterns to those of the developing kidney. These observations indicate WTs can arise from a precursor cell capable of generating the entire kidney, such as the cells of the intermediate mesoderm from which both the MM and UB are derived. Moreover, this provides an explanation for the variable histological features of mesenchymal to epithelial differentiation seen in WT. PMID:29040332

Healthy Kidneys (A Cup of Health with CDC)

Centers for Disease Control (CDC) Podcasts

Kidney disease is among the leading causes of death in the U.S. More than one in 10 people over the age of 20 are impacted by this condition, and most don’t know it. In this podcast, Nilka Rios Burrows discusses the risks for kidney disease.

Effect of turmeric and its active principle curcumin on t(3)-induced oxidative stress and hyperplasia in rat kidney: a comparison.

Science.gov (United States)

Samanta, Luna; Panigrahi, Jogamaya; Bhanja, Shravani; Chainy, Gagan B N

2010-10-01

The present study was designed to compare the potential of turmeric and its active principle curcumin on T(3)-induced oxidative stress and hyperplasia. Adult male Wistar strain rats were rendered hyperthyroid by T(3) treatment (10 μg · 100 g(-1) · day(-1) intraperitoneal for 15 days in 0.1 mM NaOH) to induce renal hyperplasia. Another two groups were treated similarly with T(3) along with either turmeric or curcumin (30 mg kg(-1) body weight day(-1) orally for 15 days). The results indicate that T(3) induces both hypertrophy and hyperplasia in rat kidney as evidenced by increase in cell number per unit area, increased protein content, tubular dilation and interstitial edema. These changes were accompanied by increased mitochondrial lipid peroxidation and superoxide dismutase activity without any change in catalase activity and glutathione content suggesting an oxidative predominance. Both turmeric and curcumin were able to restore the level of mitochondrial lipid peroxidation and superoxide dismutase activity in the present dose schedule. T(3)-induced histo-pathological changes were restored with turmeric treatment whereas curcumin administration caused hypoplasia. This may be due to lower concentration of curcumin in the whole turmeric. Thus it is hypothesized that regulation of cell cycle in rat kidney by T(3) is via reactive oxygen species and curcumin reveres the changes by scavenging them. Although the response trends are comparable for both turmeric and curcumin, the magnitude of alteration is more in the later. Turmeric in the current dose schedule is a safer bet than curcumin in normalizing the T(3)-induced hyperplasia may be due to the lower concentration of the active principle in the whole spice.

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells.

Science.gov (United States)

Nishiyama, Takahito; Izawa, Tadashi; Usami, Mami; Ohnuma, Tomokazu; Ogura, Kenichiro; Hiratsuka, Akira

2010-04-09

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process. Copyright 2009 Elsevier Inc. All rights reserved.

Periconceptional maternal one-carbon biomarkers are associated with embryonic development according to the Carnegie stages.

Science.gov (United States)

Parisi, F; Rousian, M; Koning, A H J; Willemsen, S P; Cetin, I; Steegers-Theunissen, R P M

2017-03-01

Is periconceptional maternal one-carbon (I-C) metabolism associated with embryonic morphological development in non-malformed ongoing pregnancies? Serum vitamin B12, red blood cell (RBC) folate and plasma total homocysteine (tHcy) are associated with embryonic development according to the Carnegie stages. Derangements in maternal I-C metabolism affect reproductive and pregnancy outcomes, as well as future health of the offspring. Between 2010 and 2014, women with singleton ongoing pregnancies were enrolled in a prospective periconceptional cohort study. A total of 234 pregnancies, including 138 spontaneous or IUI pregnancies with strict pregnancy dating and 96 pregnancies derived from IVF, ICSI or cryopreserved embryo transfer (IVF/ICSI pregnancies), underwent longitudinal transvaginal three-dimensional ultrasound (3D US) scans from 6+0 up to 10+2 weeks of gestation. Carnegie stages were defined using internal and external morphologic criteria in a virtual reality system. Maternal venous blood samples were collected at enrollment for serum vitamin B12, RBC folate and plasma tHcy assessment. Associations between biomarker concentrations and longitudinal Carnegie stages were investigated using linear mixed models. We performed a median of three 3D US scans per pregnancy (range 1-5) resulting in 600 good quality data sets for the Carnegie stage annotation (80.5%). Vitamin B12 was positively associated with embryonic development in the total study population (β = 0.001 (95% CI: 0.000; 0.002), P Carnegie stages only in IVF/ICSI pregnancies (β = 0.001 (95% CI: 0.0005; 0.0015), P < 0.05). In this group, low RBC folate concentrations (-2SD, 875.4 nmol/l) were associated with a 1.8-day delay (95% CI: 1.7-1.8) in development compared with high concentrations (+2SD, 2119.9 nmol/l). tHcy was negatively associated with embryonic development in the total study population (β = -0.08 (95% CI: -0.14; -0.02), P < 0.01), as well as in the IVF/ICSI subgroup (β = -0.08 (95% CI: -0

Hyperthyroidism as a clinical manifestation of a embryonal carcinoma of the testis.

Science.gov (United States)

Arrabal-Polo, M A; Jimenez-Pacheco, A; Arrabal-Martin, M; Moreno-Jimenez, J; Gutierrez-Tejero, F; Galisteo-Moya, R; Zuluaga-Gomez, A

2012-01-01

This case report describes a case of hyperthyroidism as manifestation of an embryonal carcinoma, and illustrates the causes that led to it. The case describes a 33-year-old male patient who complained of chest pain, palpitations, mild dyspnoea, and weight loss. Blood analysis reveals high levels of human chorionic gonadotropin (833818 mlU/ml), T3 (16.90 pg/ml), and T4 (7.77 ng/dl), as well as a fall of TSH (0.01 ulU/ml). Physical examination and imaging procedures confirm the occurrence of a left testicular tumour associated with numerous lung, hepatic and retroperitoneal metastases. Treatment with carbimazol and propanolol is established to manage hyperthyroidism, and an urgent orchiectomy is performed; the histologic diagnosis confirms an embryonal carcinoma (organoid type), but the patient died unexpectedly 24 hours later after having suffered sudden dyspnoea, tachypnoea, and tachyarrhythmia. Hyperthyroidism is a rare manifestation of a testicular tumour that should be borne in mind with regard to the patient's symptomatology and HCG levels.

[Outcome of living kidney donors for transplantation].

Science.gov (United States)

Lanot, Antoine; Bouvier, Nicolas; Chatelet, Valérie; Lecouf, Angélique; Tillou, Xavier; Hurault de Ligny, Bruno

2017-11-01

Nowadays, several treatments exist to treat terminal chronic renal failure. Best results for the recipients are obtained with kidney transplantation concerning mortality and quality of life. Transplantation is also the cheaper option for society. Living kidney donation raises the issue of the becoming of the donor, an absolutely healthy subject who gets to a surgical procedure. The becoming of living kidney donors has been compared with the one of controls subjects in several studies. The evaluations focused on the complications of nephrectomy in the short and long-term: kidney failure, hypertension, proteinuria, possibility of pregnancy, quality of life, and mortality. The first results did not show any risk linked to kidney donation, compared to general population. However, since 2013, kidney donors were found at higher risk for kidney failure and even for mortality, compared with controls selected like donor candidates. The risk of kidney donation is nevertheless acceptable and minimal, on the condition of rigorous selection of candidates and regular follow-up. Copyright © 2017 Société francophone de néphrologie, dialyse et transplantation. Published by Elsevier Masson SAS. All rights reserved.

Phenotype-Genotype Analysis of Chinese Patients with Early-Onset LMNA-Related Muscular Dystrophy.

Directory of Open Access Journals (Sweden)

Dandan Tan

Full Text Available This study aimed to analyze the correlation between the phenotype and genotype of Chinese patients with early-onset lamin A (LMNA-related muscular dystrophy (MD. The clinical and myopathological data of 21 Chinese pediatric patients with early-onset LMNA-related MD were collected and analyzed. LMNA gene mutation analysis was performed by direct sequencing of genomic DNA. Sublocalization of wild-type and mutant proteins were observed by immunofluorescence using cultured fibroblasts and human embryonic kidney 293 (HEK 293 cell. Seven patients were diagnosed with Emery-Dreifuss muscular dystrophy (EDMD and 14 were diagnosed with LMNA-associated congenital muscular dystrophy (L-CMD. Four biopsy specimens from the L-CMD cases exhibited inflammatory changes. Abnormal nuclear morphology was observed with both transmission electron microscopy and lamin A/C staining. We identified 10 novel and nine known LMNA gene mutations in the 21 patients. Some mutations (c.91G>A, c.94_96delAAG, c.116A>G, c.745C>T, c.746G>A, and c.1580G>C were well correlated with EDMD or L-CMD. LMNA-related MD has a common symptom triad of muscle weakness, joint contractures, and cardiac involvement, but the severity of symptoms and disease progression differ greatly. Inflammatory change in biopsied muscle is a characteristic of early-stage L-CMD. Phenotype-genotype analysis determines that some mutations are well correlated with LMNA-related MD.

Cryptorchidism as a caudal developmental field defect. A new description of cryptorchidism associated with malformations and dysplasias of the kidneys, the ureters and the spine from T10 to S5

DEFF Research Database (Denmark)

Cortes, Dina; Thorup, J M; Beck, Bjarne Lomholdt

1998-01-01

Cryptorchidism is a feature of abnormalities in the hypothalamo-pituitary-testicular axis, and almost all disorders of sexual differentiation in which a testis is present. We found cryptorchidism to be associated with malformations and dysplasias of the kidneys, the ureters and the spine from T10...... field defect. Study of cryptorchid patients exhibiting malformations or dysplasias of the kidneys, the ureters or the spine from T10 to S5 is essential in order to isolate new genetic disorders and to spot environmental factors causing cryptorchidism....

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

Science.gov (United States)

Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I; Lanciego, José L; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

2017-01-01

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

Directory of Open Access Journals (Sweden)

Eva Martínez-Pinilla

2017-10-01

Full Text Available The mechanism of action of cannabidiol (CBD, the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs; however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Experimental excess molar properties of binary mixtures of (3-amino-1-propanol + isobutanol, 2-propanol) at T = (293.15 to 333.15) K and modelling the excess molar volume by Prigogine–Flory–Patterson theory

International Nuclear Information System (INIS)

Kermanpour, F.; Niakan, H.Z.

2012-01-01

Highlights: ► Density and viscosity of binary mixtures of propanol derivatives were measured at T = (293.15 to 333.15) K. ► The excess molar properties were calculated from these experimental data and correlated by Redlich–Kister equation. ► The PFP model was applied for correlating the excess molar volumes. - Abstract: Density and viscosity of binary mixtures of (x 1 3-amino-1-propanol + x 2 isobutanol) and (x 1 3-amino-1-propanol + x 2 2-propanol) were measured over the entire composition range and from temperatures (293.15 to 333.15) K at ambient pressure. The excess molar volumes and viscosity deviations were calculated and correlated by the Redlich–Kister (RK) equation. The thermal expansion coefficient and its excess value, isothermal coefficient of excess molar enthalpy, and excess partial molar volumes were determined by using the experimental values of density and are described as a function of composition and temperature. The excess molar volumes are negative over the entire mole fraction range for both mixtures and increase with increasing temperature. The excess molar volumes obtained were correlated by the Prigogine–Flory–Patterson (PFP) model. The viscosity deviations of the binary mixtures are negative over the entire composition range and decrease with increasing temperature.

Thyroid function, reduced kidney function and incident chronic kidney disease in a community-based population: the Atherosclerosis Risk in Communities study.

Science.gov (United States)

Schultheiss, Ulla T; Daya, Natalie; Grams, Morgan E; Seufert, Jochen; Steffes, Michael; Coresh, Josef; Selvin, Elizabeth; Köttgen, Anna

2017-11-01

Reduced kidney function is a common public health problem that increases risk for a wide variety of adverse outcomes, making the identification of potentially modifiable factors associated with the development of incident chronic kidney disease (CKD) important. Alterations in the hypothalamic-pituitary-thyroid axis have been linked to reduced kidney function, but the association of thyroid function with the development of incident CKD is largely uncharacterized. Concentrations of thyroid stimulating hormone (TSH), free thyroxine (FT4), triiodothyronine (T3) and thyroid peroxidase antibody (TPOAb) were quantified in 12 785 black and white participants of the ongoing community-based prospective Atherosclerosis Risk in Communities study. Thyroid markers and clinical categories of thyroid dysfunction (euthyroidism, combined subclinical and overt hypothyroidism, combined subclinical and overt hyperthyroidism) were also evaluated for their association with reduced kidney function (estimated glomerular filtration rate kidney function at study baseline. The clinical entities hypothyroidism and hyperthyroidism were also associated with higher odds of baseline reduced kidney function, but this was not significant. However, none of the markers of thyroid function nor different clinical categories of thyroid dysfunction (hypothyroidism, hyperthyroidism or TPOAb positivity) were associated with incident CKD in adjusted analyses. Elevated TSH, FT4 and reduced T3 concentrations were associated with reduced kidney function cross-sectionally. The lack of association with the development of incident CKD suggests that altered thyroid function in the general population is not causally related to CKD development, but screening for thyroidal status may be especially relevant in persons with reduced kidney function. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Practice Patterns in the Treatment and Monitoring of Acute T Cell-Mediated Kidney Graft Rejection in Canada.

Science.gov (United States)

Leblanc, Julie; Subrt, Peter; Paré, Michèle; Hartell, David; Sénécal, Lynne; Blydt-Hansen, Tom; Cardinal, Héloïse

2018-01-01

One of the goals of the Canadian National Transplant Research Program (CNTRP) is to develop novel therapies for acute rejection that could positively affect graft outcomes with greater efficacy or less toxicity. To develop innovative management strategies for kidney graft rejection, new modalities need to be compared with current clinical practices. However, there are no standardized practices concerning the management of acute T cell-mediated rejection (TCMR). To describe clinicians' practice patterns in the diagnosis, treatment, and monitoring of acute TCMR in Canada. Survey. Canadian transplant nephrologists and transplant surgeons involved in the management of acute TCMR. We developed an anonymous, web-based survey consisting of questions related to the diagnosis, treatment, and monitoring of TCMR. The survey was disseminated on 3 occasions between June and October 2016 through the Canadian Society of Transplantation (CST) kidney group electronic mailing list. Forty-seven respondents, mostly transplant nephrologists (97%), originating from at least 18 of the 25 Canadian centers offering adult or pediatric kidney transplantation, participated in the study. Surveillance biopsies were used by 28% of respondents to screen for kidney graft rejection. High-dose steroids were used by most of the respondents to treat clinical and subclinical Banff grade 1A and 1B rejections. Nine percent (95% confidence interval [CI]: 1-17) of practitioners used lymphocyte-depleting agents as the first-line approach for the treatment of Banff grade 1B acute rejection. Eighteen percent (95% CI: 7-29) and 36% (95% CI: 8-65) of respondents reported that they would not use high-dose steroids for treating clinical and subclinical borderline rejections, respectively. Seventy percent (95% CI: 54-83) of respondents answered that there was no indication to assess histological response to treatment independent of the change in kidney function. The limitations of this study are its limited sample

27 CFR 24.293 - Wine for Government use.

Science.gov (United States)

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Wine for Government use..., DEPARTMENT OF THE TREASURY LIQUORS WINE Removal, Return and Receipt of Wine Removals Without Payment of Tax § 24.293 Wine for Government use. (a) General. Wine may be removed from bonded wine premises, free of...

Giant hydronephrosis in horseshoe kidney

International Nuclear Information System (INIS)

Huesh, I-V. Malla; Zlatareva, D.; Milenova, V.; Krasteva, R.; Bogov, B.

2016-01-01

Horseshoe kidney, also known as ren arcuatus is a congenital anomaly with incidence 1 in 500 people and it is more common in males. Usually this anomaly is asymptomatic and most of the cases are undiagnosed. This condition may contribute to upper Gl tract dyspeptic syndrome, abdominal discomfort, nephrolithiasis and frequent infections of the urinary system. Horseshoe kidney may lead to complications such as renal obstruction, recurrent inflammatory conditions and malignant diseases. The authors describe the case of 58y.o. male who had suffered acute renal failure. The patient presented with pain in the lumbar area and abode the symphysis, reduction of diuresis and fever 38° C. The laboratory findings showed slight anemic syndrome and preserved renal function. The US examination revealed low positioned right kidney with enlarged sizes and numerous cysts. The left kidney was visualized as gigantic hydronephrosis. Color and Power Doppler didn't show signal from the vessels. MRT of the abdomen and pelvis was performed with intravenous application of contrast medium. The examination showed horseshoe kidney with excessive hydro-nephrosis with massive dilation of the pyelocalyceal system and reduced parenchyma

5 CFR 293.107 - Special safeguards for automated records.

Science.gov (United States)

2010-01-01

... for automated records. (a) In addition to following the security requirements of § 293.106 of this... careless, accidental, or unintentional disclosure, modification, or destruction of identifiable personal data; (2) Minimize the risk that skilled technicians or knowledgeable persons could improperly obtain...

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Science.gov (United States)

Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

2013-01-01

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

A Chimeric NaV1.8 Channel Expression System Based on HEK293T Cell Line

Directory of Open Access Journals (Sweden)

Xi Zhou

2018-04-01

Full Text Available Among the nine voltage-gated sodium channel (NaV subtypes, NaV1.8 is an attractive therapeutic target for pain. The heterologous expression of recombinant NaV1.8 currents is of particular importance for its electrophysiological and pharmacological studies. However, NaV1.8 expresses no or low-level functional currents when transiently transfected into non-neuronal cell lines. The present study aims to explore the molecular determinants limiting its functional expression and accordingly establish a functional NaV1.8 expression system. We conducted screening analysis of the NaV1.8 intracellular loops by constructing NaV chimeric channels and confirmed that the NaV1.8 C-terminus was the only limiting factor. Replacing this sequence with that of NaV1.4, NaV1.5, or NaV1.7 constructed functional channels (NaV1.8/1.4L5, NaV1.8/1.5L5, and NaV1.8/1.7L5, respectively, which expressed high-level NaV1.8-like currents in HEK293T cells. The chimeric channel NaV1.8/1.7L5 displayed much faster inactivation of its macroscopic currents than NaV1.8/1.4L5 and NaV1.8/1.5L5, and it was the most similar to wild-type NaV1.8 expressed in ND7/23 cells. Its currents were very stable during repetitive depolarizations, while its repriming kinetic was different from wild-type NaV1.8. Most importantly, NaV1.8/1.7L5 pharmacologically resembled wild-type NaV1.8 as revealed by testing their susceptibility to two NaV1.8 selective antagonists, APETx-2 and MrVIB. NaV chimeras study showed that at least the domain 2 and domain 4 of NaV1.8 were involved in binding with APETx-2. Our study provided new insights into the function of NaV1.8 intracellular loops, as well as a reliable and convenient expression system which could be useful in NaV1.8 studies.

Characterization of the human nasal embryonic LHRH factor gene, NELF, and a mutation screening among 65 patients with idiopathic hypogonadotropic hypogonadism (IHH).

Science.gov (United States)

Miura, Kiyonori; Acierno, James S; Seminara, Stephanie B

2004-01-01

As the mouse nasal embryonic LHRH factor gene (Nelf) encodes a guidance molecule for the migration of the olfactory axon and gonadotropin-releasing hormone neurons, its human homolog, NELF, is a candidate gene for Kallmann syndrome, a disease of idiopathic hypogonadotropic hypogonadism (IHH) with anosmia or hyposmia. We report here characterization of NELF and results of mutation analysis in 65 IHH patients. Assembling EST clones, RACE, and sequencing showed that NELF mapped to 9q34.3 is composed of 16 exons and 15 introns with a 1,590-bp ORF encoding 530 amino acids. RT-PCR on a fetal brain cDNA library revealed five alternatively spliced variants. Among them, NELF-v1 has 93-94% identity at the amino acid level to mouse/rat Nelf, and four other transcripts are also highly conserved among the three species. A 3.0-kb transcript is expressed most highly in the adult and fetal brain, testis, and kidney, indicating that NELF plays a role in the function of these tissues. Mutation screening detected in a patient with IHH one novel heterozygous missense mutation (1438A>G, T480A) at the donor-splice site in exon 15 of NELF. As this mutation was not found in 100 normal control individuals, T480A may be associated with IHH. Four other novel SNPs (102C > T and 1029C > T within the coding region, and two IVS14+47C > T and IVS15+41G > A) were also identified in NELF.

Reconstructive surgery in eight children with solitary kidneys

DEFF Research Database (Denmark)

Thorup, Jørgen Mogens

1989-01-01

Within a 10-year period reconstructive urinary tract surgery has been carried out in eight children with solitary kidneys. The children were 0-5 years old. Six had unilateral renal agenesis and two had unilateral multicystic kidney. In five children ureteroneocystostomy was performed, in two of t...... months of age. Postoperatively, the renal function was subnormal (although improved) in two children; in six it was normal. The most important prognostic factors in solitary kidneys with urinary tract obstruction are infection and developmental injury.......Within a 10-year period reconstructive urinary tract surgery has been carried out in eight children with solitary kidneys. The children were 0-5 years old. Six had unilateral renal agenesis and two had unilateral multicystic kidney. In five children ureteroneocystostomy was performed, in two...

MR imaging of kidneys following extracorporeal shock wave lithotripsy

International Nuclear Information System (INIS)

Baumgartner, B.R.; Dickey, K.W.; Nelson, R.C.; Ambrose, S.S.; Walton, K.N.; Bernardino, M.E.

1986-01-01

MR images were obtained the day after extracorporeal shock wave lithotripsy (ESWL) therapy in 34 patients; the untreated kidneys served as controls. Five patients underwent ESWL of both kidneys before MR imaging. The kidneys were imaged with a spin-echo technique. Multisection coronal, sagittal, and axial images were obtained with T1-weighted pulse sequences. MR imaging studies of 39 kidneys after ESWL showed no abnormality in ten (25%) cases. The other kidneys (75%) had one or more of several findings. Small subcapsular or perinephric fluid collections were noted in ten (25%) patients. Generalized loss of corticomedullary junction (CMJ) was noted in eight (21%) cases and focal loss in 16 (24%). The more pronounced alterations in the CMJ correlated with increased numbers of shock waves received by the kidney

Embryonal rhabdomyosarcoma of the biliary tree: a case report

International Nuclear Information System (INIS)

Oh, Jong Young; Nam, Kyung Jin; Choi, Jong Chul; Park, Byung Ho; Lee, Ki Nam; Chung, Duck Hwan

1995-01-01

Rhabdomyosarcoma are reportedly the most common soft tissue sarcoma occurring in childhood, but the biliary tree is a rare site of origin for this tumor. Recently we experienced a case of embryonal rhabdomyosarcoma of the biliary tree in a 30-month-old child. Ultrasonography showed hypoechoic mass filling the dilated left intrahepatic and extrahepatic bile ducts, and CT showed hypodense mass with heterogeneous enhancement after contrast infusion. Intraoperative cholangiography showed filling defects within the dilated left intrahepatic and extrahepatic bile ducts. Postoperative MRI showed residual mass within the left intrahepatic duct which was hypointense on T1WI and hyperintense on T2WI

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

Science.gov (United States)

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-11-30

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

Science.gov (United States)

Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

2017-08-01

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

Diffusion-weighted MRI of kidneys in healthy volunteers and living kidney donors

International Nuclear Information System (INIS)

Sulkowska, K.; Palczewski, P.; Duda-Zysk, A.; Szeszkowski, W.; Wojcik, D.; Kownacka-Piotrowska, D.; Gołebiowski, M.

2015-01-01

Aim: To establish the normal apparent diffusion coefficient (ADC) values in healthy kidneys, comparing them with the literature, and assessing the correlation between ADC values, creatinine blood level, and glomerular filtration rate (GFR). Materials and methods: Twenty-four healthy volunteers and 26 living kidney donors were examined on a 1.5 T magnetic resonance imaging (MRI) unit. Two diffusion-weighted imaging (DWI) sequences were included in the study protocol (protocol 1 with 16 b-values, protocol 2 with 10 b-values) before the examination blood and urine samples were collected. The GFR was calculated using Cockcroft & Gault and MDRD (Modification of Diet In Renal Disease) formulas and the ADC values were measured separately for the cortex and medulla of each kidney by two independent observers. All statistical analyses were performed using the STATISTICA (version 10.0) software package. Data were analysed using an unpaired t-test; p<0.05 indicated a statistically significant difference. Results: The average ADC value for protocol 1 for the cortex was 2.26×10 −3  mm 2 /s, for the medulla 2.21×10 −3  mm 2 /s. In protocol 2, the respective values were 2.13×10 −3  mm 2 /s and 2.06×10 −3  mm 2 /s. Neither statistically significant interobserver differences nor correlation between ADC values, GFR, and creatinine serum level were observed. Conclusion: The reference ADC values were established. The measurements show high interobserver consistency. The differences in ADC values reported in the literature suggest dependence on the equipment and methodology and point to the necessity of obtaining ADC norms for each MRI unit. -- Highlights: •Magnetic resonance diffusion-weighted imaging of kidneys. •Apparent diffusion coefficient in healthy individuals. •Monoexponential model of diffusion

Distribution of the alphaGal- and the non-alphaGal T-antigens in the pig kidney: potential targets for rejection in pig-to-man xenotransplantation

DEFF Research Database (Denmark)

Kirkeby, Svend; Mikkelsen, Hanne B

2008-01-01

Carbohydrate antigens, present on pig vascular endothelial cells, seem to be the prime agents responsible for graft rejection, and although genetically modified animals that express less amounts of carbohydrate antigen are available, it is still useful to decide the localization of the reactive...... xenoantigens in organs contemplated for xenotransplantation. Here we compare the distribution in pig kidney of antigens important in xenograft destruction, namely the Galalpha1-3Gal (alphaGal) glycans, with the localization of the T-antigen (Galbeta1-3GalNAc). The alpha-galactose-specific lectin Griffonia...... simplicifolia isolectin 1B4 was used to detect the Galalpha1-3Gal glycans, whereas Arachis hypogaea (PNA) lectin and a monoclonal antibody (3C9) detected T-antigen. In addition, two vascular markers (anti-caveolin-1 and anti-von Willebrand factor) served to identify vascular structures of the kidney. Both...

How the embryonic chick brain twists

OpenAIRE

Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A.

2016-01-01

During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left–right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic m...

The metabolism of parathyroid hormone in kidney

International Nuclear Information System (INIS)

Hanao, Yasuhisa

1978-01-01

In order to investigate the mechanism and localization of parathyroid hormone (PTH), the degradation and the effects of calcium ion to PTH degradation in kidney, bovine PTH (b-PTH 1 - 84) and its synthetic N-terminal peptide (b-PTH 1 - 34) labeled with 125 I by Chloramine T methods ( 125 I-b-PTH 1 - 84 and 125 I-b-PTH 1 - 34) or labeled with horse radish peroxidase ( 125 I-POX-b-PTH 1 - 84 and 125 I-POX-bPTH 1-34) were used to study the disappearance from the blood stream and degradation and retention in the kidney after intravenous injections in male Wistar rats, weighing approximately 350 - 450 g. Degradation of PTH was studied in vitro, using isolated cells and homogenates of the kidney, and the effects of calcium ion to PTH degradation were furthermore studied, using our kidney perfusion system. PTH labeled with 125 I and POX was less degraded by the kidney than PTH labeled with 125 I alone. PTH 1 - 34 was more delayed in blood stream than PTH 1 - 84. Isolated intact kidney cells degrade PTH less efficiently than homogenates, indicating the prominance of microsomal degradative system in the kidney. The degradation of PTH in kidney was supposed to be controlled by calcium ion in our kidney perfusion system. (author)

Analysed cap mesenchyme track data from live imaging of mouse kidney development

Directory of Open Access Journals (Sweden)

James G. Lefevre

2016-12-01

Full Text Available This article provides detailed information on manually tracked cap mesenchyme cells from timelapse imaging of multiple ex vivo embryonic mouse kidneys. Cells were imaged for up to 18 h at 15 or 20 min intervals, and multiple cell divisions were tracked. Positional data is supplemented with a range of information including the relative location of the closest ureteric tip and a correction for drift due to bulk movement and tip growth. A subset of tracks were annotated to indicate the presence of processes attached to the ureteric epithelium. The calculations used for drift correction are described, as are the main methods used in the analysis of this data for the purpose of describing cap cell motility. The outcomes of this analysis are discussed in “Cap mesenchyme cell swarming during kidney development is influenced by attraction, repulsion, and adhesion to the ureteric tip” (A.N. Combes, J.G. Lefevre, S. Wilson, N.A. Hamilton, M.H. Little, 2016 [1].

Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

Science.gov (United States)

Shi, M; Zhao, S; Wang, Z-H; Stanley, D; Chen, X-X

2016-12-01

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H 2 O 2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv. © 2016 The Royal Entomological Society.

The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells.

Science.gov (United States)

Piekarowicz, Katarzyna; Machowska, Magdalena; Dratkiewicz, Ewelina; Lorek, Daria; Madej-Pilarczyk, Agnieszka; Rzepecki, Ryszard

2017-08-01

LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

Endothelial marker-expressing stromal cells are critical for kidney formation.

Science.gov (United States)

Mukherjee, Elina; Maringer, Katherine; Papke, Emily; Bushnell, Daniel; Schaefer, Caitlin; Kramann, Rafael; Ho, Jacqueline; Humphreys, Benjamin D; Bates, Carlton; Sims-Lucas, Sunder

2017-09-01

Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice ( Flk1 fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1 fl/fl ( Flk1 ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1 ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1 ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1 ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1 ST-/- kidneys vs. Juvenile Flk1 ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1 ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages. Copyright © 2017 the American Physiological Society.

Evaluation of anticancer peptide VEGF111b secretion in HEK293 human cells

Directory of Open Access Journals (Sweden)

Morteza Sadeghi

2017-04-01

Full Text Available Background: VEGF111b is a new isoform of vascular endothelial growth factor (VEGF recently considered as a new anticancer drug. The aim of this study was to evaluate the VEGF111b secretion from HEK293 cell wall in order to commercial production of this recombinant factor. Materials and Methods: After the design of VEGF111b sequence using OLIGO software and NCBI gene bank data, it was cloned into the pBUD.cE4.1 vector. The pBUD.VEGF111b recombinant vector was transfected into HEK293 cells using lipofectamine kit. Forty-eight hours after the transfection the production of VEGF111b was estimated by Western blotting and Human anti VEGF antibody. The VEGF111b secretion into cell culture and cell lysate extract was measured using ELISA. Results: The correct cloning of VEGF111b into pBUD.cE4.1vector was confirmed using enzymatic digestion and gel electrophoresis. The observed production of recombinant peptide in HEK293 was confirmed with 12KDa band in cell lysate extract of Western blotting. The ELISA results at 450 nanometer absorbance for cell culture media and cell lysate extract were 19.20±2.81 pg/ml and 32.87±7.42 pg/ml, respectively. However, no VEGF111b expression was observed in negative controls. Conclusion: The findings of this study indicate the powerful ability of transformation and secretion of VEGF111b from HEK293 cell wall to cell culture media with no breaking and proteolytic digestion. It seems that the commercial production and purification of this therapeutic peptide from HEK293 cell culture would be possible with high efficiency.

Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network

Science.gov (United States)

Chevalier, Marc; Toporikova, Natalia; Simmers, John; Thoby-Brisson, Muriel

2016-01-01

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons. DOI: http://dx.doi.org/10.7554/eLife.16125.001 PMID:27434668

Quantitative arterial spin labelling perfusion measurements in rat models of renal transplantation and acute kidney injury at 3 T

Energy Technology Data Exchange (ETDEWEB)

Zimmer, Fabian; Schad, Lothar R.; Zoellner, Frank G. [Heidelberg Univ., Mannheim (Germany). Computer Assisted Clinical Medicine; Klotz, Sarah; Hoeger, Simone; Yard, Benito A.; Kraemer, Bernhard K. [Heidelberg Univ., Mannheim (Germany). Dept. of Medicine V

2017-05-01

To employ ASL for the measurement of renal cortical perfusion in particular renal disorders typically associated with graft loss and to investigate its potential to detect and differentiate the related functional deterioration i.e., in a setting of acute kidney injury (AKI) as well as in renal grafts showing acute and chronic transplant rejection. 14 Lewis rats with unilateral ischaemic AKI and 43 Lewis rats with renal grafts showing acute or chronic rejections were used. All ASL measurements in this study were performed on a 3 T MR scanner using a FAIR True-FISP approach to assess renal blood flow (RBF). Perfusion maps were calculated and the cortical blood flow was determined using a region-of-interest based analysis. RBF of healthy and AKI kidneys as well as of both rejection models, were compared. In a subsample of 20 rats, creatinine clearance was measured and correlated with cortical perfusion. RBF differs significantly between healthy and AKI kidneys (P < 0.001) with a mean difference of 213 ± 80 ml/100 g/min. Renal grafts with chronic rejections show a significantly higher (P < 0.001) mean cortical perfusion (346 ± 112 ml/100 g/min) than grafts with acute rejection (240 ± 66 ml/100 g/min). Both transplantation models have a significantly (P < 0.001) lower perfusion than healthy kidneys. Renal creatinine clearance is significantly correlated (R = 0.85, P < 0.001) with cortical blood flow. Perfusion measurements with ASL have the potential to become a valuable diagnostic tool, regarding the detection of renal impairment and the differentiation of disorders that lead to a loss of renal function and that are typically associated with graft loss.

Quantitative arterial spin labelling perfusion measurements in rat models of renal transplantation and acute kidney injury at 3 T

International Nuclear Information System (INIS)

Zimmer, Fabian; Schad, Lothar R.; Zoellner, Frank G.; Klotz, Sarah; Hoeger, Simone; Yard, Benito A.; Kraemer, Bernhard K.

2017-01-01

To employ ASL for the measurement of renal cortical perfusion in particular renal disorders typically associated with graft loss and to investigate its potential to detect and differentiate the related functional deterioration i.e., in a setting of acute kidney injury (AKI) as well as in renal grafts showing acute and chronic transplant rejection. 14 Lewis rats with unilateral ischaemic AKI and 43 Lewis rats with renal grafts showing acute or chronic rejections were used. All ASL measurements in this study were performed on a 3 T MR scanner using a FAIR True-FISP approach to assess renal blood flow (RBF). Perfusion maps were calculated and the cortical blood flow was determined using a region-of-interest based analysis. RBF of healthy and AKI kidneys as well as of both rejection models, were compared. In a subsample of 20 rats, creatinine clearance was measured and correlated with cortical perfusion. RBF differs significantly between healthy and AKI kidneys (P < 0.001) with a mean difference of 213 ± 80 ml/100 g/min. Renal grafts with chronic rejections show a significantly higher (P < 0.001) mean cortical perfusion (346 ± 112 ml/100 g/min) than grafts with acute rejection (240 ± 66 ml/100 g/min). Both transplantation models have a significantly (P < 0.001) lower perfusion than healthy kidneys. Renal creatinine clearance is significantly correlated (R = 0.85, P < 0.001) with cortical blood flow. Perfusion measurements with ASL have the potential to become a valuable diagnostic tool, regarding the detection of renal impairment and the differentiation of disorders that lead to a loss of renal function and that are typically associated with graft loss.

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

Science.gov (United States)

Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

2015-02-15

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

MR imaging of adult glomerulocystic kidney disease

International Nuclear Information System (INIS)

Egashira, K.; Nakata, H.; Hashimoto, O.; Kaizu, K.; University of Occupational and Environmental Health School of Medicine, Kitakyushu

1991-01-01

A 59-year-old man with hypertension and severe renal dysfunction was diagnosed as having adult glomerulocystic kidney disease. MR imaging of the kidney showed a diffuse reduction of the intensity of the renal cortex with a loss of normal cortico-medullary differentiation of T1-weighted images. Numerous small cortical cysts were also demonstrated. These MR findings complemented the results of the biopsy and were useful for making a definitive diagnosis. (orig.)

Kidney tubular-cell secretion of osteoblast growth factor is increased by kaempferol: a scientific basis for "the kidney controlling the bone" theory of Chinese medicine.

Science.gov (United States)

Long, Mian; Li, Shun-xiang; Xiao, Jiang-feng; Wang, Jian; Lozanoff, Scott; Zhang, Zhi-guang; Luft, Benjamin J; Johnson, Francis

2014-09-01

To study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone". Kaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis. Kaempferol was found to increase OK cell growth (Pkaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells. This lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.

36 CFR 293.16 - Special provisions governing the Boundary Waters Canoe Area Wilderness, Superior National Forest...

Science.gov (United States)

2010-07-01

... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Special provisions governing the Boundary Waters Canoe Area Wilderness, Superior National Forest, Minnesota. 293.16 Section 293.16 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE WILDERNESS-PRIMITIVE AREAS...

Healthy Kidneys (A Cup of Health with CDC)

Centers for Disease Control (CDC) Podcasts

2015-03-12

Kidney disease is among the leading causes of death in the U.S. More than one in 10 people over the age of 20 are impacted by this condition, and most don’t know it. In this podcast, Nilka Rios Burrows discusses the risks for kidney disease.  Created: 3/12/2015 by MMWR.   Date Released: 3/12/2015.

Kidney transplant

Science.gov (United States)

... always take your medicine as directed. Alternative Names Renal transplant; Transplant - kidney Patient Instructions Kidney removal - discharge Images Kidney anatomy Kidney - blood and urine flow Kidneys Kidney transplant - ...

Synthesis and in Vitro Antiproliferative Evaluation of Some B-norcholesteryl Benzimidazole and Benzothiazole Derivatives

Directory of Open Access Journals (Sweden)

Jianguo Cui

2015-04-01

Full Text Available Taking orostanal (a compound from a Japanese marine sponge, Stelletta hiwasaensis as a lead compound, some novel B-norcholesteryl benzimidazole and benzothiazole derivatives were synthesized. The antiproliferative activity of the compounds against human cervical carcinoma (HeLa, human lung carcinoma (A549, human liver carcinoma cells (HEPG2 and normal kidney epithelial cells (HEK293T was assayed. The results revealed that the benzimidazole group was a better substituent than benzothiazole group for increasing the antiproliferative activity of compounds. 2-(3β′-Acetoxy-5β′-hydroxy-6′-B-norcholesterylbenzimidazole (9b with the structure of 6-benzimidazole displays the best antiproliferative activity to the cancer cells in all compounds, but is almost inactive to normal kidney epithelial cells (HEK293T. The assay of compound 9b to cancer cell apoptosis by flow cytometry showed that the compound was able to effectively induce cancer cell apoptosis. The research provided a theoretical reference for the exploration of new anti-cancer agents and may be useful for the design of novel chemotherapeutic drugs.

76 FR 67418 - Request for Comments on NIST Special Publication 500-293, US Government Cloud Computing...

Science.gov (United States)

2011-11-01

...-1659-01] Request for Comments on NIST Special Publication 500-293, US Government Cloud Computing... Publication 500-293, US Government Cloud Computing Technology Roadmap, Release 1.0 (Draft). This document is... (USG) agencies to accelerate their adoption of cloud computing. The roadmap has been developed through...

Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A.

Science.gov (United States)

Somanna, Naveen K; Mani, Indra; Tripathi, Satyabha; Pandey, Kailash N

2018-04-01

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using 125 I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

Science.gov (United States)

Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

2009-10-30

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

Thyroid Disorders and Chronic Kidney Disease

Directory of Open Access Journals (Sweden)

Mohamed Mohamedali

2014-01-01

Full Text Available Thyroid hormones play a very important role regulating metabolism, development, protein synthesis, and influencing other hormone functions. The two main hormones produced by the thyroid are triiodothyronine (T3 and thyroxine (T4. These hormones can also have significant impact on kidney disease so it is important to consider the physiological association of thyroid dysfunction in relation to chronic kidney disease (CKD. CKD has been known to affect the pituitary-thyroid axis and the peripheral metabolism of thyroid hormones. Low T3 levels are the most common laboratory finding followed by subclinical hypothyroidism in CKD patients. Hyperthyroidism is usually not associated with CKD but has been known to accelerate it. One of the most important links between thyroid disorders and CKD is uremia. Patients who are appropriately treated for thyroid disease have a less chance of developing renal dysfunction. Clinicians need to be very careful in treating patients with low T3 levels who also have an elevation in TSH, as this can lead to a negative nitrogen balance. Thus, clinicians should be well educated on the role of thyroid hormones in relation to CKD so that proper treatment can be delivered to the patient.

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

Science.gov (United States)

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

Kidney involvement in MELAS syndrome: Description of 2 cases.

Science.gov (United States)

Alcubilla-Prats, Pau; Solé, Manel; Botey, Albert; Grau, Josep Maria; Garrabou, Glòria; Poch, Esteban

2017-04-21

MELAS syndrome -myopathy, encephalopathy, lactic acidosis and stroke-like episodes- is a maternally-inherited mitochondrial cytopathy related to several mitochondrial DNA mutations, with the A3243G mutation in tRNA Leu gene being the most frequent of them. Apart from its typical symptomatology, patients usually exhibit a maternally-inherited history of neurosensory deafness and insulin-dependent type 2 diabetes mellitus (T2DM). Recent studies have shown that few patients carrying a A3243G mutation also suffer from renal dysfunction, usually in form of focal segmental glomerulosclerosis (FSGS). In this study we examine kidney involvement in 2 unrelated patients with a A3243G mutation by genetic testing. Both have a maternally-inherited neurosensory deafness and insulin-dependent T2DM. A renal biopsy was performed in both patients. One patient developed nephrotic proteinuria and renal insufficiency, with FSGS findings being observed in the kidney biopsy, whereas the other suffered from mild proteinuria and renal insufficiency, with non-specific glomerular changes. The presence of FSGS or other kidney involvement accompanied by hereditary neurosensory deafness and T2DM could be suggestive of a A3243G tRNA Leu mutation and should prompt a genetic testing and an evaluation of potential extrarenal involvement. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

The Long Intron 1 of Growth Hormone Gene from Reeves’ Turtle (Chinemys reevesii Correlates with Negatively Regulated GH Expression in Four Cell Lines

Directory of Open Access Journals (Sweden)

Wen-Sheng Liu

2016-04-01

Full Text Available Turtles grow slowly and have a long lifespan. Ultrastructural studies of the pituitary gland in Reeves’ turtle (Chinemys reevesii have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (GH cells than other animal species in summer and winter. C. reevesii GH gene was cloned and species-specific similarities and differences were investigated. The full GH gene sequence in C. reevesii contains 8517 base pairs (bp, comprising five exons and four introns. Intron 1 was found to be much longer in C. reevesii than in other species. The coding sequence (CDS of the turtle’s GH gene, with and without the inclusion of intron 1, was transfected into four cell lines, including DF-1 chicken embryo fibroblasts, Chinese hamster ovary (CHO cells, human embryonic kidney 293FT cells, and GH4C1 rat pituitary cells; the turtle growth hormone (tGH gene mRNA and protein expression levels decreased significantly in the intron-containing CDS in these cell lines, compared with that of the corresponding intronless CDS. Thus, the long intron 1 of GH gene in Reeves’ turtle might correlate with downregulated gene expression.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

International Nuclear Information System (INIS)

Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang; Xu Zhihan

2006-01-01

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to β 2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively

Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression

International Nuclear Information System (INIS)

Hakelien, Anne-Mari; Gaustad, Kristine G.; Taranger, Christel K.; Skalhegg, Bjorn S.; Kuentziger, Thomas; Collas, Philippe

2005-01-01

We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, ∼2500 genes are upregulated >3-fold, of which ∼900 are also expressed in Jurkat cells. Concomitantly, ∼1500 genes are downregulated or repressed, of which ∼500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages (∼80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells

Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish

OpenAIRE

Xiang, Fan; Hagos, Engda G.; Xu, Bo; Sias, Christina; Kawakami, Koichi; Burdine, Rebecca D.; Dougan, Scott T.

2007-01-01

In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce thei...

Comparison of glomerular filtration rates derived from dynamic renal imaging and dual sample clearance in kidney transplantation

International Nuclear Information System (INIS)

Zhu Yangjun; Li Linfa; Zhang Jun; Du Xiaoying

2007-01-01

Objective: Glomerular filtration rate (GFR) is one of the valuable indictors of allograft status after kidney transplantation. The feasibility of an easy measurement of GFR by dynamic renal imaging (dGFR) was assessed in comparison to a classical dual plasma sample method (tGFR) in the current study for evaluating the function of grafting kidney. Methods: In 73 patients of kidney transplantation, both dynamic renal imaging and dual sample GFR measurement were undertaken after 99 Tc m -DTPA injection. The correlation between dGFR and tGFR, both being standardized according to surface area, was analyzed and the linear regression equation was obtained. Results: The mean dGFR was slightly lower than that of tGFR (t=-2.010, P<0.05). The dGFR correlated significantly with tGFR (r=0.759, P< 0.01). The linear regression equation was tGFR=0.6455 x dGFR + 25.514. Conclusion: The dGFR correlated well with tGFR and they were accurate in evaluating the filtration function of transplanted kidney. (authors)

Genetic basis of kidney cancer: Role of genomics for the development of disease-based therapeutics

OpenAIRE

Linehan, W. Marston

2012-01-01

Kidney cancer is not a single disease; it is made up of a number of different types of cancer, including clear cell, type 1 papillary, type 2 papillary, chromophobe, TFE3, TFEB, and oncocytoma. Sporadic, nonfamilial kidney cancer includes clear cell kidney cancer (75%), type 1 papillary kidney cancer (10%), papillary type 2 kidney cancer (including collecting duct and medullary RCC) (5%), the microphalmia-associated transcription (MiT) family translocation kidney cancers (TFE3, TFEB, and MITF...

Outcomes of dual adult kidney transplants in the United States: an analysis of the OPTN/UNOS database.

Science.gov (United States)

Gill, Jagbir; Cho, Yong W; Danovitch, Gabriel M; Wilkinson, Alan; Lipshutz, Gerald; Pham, Phuong-Thu; Gill, John S; Shah, Tariq; Bunnapradist, Suphamai

2008-01-15

The organ shortage has resulted in increased use of kidneys from expanded criteria donors (ECD). For ECD kidneys unsuitable for single use, dual kidney transplants (DKT) may be possible. There are limited data comparing outcomes of DKT to single kidney ECD transplants, making it unclear where DKT fits in the current allocation scheme. Our purpose was to compare outcomes of DKT and ECD transplants in the United States. From 2000 to 2005, a total of 625 DKT, 7686 single kidney ECD, and 6,044 SCD transplants from donors aged>or=50 years were identified from the Organ Procurement and Transplantation Network/United Network for Organ Sharing data. Allograft survival was the primary outcome. DKT comprised 4% of kidney transplants from donors aged>or=50 years. Compared to the ECD donor group, the DKT donor group was older (mean age 64.6+/-7.7 years vs. 59.9+/-6.2 years) and consisted of more African Americans (13.1% vs. 9.9%), and more diabetic donors (16.3% vs. 10.4%; PDKT (22.2+/-9.7 hr), but rates of delayed graft function were lower (29.3%) compared to ECD transplants (33.6%, P=0.03). Three-year overall graft survival was 79.8% for DKT and 78.3% for ECD transplants. DKT were infrequent and had outcomes comparable to ECD transplants, despite the use of organs from higher risk donors. With a more upfront approach to DKT by offering this option to patients at the time of wait-listing as part of an ECD algorithm, we may be able to further optimize outcomes of DKT and minimize discard of potential organs.

Dispositional optimism and coping strategies in patients with a kidney transplant.

Science.gov (United States)

Costa-Requena, Gemma; Cantarell-Aixendri, M Carmen; Parramon-Puig, Gemma; Serón-Micas, Daniel

2014-01-01

 Dispositional optimism is a personal resource that determines the coping style and adaptive response to chronic diseases. The aim of this study was to assess the correlations between dispositional optimism and coping strategies in patients with recent kidney transplantation and evaluate the differences in the use of coping strategies in accordance with the level of dispositional optimism.  Patients who were hospitalised in the nephrology department were selected consecutively after kidney transplantation was performed. The evaluation instruments were the Life Orientation Test-Revised, and the Coping Strategies Inventory. The data were analysed with central tendency measures, correlation analyses and means were compared using Student’s t-test.   66 patients with a kidney transplant participated in the study. The coping styles that characterised patients with a recent kidney transplantation were Social withdrawal and Problem avoidance. Correlations between dispositional optimism and coping strategies were significant in a positive direction in Problem-solving (p<.05) and Cognitive restructuring (p<.01), and inversely with Self-criticism (p<.05). Differences in dispositional optimism created significant differences in the Self-Criticism dimension (t=2.58; p<.01).  Dispositional optimism scores provide differences in coping responses after kidney transplantation. Moreover, coping strategies may influence the patient’s perception of emotional wellbeing after kidney transplantation.

Physical properties of the binary systems methylcyclopentane with ketones (acetone, butanone and 2-pentanone) at T = (293.15, 298.15, and 303.15) K. New UNIFAC-VISCO interaction parameters

Energy Technology Data Exchange (ETDEWEB)

Gonzalez, B. [Department of Chemical Engineering, University of Vigo, Lagoas-Marconende, s/n Apartado 874, 36200 Vigo (Spain); Dominguez, A. [Department of Chemical Engineering, University of Vigo, Lagoas-Marconende, s/n Apartado 874, 36200 Vigo (Spain); Tojo, J. [Department of Chemical Engineering, University of Vigo, Lagoas-Marconende, s/n Apartado 874, 36200 Vigo (Spain)]. E-mail: jtojo@uvigo.es

2006-06-15

In this work, the physical properties, dynamic viscosities, densities, and speed of sound have been measured over the whole composition range and atmospheric pressure for the binary mixtures (methylcyclopentane with acetone, butanone, and 2-pentanone) at several temperatures T = (293.15, 298.15, and 303.15) K along with the properties of the pure components. Excess molar volumes, isentropic compressibility, deviations in isentropic compressibility and viscosity deviation for the binary systems at the above-mentioned temperatures were calculated and fitted to the Redlich-Kister equation to determine the fitting parameters and the root-mean-square deviations. The UNIQUAC equation was used to correlate the experimental viscosity data. The UNIFAC-VISCO method and ASOG-VISCO method, based on contribution groups, were used to predict the dynamic viscosities of the binary mixtures. The interaction parameters of cycloalkanes with ketones (CH{sub cy}/CO) have been determined for their application in the predictive UNIFAC-VISCO method.

Four decades of kidney transplantation in Cuba.

Science.gov (United States)

Alfonzo, Jorge P

2013-01-01

This article describes the background, beginnings, development, evolution and outcomes of kidney transplantation in Cuba. Nephrology as a medical specialty in Cuba began in 1962 and was formalized in 1966. Conditions were created to implement renal replacement therapy (including transplants), bring nephrology care to the entire country and train human resources who would assume this responsibility, making Cuba one of the first countries with a comprehensive program for renal patient care. After three unsuccessful cadaveric-donor kidney transplantations in 1968-69, the ensuing history of kidney transplantation can be summarized in the following three stages. 1970-1975: In January 1970, cadaveric-donor kidney transplantation began at the Nephrology Institute. That year, 17 kidney transplantations were performed; four of these patients lived with functional kidneys for 15-25 years; 10-year graft survival was 23.5% (Kaplan-Meier survival curve); HLA typing began in 1974. By December 1975, 170 grafts had been done in three hospitals. 1976-1985: Seven transplantation centers performed 893 grafts during this period. HLA-DR typing was introduced in 1976 and the National Histocompatibility Laboratory Network was founded in 1978. The first related living-donor kidney transplantation was done in 1979. 1986-2011: The National Kidney Transplantation Coordinating Center and the National Kidney Transplantation Program were created in 1986; the first combined kidney-pancreas transplantation was performed the same year. In 1990, cyclosporine and the Cuban monoclonal antibody IOR-T3 were introduced for immunosuppression to prevent rejection, as were other Cuban products (hepatitis B vaccine and recombinant human erythropoietin) for transplant patients. By December 2011, the cumulative number of transplants was 4636 (384 from related living donors). With over 40 years of experience, kidney transplantation is now well established in Cuba; it is free and universally accessible, on the

Altered glucose transport to utero-embryonic unit in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

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Arnab, Banerjee; Amitabh, Krishna

2011-02-10

The aim of this study was to compare the changes in concentration of glucose and glucose transporters (GLUTs) in the utero-embryonic unit, consisting of decidua, trophoblast and embryo, during delayed and non-delayed periods to understand the possible cause of delayed embryonic development in Cynopterus sphinx. The results showed a significantly decreased concentration of glucose in the utero-embryonic unit due to decline in the expression of insulin receptor (IR) and GLUT 3, 4 and 8 proteins in the utero-embryonic unit during delayed period. The in vitro study showed suppressive effect of insulin on expression of GLUTs 4 and 8 in the utero-embryonic unit and a significant positive correlation between the decreased amount of glucose consumed by the utero-embryonic unit and decreased expression of GLUTs 4 (r=0.99; psphinx. Increased supply of fatty acid to the delayed embryo may be responsible for its survival under low glucose condition but unable to promote embryonic development in C. sphinx. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Kidney adysplasia and variable hydronephrosis, a new mutation affecting the odd-skipped related 1 gene in the mouse, causes variable defects in kidney development and hydronephrosis.

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Davisson, Muriel T; Cook, Susan A; Akeson, Ellen C; Liu, Don; Heffner, Caleb; Gudis, Polyxeni; Fairfield, Heather; Murray, Stephen A

2015-06-15

Many genes, including odd-skipped related 1 (Osr1), are involved in regulation of mammalian kidney development. We describe here a new recessive mutation (kidney adysplasia and variable hydronephrosis, kavh) in the mouse that leads to downregulation of Osr1 transcript, causing several kidney defects: agenesis, hypoplasia, and hydronephrosis with variable age of onset. The mutation is closely associated with a reciprocal translocation, T(12;17)4Rk, whose Chromosome 12 breakpoint is upstream from Osr1. The kavh/kavh mutant provides a model to study kidney development and test therapies for hydronephrosis. Copyright © 2015 the American Physiological Society.

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

2012-01-01

DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Van Rossom, Sofie [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Op de Beeck, Ken [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Franssens, Vanessa; Swinnen, Erwin [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Schepers, Anne [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Ghillebert, Ruben; Caldara, Marina [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Van Camp, Guy [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Winderickx, Joris, E-mail: guy.vancamp@ua.ac.be, E-mail: joris.winderickx@bio.kuleuven.be [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium)

2012-07-25

DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

In vitro and in vivo assessment of nanotoxicity of CdS quantum dot/aminopolysaccharide bionanoconjugates.

Science.gov (United States)

de Carvalho, S M; Mansur, A A P; Mansur, H S; Guedes, M I M C; Lobato, Z I P; Leite, M F

2017-02-01

The nanotoxicity of Cd-containing quantum dots (QDs) for biomedical applications is very controversial and not completely understood. In this study, we evaluated the cytotoxicity of surface-biofunctionalized CdS QDs with chitosan directly synthesized via aqueous route at room temperature. These core-shell CdS-chitosan nanoconjugates showed different degrees of cytotoxic responses using MTT cell proliferation assay toward three human cell cultures, human osteosarcoma cell line (SAOS), non-Hodgkin's B cell lymphoma (Toledo), and human embryonic kidney cell line (HEK293T), under three exposure times (1, 3, and 5days) and three colloidal concentrations (10nM, 50nM, and 100nM). The results clearly demonstrated that the CdS QDs, regardless to the fact that they were coated with a biocompatible aminopolysaccharide shell, induced a severe dose- and time-dependent inhibition of cell viability. In addition, the HEK293T and SAOS cell lines showed much more sensitive response compared to Toledo, which indicated that the cytotoxicity was also cell-type dependent. The exceptional resistance of Toledo cells to toxic effects of CdS nanoconjugates even at severe test conditions was assigned to specific role of B-lineage cells of the immune defense system. Remarkably, no conclusive evidence of toxicity of CdS nanoconjugates was observed in vivo using intravenous injections of CdS nanoconjugates in BALB/c mouse animal models for 30days, but localized fluorescence was detected in ex-vivo liver tissue samples. Therefore, these results prove that there is no guarantee of "risk-free" use of CdS nanoconjugates for in vivo applications, even when functionalized with biopolymer ligands, as they can pose an excessive threat due to unpredicted and uncorrelated responses under in vitro and in vivo biological assays with highly toxic cadmium ions. Copyright © 2016 Elsevier B.V. All rights reserved.

The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

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Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

2016-04-01

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle. © 2016 Authors; published by Portland Press Limited.

Bardoxolone Methyl Improves Kidney Function in Patients with Chronic Kidney Disease Stage 4 and Type 2 Diabetes: Post-Hoc Analyses from Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes Study

Science.gov (United States)

Chin, Melanie P.; Bakris, George L.; Block, Geoffrey A.; Chertow, Glenn M.; Goldsberry, Angie; Inker, Lesley A.; Heerspink, Hiddo J.L.; O'Grady, Megan; Pergola, Pablo E.; Wanner, Christoph; Warnock, David G.; Meyer, Colin J.

2018-01-01

Background Increases in measured inulin clearance, measured creatinine clearance, and estimated glomerular filtration rate (eGFR) have been observed with bardoxolone methyl in 7 studies enrolling approximately 2,600 patients with type 2 diabetes (T2D) and chronic kidney disease (CKD). The largest of these studies was Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes (BEACON), a multinational, randomized, double-blind, placebo-controlled phase 3 trial which enrolled patients with T2D and CKD stage 4. The BEACON trial was terminated after preliminary analyses showed that patients randomized to bardoxolone methyl experienced significantly higher rates of heart failure events. We performed post-hoc analyses to characterize changes in kidney function induced by bardoxolone methyl. Methods Patients in ­BEACON (n = 2,185) were randomized 1: 1 to receive once-daily bardoxolone methyl (20 mg) or placebo. We compared the effects of bardoxolone methyl and placebo on a post-hoc composite renal endpoint consisting of ≥30% decline from baseline in eGFR, eGFR <15 mL/min/1.73 m2, and end-stage renal disease (ESRD) events (provision of dialysis or kidney transplantation). Results Consistent with prior studies, patients randomized to bardoxolone methyl experienced mean increases in eGFR that were sustained through study week 48. Moreover, increases in eGFR from baseline were sustained 4 weeks after cessation of treatment. Patients randomized to bardoxolone methyl were significantly less likely to experience the composite renal endpoint (hazards ratio 0.48 [95% CI 0.36–0.64]; p < 0.0001). Conclusions Bardoxolone methyl preserves kidney function and may delay the onset of ESRD in patients with T2D and stage 4 CKD. PMID:29402767

Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients

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Sester Urban

2008-06-01

Full Text Available Abstract Background Cytomegalovirus (CMV seronegative recipients (R- of kidney transplants (KT from seropositive donors (D+ are at higher risk for CMV replication and ganciclovir(GCV-resistance than CMV R(+. We hypothesized that low CMV-specific T-cell responses are associated with increased risk of CMV replication in R(+-patients with D(+ or D(- donors. Methods We prospectively evaluated 73 consecutive KT-patients [48 R(+, 25 D(+R(-] undergoing routine testing for CMV replication as part of a preemptive strategy. We compared CMV-specific interferon-γ (IFN-γ responses of CD4+CD3+ lymphocytes in peripheral blood mononuclear cells (PBMC using three different antigen preparation (CMV-lysate, pp72- and pp65-overlapping peptide pools using intracellular cytokine staining and flow cytometry. Results Median CD4+ and CD8+T-cell responses to CMV-lysate, pp72- and pp65-overlapping peptide pools were lower in D(+R(- than in R(+patients or in non-immunosuppressed donors. Comparing subpopulations we found that CMV-lysate favored CD4+- over CD8+-responses, whereas the reverse was observed for pp72, while pp65-CD4+- and -CD8+-responses were similar. Concurrent CMV replication in R(+-patients was associated with significantly lower T-cell responses (pp65 median CD4+ 0.00% vs. 0.03%, p = 0.001; CD8+ 0.01% vs. 0.03%; p = 0.033. Receiver operated curve analysis associated CMV-pp65 CD4+ responses of > 0.03% in R(+-patients with absence of concurrent (p = 0.003 and future CMV replication in the following 8 weeks (p = 0.036. GCV-resistant CMV replication occurred in 3 R(+-patients (6.3% with pp65- CD4+ frequencies Conclusion The data suggest that pp65-specific CD4+ T-cells might be useful to identify R(+-patients at increased risk of CMV replication. Provided further corroborating evidence, CMV-pp65 CD4+ responses above 0.03% in PBMCs of KT patients under stable immunosuppression are associated with lower risk of concurrent and future CMV replication during the

The relationship between serum and urinary Fetuin-A levels and kidney stone formation among kidney stone patients.

Science.gov (United States)

Mehrsai, Abdolrasoul; Guitynavard, Fateme; Nikoobakht, Mohammad Reza; Gooran, Shahram; Ahmadi, Ayat

2017-01-01

Mineralization inhibitors are required to prevent the precipitation of minerals and inhibit the formation of kidney stones and other ectopic calcifications. In laboratory studies, Fetuin-A as a glycoprotein has inhibited hydroxyapatite precipitation in calcium and phosphate supersaturated solutions; however, information about patients with kidney stones is limited. The aim of this study was to investigate the association of serum and urinary Fetuin-A levels with calcium oxalate kidney stones. In this case-control study, 30 patients with kidney stones and 30 healthy individuals without any history of urolithiasis who were referred to the urology ward of Sina Hospital of Tehran, Iran, in 2015 were entered into the study. All patients underwent computerized tomography scans. After collecting demographic information, serum and urine levels of Fetuin-A and some other calcification inhibitors and promoters, were measured and compared using T-test, Mann-Whitney and logistic regression between the two study groups. Patients with kidney stones, on average, had lower levels of Serum Fetuin-A (1522.27 ±755.39 vs. 1914.64 ±733.76 μg/ml; P = 0.046) as well as lower levels of Urine Fetuin-A (944.62 ±188.5 vs. 1409.68 ±295.26 μg/ml; P <0.001). Multivariate logistic analysis showed that urinary calcium and serum creatinine are the risk factors and Fetuin-A is a urinary protective factor for kidney stones. PFC Our study showed that patients with kidney stones had lower serum and urinary levels of Fetuin-A. In the logistic regression model, urinary Fetuin-A was reported as a protective factor for kidney stones.

Lysosomal trafficking of {beta}-catenin induced by the tea polyphenol epigallocatechin-3-gallate

Energy Technology Data Exchange (ETDEWEB)

Dashwood, Wan-Mohaiza [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Carter, Orianna [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Al-Fageeh, Mohamed [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Li, Qingjie [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Dashwood, Roderick H. [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States)]. E-mail: Rod.Dashwood@oregonstate.edu

2005-12-11

{beta}-Catenin is a cadherin-binding protein involved in cell-cell adhesion, which also functions as a transcriptional activator when complexed in the nucleus with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of proteins. There is considerable interest in mechanisms that down-regulate {beta}-catenin, since this provides an avenue for the prevention of colorectal and other cancers in which {beta}-catenin is frequently over-expressed. We show here that physiologically relevant concentrations of the tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited {beta}-catenin/TCF-dependent reporter activity in human embryonic kidney 293 cells transfected with wild type or mutant {beta}-catenins, and there was a corresponding decrease in {beta}-catenin protein levels in the nuclear, cytosolic and membrane-associated fractions. However, {beta}-catenin accumulated as punctate aggregates in response to EGCG treatment, including in human colon cancer cells over-expressing {beta}-catenin endogenously. Confocal microscopy studies revealed that the aggregated {beta}-catenin in HEK293 cells was extra-nuclear and co-localized with lysosomes, suggesting that EGCG activated a pathway involving lysosomal trafficking of {beta}-catenin. Lysosomal inhibitors leupeptin and transepoxysuccinyl-L-leucylamido(4-guanido)butane produced an increase in {beta}-catenin protein in total cell lysates, without a concomitant increase in {beta}-catenin transcriptional activity. These data provide the first evidence that EGCG facilitates the trafficking of {beta}-catenin into lysosomes, presumably as a mechanism for sequestering {beta}-catenin and circumventing further nuclear transport and activation of {beta}-catenin/TCF/LEF signaling.

Lysosomal trafficking of β-catenin induced by the tea polyphenol epigallocatechin-3-gallate

International Nuclear Information System (INIS)

Dashwood, Wan-Mohaiza; Carter, Orianna; Al-Fageeh, Mohamed; Li, Qingjie; Dashwood, Roderick H.

2005-01-01

β-Catenin is a cadherin-binding protein involved in cell-cell adhesion, which also functions as a transcriptional activator when complexed in the nucleus with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of proteins. There is considerable interest in mechanisms that down-regulate β-catenin, since this provides an avenue for the prevention of colorectal and other cancers in which β-catenin is frequently over-expressed. We show here that physiologically relevant concentrations of the tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited β-catenin/TCF-dependent reporter activity in human embryonic kidney 293 cells transfected with wild type or mutant β-catenins, and there was a corresponding decrease in β-catenin protein levels in the nuclear, cytosolic and membrane-associated fractions. However, β-catenin accumulated as punctate aggregates in response to EGCG treatment, including in human colon cancer cells over-expressing β-catenin endogenously. Confocal microscopy studies revealed that the aggregated β-catenin in HEK293 cells was extra-nuclear and co-localized with lysosomes, suggesting that EGCG activated a pathway involving lysosomal trafficking of β-catenin. Lysosomal inhibitors leupeptin and transepoxysuccinyl-L-leucylamido(4-guanido)butane produced an increase in β-catenin protein in total cell lysates, without a concomitant increase in β-catenin transcriptional activity. These data provide the first evidence that EGCG facilitates the trafficking of β-catenin into lysosomes, presumably as a mechanism for sequestering β-catenin and circumventing further nuclear transport and activation of β-catenin/TCF/LEF signaling

The 'ventral organs' of Pycnogonida (Arthropoda) are neurogenic niches of late embryonic and post-embryonic nervous system development.

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Brenneis, Georg; Scholtz, Gerhard

2014-01-01

Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions - traditionally designated as 'ventral organs' - detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons - as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient posterior

Fusion of the SUMO/Sentrin-specific protease 1 gene SENP1 and the embryonic polarity-related mesoderm development gene MESDC2 in a patient with an infantile teratoma and a constitutional t(12;15)(q13;q25).

NARCIS (Netherlands)

Veltman, I.M.; Basten-Vreede, L.A.J.; Cheng, J.; Looijenga, L.H.J.; Janssen, H.A.P.; Schoenmakers, E.F.P.M.; Yeh, E.T.; Geurts van Kessel, A.H.M.

2005-01-01

Recently, we identified a patient with an infantile sacrococcygeal teratoma and a constitutional t(12;15)(q13;q25). Here, we show that, as a result of this chromosomal translocation, the SUMO/Sentrin-specific protease 1 gene (SENP1) on chromosome 12 and the embryonic polarity-related mesoderm

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

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The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

Peculiarities of Embryonic and Post-Embryonic Development of Оesophagostomum dentatum (Nematoda, Strongylidae Larvae Cultured in Vitro

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Yevstafieva V. А.

2017-02-01

Full Text Available Morphometric peculiarities of the development of Оesophagostomum dentatum Rudolphi, 1803 from egg to infective larva were studied under laboratory conditions at various temperatures. The determined optimum temperature for embryonic and post-embryonic development of О. dentatum larvae from domestic pig (Sus scrofa domesticus Linnaeus, 1758 is 22 °С. At this temperature, 81 % of larvae develop to the third stage (L3 on the 10th day. Temperatures of 24 °С and 20 °С are less favorable for the development of the nematode, at those temperatures only 67 and 63 % of larvae, respectively, reached infective stage by the 10th day of cultivation. Embryonic development of О. dentatum eggs is characterized by their lengthening (by 8.87-9.50 %, р < 0.01 and widening (by 6.77-9.35 %, р < 0.05-0.01, and post-embryonic larval development is associated with lengthening (by 4.59-17.33 %, р < 0.01-0.001.

It was/wasn't everything I had imagined: advantages and disadvantages after kidney transplantation.

Science.gov (United States)

Santos, Bianca Pozza Dos; Viegas, Aline da Costa; Feijó, Aline Machado; Lise, Fernanda; Schwartz, Eda

2016-10-31

To know the advantages and difficulties that people with chronic kidney disease experience after renal transplantation. A qualitative and descriptive study with 20 kidney transplant patients in a city in Southern Brazil, from May to July of 2013. Semi-structured interviews were used, analyzed according to the critical incident technique. The main advantages were presented in the similarity to "normal" living: advantages resulting from the kidney transplant category, related to the patient's discharge from dialysis, food and water restriction, among others. The difficulties were presented in the permanent chronic condition and the need for care category. The advantages and disadvantages depend on each person's experience. The health professionals need to understand and promote health actions that promote the uniqueness and context of renal transplant.

Photobiomodulation on KATP Channels of Kir6.2-Transfected HEK-293 Cells

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Fu-qing Zhong

2014-01-01

Full Text Available Background and Objective. ATP-sensitive potassium (KATP channel couples cell metabolism to excitability. To explore role of KATP channels in cellular photobiomodulation, we designed experiment to study effect of low intensity 808 nm laser irradiation on the activity of membrane KATP channel. Study Design/Materials and Methods. Plasmids encoding Kir6.2 was constructed and heterologously expressed in cultured mammalian HEK-293 cells. The patch-clamp and data acquisition systems were used to record KATP channel current before and after irradiation. A laser beam of Ga-As 808 nm at 5 mW/cm2 was used in experiments. A one-way ANOVA test followed by a post hoc Student-Newman-Keuls test was used to assess the statistical differences between data groups. Results. Obvious openings of KATP channels of Kir6.2-transfected HEK-293 cells and excised patches were recorded during and after low intensity 808 nm laser irradiation. Compared with the channels that did not undergo irradiation, open probability, current amplitude, and dwell time of KATP channels after irradiation improved. Conclusions. Low intensity 808 nm laser irradiation may activate membrane KATP channels of Kir6.2-transfected HEK-293 cells and in excised patches.

Fanconi anemia complementation group A (FANCA) localizes to centrosomes and functions in the maintenance of centrosome integrity.

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Kim, Sunshin; Hwang, Soo Kyung; Lee, Mihee; Kwak, Heejin; Son, Kook; Yang, Jiha; Kim, Sung Hak; Lee, Chang-Hun

2013-09-01

Fanconi anemia (FA) proteins are known to play roles in the cellular response to DNA interstrand cross-linking lesions; however, several reports have suggested that FA proteins play additional roles. To elucidate novel functions of FA proteins, we used yeast two-hybrid screening to identify binding partners of the Fanconi anemia complementation group A (FANCA) protein. The candidate proteins included never-in-mitosis-gene A (NIMA)-related kinase 2 (Nek2), which functions in the maintenance of centrosome integrity. The interaction of FANCA and Nek2 was confirmed in human embryonic kidney (HEK) 293T cells. Furthermore, FANCA interacted with γ-tubulin and localized to centrosomes, most notably during the mitotic phase, confirming that FANCA is a centrosomal protein. Knockdown of FANCA increased the frequency of centrosomal abnormalities and enhanced the sensitivity of U2OS osteosarcoma cells to nocodazole, a microtubule-interfering agent. In vitro kinase assays indicated that Nek2 can phosphorylate FANCA at threonine-351 (T351), and analysis with a phospho-specific antibody confirmed that this phosphorylation occurred in response to nocodazole treatment. Furthermore, U2OS cells overexpressing the phosphorylation-defective T351A FANCA mutant showed numerical centrosomal abnormalities, aberrant mitotic arrest, and enhanced nocodazole sensitivity, implying that the Nek2-mediated T351 phosphorylation of FANCA is important for the maintenance of centrosomal integrity. Taken together, this study revealed that FANCA localizes to centrosomes and is required for the maintenance of centrosome integrity, possibly through its phosphorylation at T351 by Nek2. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

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Rolletschek Alexandra

2009-06-01

Full Text Available Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. Conclusion In embryonic stem cells where (anti-proliferative p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

[Kidney transplantation epidemiology in France].

Science.gov (United States)

Hiesse, Christian

2013-11-01

Kidney transplantation activity in France is among the most important worldwide: in 2011, 2976 transplants have been performed (47.5 per million population), and the number of patients living with a functional graft is estimated around 30,000, representing 44.7% of all patients (n = 67,270) treated for end-stage renal failure. However, the rate of preemptive kidney transplants remains very low, only 3.3% of incident patients starting renal replacement therapy. The analysis of demand showed a progressive increase in recent years, as demonstrated by the registration rate on the kidney transplantation waiting list, increasing by 5% yearly between 2006 and 2010, but with huge differences according to age categories and regional registration areas, reflecting discrepant appreciations in indications for kidney transplantation. The median waiting time between registration and transplantation increased progressively in recent years, reaching 22.3 months with considerable variations according to regional areas and transplantation teams. Kidney transplantation activity, while increasing continuously, is far to cover the rising demand, and inexorably patients accumulate on the waiting list (around 9000 patients were registered by January 2012). This situation is the consequence of insufficient organ procurement activity. The deceased organ procurement rate remained high: 1572 harvested donors in 2011 (24.1 per million population), but the proportion of older donors rose in recent years, to reach the rate of 26% of donors older than 65 years in 2011. The procurement activity of donors after cardiac arrest was reintroduced in 2006, but increased slowly: 65 transplants were performed in 2011 using kidney procured in non heart-beating donors. The living donor kidney transplantation activity has markedly increased recently: 302 living donor transplantations were performed in 2011, representing 10.1% of the kidney transplantations. Facing the predictable increase in the number of

The kidney and type 2 diabetes mellitus: therapeutic implications of SGLT2 inhibitors.

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Weir, Matthew R

2016-01-01

Understanding the role of the kidneys in type 2 diabetes mellitus (T2DM) has taken on an increased importance in recent years with the arrival of sodium-glucose co-transporter 2 (SGLT2) inhibitors - antihyperglycemic agents (AHAs) that specifically target the kidneys. This review includes an update on the physiology of the kidneys, their role in the pathophysiology of T2DM, and the mechanisms implicated in the development and progression of diabetic kidney disease, such as glomerular hyperfiltration and inflammation. It also discusses renal issues that could influence the choice of AHA for patients with T2DM, including special populations such as patients with concomitant chronic kidney disease. The most recent data published on the clinical efficacy and safety of the SGLT2 inhibitors canagliflozin, dapagliflozin, and empagliflozin and their effects on renal function are presented, showing how the renally mediated mechanisms of action of these agents translate into clinical benefits, including the potential for renoprotection. The observed positive effects of these agents on measures such as glucose control, estimated glomerular filtration rate, albumin-to-creatinine ratio, blood pressure, and body weight in patients both with and without impaired renal function suggest that SGLT2 inhibitors represent an important extension to the diabetes treatment armamentarium.

Simple Kidney Cysts

Science.gov (United States)

... Solitary Kidney Your Kidneys & How They Work Simple Kidney Cysts What are simple kidney cysts? Simple kidney cysts are abnormal, fluid-filled ... that form in the kidneys. What are the kidneys and what do they do? The kidneys are ...

Taurine Inhibits K+-Cl− Cotransporter KCC2 to Regulate Embryonic Cl− Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway*

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Inoue, Koichi; Furukawa, Tomonori; Kumada, Tatsuro; Yamada, Junko; Wang, Tianying; Inoue, Rieko; Fukuda, Atsuo

2012-01-01

GABA inhibits mature neurons and conversely excites immature neurons due to lower K+-Cl− cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl− homeostasis, which is required for normal brain development. PMID:22544747

Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

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Amanda Fraga

Full Text Available Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3 and hexokinase (HexA genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

Science.gov (United States)

Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson

2013-01-01

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen. PMID:23750237

CKD in diabetes: diabetic kidney disease versus nondiabetic kidney disease.

Science.gov (United States)

Anders, Hans-Joachim; Huber, Tobias B; Isermann, Berend; Schiffer, Mario

2018-06-01

The increasing global prevalence of type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) has prompted research efforts to tackle the growing epidemic of diabetic kidney disease (DKD; also known as diabetic nephropathy). The limited success of much of this research might in part be due to the fact that not all patients diagnosed with DKD have renal dysfunction as a consequence of their diabetes mellitus. Patients who present with CKD and diabetes mellitus (type 1 or type 2) can have true DKD (wherein CKD is a direct consequence of their diabetes status), nondiabetic kidney disease (NDKD) coincident with diabetes mellitus, or a combination of both DKD and NDKD. Preclinical studies using models that more accurately mimic these three entities might improve the ability of animal models to predict clinical trial outcomes. Moreover, improved insights into the pathomechanisms that are shared by these entities - including sodium-glucose cotransporter 2 (SGLT2) and renin-angiotensin system-driven glomerular hyperfiltration and tubular hyper-reabsorption - as well as those that are unique to individual entities might lead to the identification of new treatment targets. Acknowledging that the clinical entity of CKD plus diabetes mellitus encompasses NDKD as well as DKD could help solve some of the urgent unmet medical needs of patients affected by these conditions.

Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

International Nuclear Information System (INIS)

Mochalova, Larisa; Gambaryan, Alexandra; Romanova, Julia; Tuzikov, Alexander; Chinarev, Alexander; Katinger, Dietmar; Katinger, Herman; Egorov, Andrej; Bovin, Nicolai

2003-01-01

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac α2-6Galβ1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acα2-6Galβ1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses

Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

Science.gov (United States)

Chen, Chien-Hong; Li, Yi; Hu, Yeshu; An, Li-You; Yang, Lan; Zhang, Jifeng; Chen, Y. Eugene

2015-01-01

Abstract The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. PMID:26579970

Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis.

Science.gov (United States)

Kenyon, Emma J; Luijten, Monique N H; Gill, Harmeet; Li, Nan; Rawlings, Matthew; Bull, James C; Hadzhiev, Yavor; van Steensel, Maurice A M; Maher, Eamonn; Mueller, Ferenc

2016-07-08

Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish. flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development. In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic

Cloning and characterization of a novel human zinc finger gene, hKid3, from a C2H2-ZNF enriched human embryonic cDNA library

International Nuclear Information System (INIS)

Gao Li; Sun Chong; Qiu Hongling; Liu Hui; Shao Huanjie; Wang Jun; Li Wenxin

2004-01-01

To investigate the zinc finger genes involved in human embryonic development, we constructed a C 2 H 2 -ZNF enriched human embryonic cDNA library, from which a novel human gene named hKid3 was identified. The hKid3 cDNA encodes a 554 amino acid protein with an amino-terminal KRAB domain and 11 carboxyl-terminal C 2 H 2 zinc finger motifs. Northern blot analysis indicates that two hKid3 transcripts of 6 and 8.5 kb express in human fetal brain and kidney. The 6 kb transcript can also be detected in human adult brain, heart, and skeletal muscle while the 8.5 kb transcript appears to be embryo-specific. GFP-fused hKid3 protein is localized to nuclei and the ZF domain is necessary and sufficient for nuclear localization. To explore the DNA-binding specificity of hKid3, an oligonucleotide library was selected by GST fusion protein of hKid3 ZF domain, and the consensus core sequence 5'-CCAC-3' was evaluated by competitive electrophoretic mobility shift assay. Moreover, The KRAB domain of hKid3 exhibits transcription repressor activity when tested in GAL4 fusion protein assay. These results indicate that hKid3 may function as a transcription repressor with regulated expression pattern during human development of brain and kidney

Management of patients with chronic kidney disease

African Journals Online (AJOL)

management of the complications of CKD, e.g. renal anaemia, ... ARTICLE. Management of patients with chronic kidney disease. T Gerntholtz,1 FCP (SA); G Paget,2 ..... Telmisartan, ramipril, or both in patients at high risk for vascular events.

Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

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Ryutaro Hirasawa

Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Kidney Problems

Science.gov (United States)

... our e-newsletter! Aging & Health A to Z Kidney Problems Basic Facts & Information The kidneys are two ... kidney (renal) diseases are called nephrologists . What are Kidney Diseases? For about one-third of older people, ...

Laparoscopic treatment of UPJ obstruction in ectopic pelvic kidneys in children

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A. Marte

2012-10-01

Full Text Available Aims: to assess the feasibility and safety of a laparoscopic approach to uPJ obstruction (uPJo in ectopic pelvic kidneys. Material and Methods: in a retrospective analysis we selected 14 children, aged 6months to 17 years, 12 males, 2 females, who had been treated in our department between January 2004 and June 2011. 9 patients presented ureteropelvic junction obstruction (in 3 cases pelvic stones coexisted with normal/moderately reduced (≥25% relative function at radionuclide scan (MAg3, 3 nonfunctioning kidneys associated or not to hypertension, 2 congenital hypo-dysplastic kidneys. the evaluation of each patient involved the medical history, ultrasound examination, VCug, MAg3 diuresis renogram and Mri in some cases. of the patients presenting uPJo, 5 underwent dismembered pyeloplasty with pyelolithotomy, if required, and 4 pelvic derotation with straightening of the uretero-pelvic junction. A previous cystoscopic placement of a double J stent was utilized. this facilitated the identification and dissection around the pelvis. With the patient in trendelenburg position we utilized an umbilical trocar and two trocar in the right and left iliac fossae; an additional trocar, when required, was inserted more cephalad on the midclavear line contralaterally to the lesion. the derotation of ureteropelvic junction was obtained by freeing the kidney’s lower pole and by placing intraperitoneally the junction protected with a double J stent. this was obtained by suturing the peritoneum behind the ureteropelvic junction resulting in a forward rotation of the major axis of the kidney and a straightening of the junction. the 5 patients presenting nonfunctioning ectopic kidneys underwent laparoscopic nephrectomy. While the removal of congenital hypoplasic kidneys resulted easy, the removal of nonfunctioning kidneys was more difficult due to their complex vascular situation and for the embryonic disposition. Results: the operating time varied between 40 to

Acute renal failure secondary to rhabdomyolysis; MR imaging of the kidney

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Kim, S.H.; Han, M.C.; Kim, S.; Lee, J.S. (Dept. of Radiology and Dept. of Internal Medicine, Seoul National Univ., Coll. of Medicine (Korea, Republic of))

1992-11-01

MR imaging of the kidney was performed in 6 patients with acute renal failure (ARF) secondary to rhabdomyolysis caused by snake bite (n = 4), crush injury (n = 1), and carbon monoxide poisoning (n = 1). A test for urine myoglobin was positive in all 6 patients and MR imaging was done 6 to 18 days after the causative event of the rhabdomyolysis. MR images in all 6 patients showed globular swelling of the kidneys, preserved corticomedullary contrast on T1-weighted images, and obliteration of corticomedullary contrast on T2-weighted images. Unlike other medical renal diseases in which corticomedullary contrast is lost on T1-weighted images, preservation of the corticomedullary contrast on T1-weighted MR images with globular renal swelling was a constant finding in patients with ARF secondary to rhabdomyolysis. (orig.).

Aging has small effects on initial ischemic acute kidney injury development despite changing intrarenal immunologic micromilieu in mice.

Science.gov (United States)

Jang, Hye Ryoun; Park, Ji Hyeon; Kwon, Ghee Young; Park, Jae Berm; Lee, Jung Eun; Kim, Dae Joong; Kim, Yoon-Goo; Kim, Sung Joo; Oh, Ha Young; Huh, Wooseong

2016-02-15

Inflammatory process mediated by innate and adaptive immune systems is a major pathogenic mechanism of renal ischemia-reperfusion injury (IRI). There are concerns that organ recipients may be at increased risk of developing IRI after receiving kidneys from elder donors. To reveal the effects of aging on the development of renal IRI, we compared the immunologic micromilieu of normal and postischemic kidneys from mice of three different ages (9 wk, 6 mo, and 12 mo). There was a higher number of total T cells, especially effector memory CD4/CD8 T cells, and regulatory T cells in the normal kidneys of old mice. On day 2 after IRI, the proportion of necrotic tubules and renal functional changes were comparable between groups although old mice had a higher proportion of damaged tubule compared with young mice. More T cells, but less B cells, trafficked into the postischemic kidneys of old mice. The infiltration of NK T cells was similar across the groups. Macrophages and neutrophils were comparable between groups in both normal kidneys and postischemic kidneys. The intrarenal expressions of TNF-α and VEGF were decreased in normal and postischemic kidneys of aged mice. These mixed effects of aging on lymphocytes and cytokines/chemokines were not different between the two groups of old mice. Our study demonstrates that aging alters the intrarenal micromilieu but has small effects on the development of initial renal injury after IRI. Further study investigating aging-dependent differences in the repair process of renal IRI may be required. Copyright © 2016 the American Physiological Society.

A QUANTITATIVE METHOD FOR ANALYSING 3-D BRANCHING IN EMBRYONIC KIDNEYS: DEVELOPMENT OF A TECHNIQUE AND PRELIMINARY DATA

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Gabriel Fricout

2011-05-01

Full Text Available The normal human adult kidney contains between 300,000 and 1 million nephrons (the functional units of the kidney. Nephrons develop at the tips of the branching ureteric duct, and therefore ureteric duct branching morphogenesis is critical for normal kidney development. Current methods for analysing ureteric branching are mostly qualitative and those quantitative methods that do exist do not account for the 3- dimensional (3D shape of the ureteric "tree". We have developed a method for measuring the total length of the ureteric tree in 3D. This method is described and preliminary data are presented. The algorithm allows for performing a semi-automatic segmentation of a set of grey level confocal images and an automatic skeletonisation of the resulting binary object. Measurements of length are automatically obtained, and numbers of branch points are manually counted. The final representation can be reconstructed by means of 3D volume rendering software, providing a fully rotating 3D perspective of the skeletonised tree, making it possible to identify and accurately measure branch lengths. Preliminary data shows the total length estimates obtained with the technique to be highly reproducible. Repeat estimates of total tree length vary by just 1-2%. We will now use this technique to further define the growth of the ureteric tree in vitro, under both normal culture conditions, and in the presence of various levels of specific molecules suspected of regulating ureteric growth. The data obtained will provide fundamental information on the development of renal architecture, as well as the regulation of nephron number.

Kidney recipients experiences before during and after kidney transplantation

DEFF Research Database (Denmark)

Nielsen, Charlotte

Background Kidney transplantation is considered to be the best treatment for terminal renal insufficiency. Kidney transplant patients report higher quality of life because they avoid regular dialysis treatment that causes side effects, complications, restrictions and limitations in their daily...... and after the kidney transplant, through outpatient visits and during possible hospitalization, which can occur due to complications or disease progression. Objective To explore the coherence of the kidney transplant process in order to explain the lived experiences of kidney recipients before, during...... and after kidney transplantation. Method Participant observation and semi-structured individual interviews was conducted with kidney recipients before, during and after kidney transplantation. Data analysis is inspired by Ricoeur's interpretation theory on three levels: Naive reading; structural analysis...

Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

Science.gov (United States)

Durand, Maxim; Dubois, Florian; Dejou, Cécile; Durand, Eugénie; Danger, Richard; Chesneau, Mélanie; Brosseau, Carole; Guerif, Pierrick; Soulillou, Jean-Paul; Degauque, Nicolas; Eliaou, Jean-François; Giral, Magali; Bonnefoy, Nathalie; Brouard, Sophie

2018-05-01

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Possible presence of common tyvelose-containing glycans in Trichinella L1 larvae and embryonated eggs of several nematodes

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Dea-Ayuela M.A.

2001-06-01

Full Text Available A monoclonal antibody (mAb US4 recognising an epitope containing tyvelose within the T. spiralis L-1 muscle larvae (TSL-1 antigens was tested in western-blot against various antigenic preparations from different stages of the following nematodes: T. spiralis (L1,adult, T. muris (egg, L1, L3, adult, Ascaris suum (egg, adult, Toxocara canis (egg, adult, Anisakis simplex (L3 and Haemochus contortus (egg. Positive reaction was present in antigen preparations from L1 larvae of T. spiralis and T. muris and from embryonated eggs of T. muris, A. suum, T. canis and H. conlortus.

The 'ventral organs' of Pycnogonida (Arthropoda are neurogenic niches of late embryonic and post-embryonic nervous system development.

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Georg Brenneis

Full Text Available Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i immunolabeling, (ii histology and (iii scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida, the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions - traditionally designated as 'ventral organs' - detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult replenishment of olfactory neurons - as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two

The value of MR perfusion weighted imaging in normal and abnormal kidneys

International Nuclear Information System (INIS)

Shi Hao; Ding Hongyu; Duan Ruiping; Sun Yongping; Xing Yiyong

2008-01-01

Objective: To explore the characteristics and the clinical application of MR perfusion weighted imaging (PWI) in the normal kidneys and the renal diseases. Methods: Thirty-one subjects including 9 cases without urinary diseases, 14 cases with renal carcinoma, 6 cases with renal cyst and 2 cases with renal tuberculosis who had been examined with T 1 WI, T 2 WI and PWI were analyzed retrospectively. All the data were processed by a workstation to obtain time-signal intensity curves, color perfusion maps and relative perfusion value. The relative renal blood volume (RBV), relative renal blood flow (RBF), mean transition time (MTY) and the time to peak (TTP) in the normal renal cortex and medulla and the renal lesions were calculated. Comparisons between the right and the left normal kidneys, and between the cortex and the medulla of the normal kidneys were performed using t test, and comparisons between the normal and the abnormal kidneys were performed using q test. Results: Relative RBV and relative RBF of the cortex were 1.33±0.08 and 1.44±0.09 respectively, and for medulla were 0.58± 0.05 and 0.78±0.13 respectively (t=9.2241 and 5.0336, P 0.05). The values of relative RBF of the renal carcinoma (1.35±0.34) were significantly higher than that of the normal tissues (1.02±0.06) (q=3.0882, P< 0.01). Conclusion: PWI is able to demonstrate the hemodynamic change of the normal renal tissues and the renal lesions, and it maybe an ideal method for showing the functional changes of the kidney and for differentiating the renal diseases. (authors)

Magnetisation transfer MR imaging of the kidney: evaluation at 3.0 T in association with renal function

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Ito, Katsuyoshi [Kawasaki Medical School, Department of Diagnostic Radiology, Kurashiki, Okayama (Japan); Kurashiki Daiichi Hospital, Department of Radiology, Kurashiki, Okayama (Japan); Hayashida, Minoru; Izumitani, Shogo; Fujimine, Tomoko; Onishi, Takeo; Genba, Katsuhiro [Kurashiki Daiichi Hospital, Department of Radiology, Kurashiki, Okayama (Japan)

2013-08-15

To evaluate the feasibility of using magnetisation transfer (MT) MRI of the kidney at 3.0 T to assess renal function. Forty-four patients who underwent abdominal MRI on a 3.0-T system including gradient-echo (GRE) sequences with and without MT pulse were included. In each patient, MT ratio (MTR) of the renal cortex and medulla was measured by using regions of interest (ROIs) placed on the MTR map image. Regression analysis showed good correlation between estimated glomerular filtration rate (eGFR) and MTR of the renal cortex (r = -0.645, P < 0.0001). Among 44 patients, 22 were categorised as the normal renal function group and 22 were classified as the decreased eGFR group. The mean MTR of the renal cortex in patients with decreased eGFR (mean MTR, 30.7 {+-} 3.2 %) was significantly higher (P < 0.0001) than that in patients with normal renal function (mean MTR, 25.3 {+-} 2.2 %), although the mean MTRs of the renal medulla in the two groups were not significantly different. There was good correlation between eGFR and MTR of the renal cortex derived from MT MRI at 3.0 T. This technique may have the potential to evaluate the degree of renal function non-invasively in patients with renal impairment. (orig.)

Kidney Disease

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... Staying Safe Videos for Educators Search English Español Kidney Disease KidsHealth / For Teens / Kidney Disease What's in ... Coping With Kidney Conditions Print What Do the Kidneys Do? You might never think much about some ...

T-Helper Subsets Cytokine Production in Kidney Transplant Recipients: Diverting Influences and Impact on Graft Outcome

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Hassan A

2000-01-01

Full Text Available A large body of evidence suggests the existence of functionally polarized human T-helper responses based on their profile of cytokine secretion. Human T-helper cell clones can be divided into two mutually exclusive subsets, T-helper cell 1 (Th1 and T-helper cell 2 (Th2. Substantial work in several animal models has demonstrated that allograft rejection is associated with enhanced Th1 activity and tolerance with enhanced Th2. Some studies have not been consistent with this association. In this study, gamma interferon (INF-y and interleukin 4 (IL-4 levels (as indicators of Th1 and Th2 activity, respectively were assayed in supernatant of cultured peripheral lymphocytes. The levels of these cytokines were compared between a study group of 26 stable kidney transplant recipients immunosuppressed with cyclosporine A, corticosteroids and azathioprine or mycophenolate mofetil, and a control group of 10 healthy blood donors. The mean INF-γ and IL-4 levels in the control group were considered as the cutoff levels for comparison. Our results showed that 25/26 of the study patients (96% had low levels of INF-γ compared to 4/10 of the control subjects (40%, (P< 0.05. However, the IL-4 level was high in 10/26 of the study patients (38% and 3/10 of the control subjects (30%, not a statistically significant difference, (P>0.05. In conclusion: These results suggest that well-established graft tolerance may be mediated via depressed Th1 activity rather than enhanced Th2 activity.

Modification of Measures of Acute Kidney Injury to Risk Stratify Combat Casualties

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2017-08-26

REPORT TYPE 08/26/2017 Poster 4. TJTLE AND SUBTITLE t\\.1odification of l’vfeasures,of Acute Kidney Injury to Risk Stratify Cotnbat Casualties 6...profiles and potential future conflicts , identifying acute kidney injury (AKI) early can help us determine the need for rapidity of evacuation

Lgr5+ve Stem/Progenitor Cells Contribute to Nephron Formation during Kidney Development

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Nick Barker

2012-09-01

Full Text Available Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5+ve cells via in vivo lineage tracing. The appearance and localization of Lgr5+ve cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle’s loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

OpenAIRE

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil; Butler, Peter C.

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in ...

Common structural basis for constitutive activity of the ghrelin receptor family

DEFF Research Database (Denmark)

Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

2004-01-01

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand......-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...

Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

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Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

2009-12-25

Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice

DEFF Research Database (Denmark)

Bisgaard, Line S; Bosteen, Markus H; Fink, Lisbeth N

2016-01-01

Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosc......Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis...... aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 μg/kg, s.......c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68+F4/80-), CD4+, and CD8+ T-cells, suggesting that moderate uremia induced kidney inflammation...

Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues.

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Hughes, Mark A; Brennan, Paul M; Bunting, Andrew S; Cameron, Katherine; Murray, Alan F; Shipston, Mike J

2014-05-01

Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us toward creating intelligent neuroprostheses. A fundamental prerequisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line [Lund human mesencephalic (LUHMES)] to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to prepattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, human embryonal kidney (HEK) 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this coculture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion, and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning. Copyright © 2013 Wiley Periodicals, Inc.

Isolation of a primate embryonic stem cell line.

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Thomson, J A; Kalishman, J; Golos, T G; Durning, M; Harris, C P; Becker, R A; Hearn, J P

1995-01-01

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, st...

[The French Chronic Kidney Disease-Renal Epidemiology and Information Network (CKD-REIN) cohort study: To better understand chronic kidney disease].

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Stengel, Bénédicte; Combe, Christian; Jacquelinet, Christian; Briançon, Serge; Fouque, Denis; Laville, Maurice; Frimat, Luc; Pascal, Christophe; Herpe, Yves-Édouard; Morel, Pascal; Deleuze, Jean-François; Schanstra, Joost P; Pisoni, Ron L; Robinson, Bruce M; Massy, Ziad A

2016-04-01

Preserving kidney function and improving the transition from chronic kidney disease to end stage is a research and healthcare challenge. The national Chronic Kidney Disease-Renal Epidemiology and Information Network (CKD-REIN) cohort was established to identify the determinants, biomarkers and practice patterns associated with chronic kidney disease outcomes. The study will include more than 3000 adult patients with moderate to advanced chronic kidney disease from a representative sample of 40 nephrology clinics with respect to regions and legal status, public or private. Patients are recruited during a routine visit and followed for 5 years, before and after starting renal replacement therapy. Patient-level clinical, biological, and lifestyle data are collected annually, as well as provider-level data on clinical practices, coordinated with the International Chronic Kidney Disease Outcomes and Practice Pattern Study. Blood and urine samples are stored in a biobank. Major studied outcomes include survival, patient-reported outcomes, disease progression and hospitalizations. More than 13,000 eligible patients with chronic kidney disease were identified, 60% with stage 3 and 40% with stage 4. Their median age is 72 years [interquartile range, 62-80 years], 60% are men and 38% have diabetes. By the end of December 2015, 2885 patients were included. The CKD-REIN cohort will serve to improve our understanding of chronic kidney disease and provide evidence to improve patient survival and quality of life as well as health care system performances. Copyright © 2016 Association Société de néphrologie. All rights reserved.

Kidney pain (image)

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A kidney stone is a solid piece of material that forms in a kidney. Kidney stones may be the size of sand or ... A kidney stone is a solid piece of material that forms in a kidney. Kidney stones may be the ...

Correlation of diffusion-weighted MRI and gross anatomy of rat kidneys

International Nuclear Information System (INIS)

Chen Rongfeng; Wu Xiaomei; Chen Xiaoyan; Deng Yu; Li Xinchun; He Jianxun; Li Huiming

2011-01-01

Objective: To investigate the feasibility and accuracy of diffusion-weighted MRI in differentiating the cortico-medullary layers of rat kidneys. Methods: Twelve rats underwent MRI using a 1.5 Tesla system including DWI of various b-values, T1WI and T 2 WI sequences. The MR characteristics and thickness of renal cortico-medullary layers compared to those of the gross anatomical layers. Results: On the longitudinal anatomical sections of the kidneys, four parenchymal layers of cortex (CO), the outer (OS) and inner (IS) stripes of the outer medulla (OM), the inner medulla (IM) and renal pelvis could be clearly recognized. The numbers of layers visible on MRI varied with different pulse sequences. Single layers of cortex and medulla were visible on T 1 WI. CO, OM and IM were delineated on T 2 WI. CO, OS, IS and IM were clearly identified on DWI and ADC maps. DWI was significantly superior to T 1 WI and T 2 WI for displaying the renal parenchymal layers (P 0.05) from the measurements on DWI (CO: 1.39±0.15 mm, OS: 1.01±0.17 mm, IS: 1.11±0.19 mm, IM: 1.06±0.10 mm). Different b-values of DWI did not show significant difference in depiction of the parenchyma (P>0.05). Conclusion: Four parenchymal layers shown on DWI correlated well with gross anatomical structure and may be used in imaging study of rat kidneys. (authors)

Bioaccumulation and Depuration of Copper in the Kidney and Liver of a Freshwater Fish, Capoeta fusca

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Borhan Mansouri

2016-07-01

Full Text Available Background: This study aims to investigate the patterns of bioaccumulation and depuration of copper in the selected kidney and liver of Capoeta fusca. Methods: The fish were collected between September and November 2010 from a qanat in Birjand. They were exposed to two types treatments with copper (0.25 and 0.75 mg/L for a period of 41 days. The fish under study were exposed to the above-mentioned sub-lethal concentrations separately for 14 and 21 days (accumulation period. At the end of this period, the remaining fish were kept in tap water (elimination period for 31 and 41 days. Results: The findings showed that the accumulation of copper in lower and higher sub-lethal concentrations was higher in kidney as the mean accumulation of copper on day 21 was 1.9±0.1 μg/g and 2.93±0.47 μg/g respectively, in 0.25 μg/g and 0.75 μg/g concentrations. On the other hand, the results also showed that the depuration level of copper in the given concentrations was higher in liver than kidney. The bioaccumulation and depuration of copper significantly increased in the kidney and liver of C. fusca (P<0.01. Conclusion: Based on the present work, it is concluded that C. fusca has a potential for the rapid accumulation and depuration of copper in freshwater. Also, the results indicate that the fish C. fusca, as representative fish species in the East of Iran, can be a useful bioindicator organism of water contamination with copper.

Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

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Tian, Xinghui; Kaufman, Dan S

2008-07-01

Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression.

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Miedlich, Susanne U; Taya, Manisha; Young, Melissa Rasar; Hammes, Stephen R

2017-06-15

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

What is the best MR sepuence to evaluate normal structures of kidney?

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Chung, Hwan Hoon; Seol, Young Hae; Park, Chul Min; Kim, Jung Hyuk; Kim, Yun Hwan; Lee, Nam Jun; Chung, Kyoo Byung; Suh, Won Hyuk [Korea Univ. College of Medicine, Seoul (Korea, Republic of)

2001-01-01

To determine the best MR sequence for evaluation of the anatomical structures of normal kidney. Twenty normal volunteers (M:F = 15:5) took part in this study, and for each, seven sequences were performed. The T1 weighed sequences were conventional spin echo T1 (Conv-SET1), turbo spin echo T1 (TSET1), and fast low angle shot (FLASH), while the T2 weighted sequences were turbo spin echo T2 (TSET2), half-Fourier acquisition single -shot turbo spin echo (HASTE), true-fast imaging with steadystate precession (True-FISP), and echoplanar imaging (EPI). The study involved quantitative and puantitative analysis. In quantitative analysis, CNRs between cortex and adjacent fat tissue, and between cortex and medulla were calculated from SNR (signal to noise ratio), and the CNRs of sequences were statistcally compared. In quantative analysis, three radiologists collectively evaluated kidney outline, cortico-medullary division, the renal vessels, the pelvis/ureter, and artifacts. For each sequence a grade was assigned, and for each parameter the grades were compared. Between cortex and adjacent fat, the highest CNR was shown by TSET1, followed by Conv-SET1, while among T2 sequences, the CNR shown by TSET2 was highest. Between cortex and medulla, the CNR demonstarted by the three T1 sequences showed no statistically significant differance. Among T2 sequences, however, HASTE showed the highest CNR, followed by EPI, and sttistcally, the findings for these two were significantly differnt from those of other T2 sepuences. Among T1 sequences, FLASH provided the best kidney outline, though among T2-sequences there was no statiscally significant difference. FLASH was also the best for cortico-medullary distinstion, while for tis purpose the best T2 sequence was HASTE. True -FISP was best for the evaluation of renal vessels, and HASTE for evaulating the pelvis and ureter. Artifacts were most prominent on Conv SET1. For evaluating the shape of the kidney, the best T2 sequence was TSET2

What is the best MR sepuence to evaluate normal structures of kidney?

International Nuclear Information System (INIS)

Chung, Hwan Hoon; Seol, Young Hae; Park, Chul Min; Kim, Jung Hyuk; Kim, Yun Hwan; Lee, Nam Jun; Chung, Kyoo Byung; Suh, Won Hyuk

2001-01-01

To determine the best MR sequence for evaluation of the anatomical structures of normal kidney. Twenty normal volunteers (M:F = 15:5) took part in this study, and for each, seven sequences were performed. The T1 weighed sequences were conventional spin echo T1 (Conv-SET1), turbo spin echo T1 (TSET1), and fast low angle shot (FLASH), while the T2 weighted sequences were turbo spin echo T2 (TSET2), half-Fourier acquisition single -shot turbo spin echo (HASTE), true-fast imaging with steadystate precession (True-FISP), and echoplanar imaging (EPI). The study involved quantitative and puantitative analysis. In quantitative analysis, CNRs between cortex and adjacent fat tissue, and between cortex and medulla were calculated from SNR (signal to noise ratio), and the CNRs of sequences were statistcally compared. In quantative analysis, three radiologists collectively evaluated kidney outline, cortico-medullary division, the renal vessels, the pelvis/ureter, and artifacts. For each sequence a grade was assigned, and for each parameter the grades were compared. Between cortex and adjacent fat, the highest CNR was shown by TSET1, followed by Conv-SET1, while among T2 sequences, the CNR shown by TSET2 was highest. Between cortex and medulla, the CNR demonstarted by the three T1 sequences showed no statistically significant differance. Among T2 sequences, however, HASTE showed the highest CNR, followed by EPI, and sttistcally, the findings for these two were significantly differnt from those of other T2 sepuences. Among T1 sequences, FLASH provided the best kidney outline, though among T2-sequences there was no statiscally significant difference. FLASH was also the best for cortico-medullary distinstion, while for tis purpose the best T2 sequence was HASTE. True -FISP was best for the evaluation of renal vessels, and HASTE for evaulating the pelvis and ureter. Artifacts were most prominent on Conv SET1. For evaluating the shape of the kidney, the best T2 sequence was TSET2

Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

DEFF Research Database (Denmark)

Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer

2013-01-01

Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought...... not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants....

Guidance Cue Netrin-1 and the Regulation of Inflammation in Acute and Chronic Kidney Disease

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Punithavathi Ranganathan

2014-01-01

Full Text Available Acute kidney injury (AKI is a common problem in the hospital setting and intensive care unit. Despite improved understanding, there are no effective therapies available to treat AKI. A large body of evidence strongly suggests that ischemia reperfusion injury is an inflammatory disease mediated by both adaptive and innate immune systems. Cell migration also plays an important role in embryonic development and inflammation, and this process is highly regulated to ensure tissue homeostasis. One such paradigm exists in the developing nervous system, where neuronal migration is mediated by a balance between chemoattractive and chemorepulsive signals. The ability of the guidance molecule netrin-1 to repulse or abolish attraction of neuronal cells expressing the UNC5B receptor makes it an attractive candidate for the regulation of inflammatory cell migration. Recent identification of netrin-1 as regulators of immune cell migration has led to a large number of studies looking into how netrin-1 controls inflammation and inflammatory cell migration. This review will focus on recent advances in understanding netrin-1 mediated regulation of inflammation during acute and chronic kidney disease and whether netrin-1 and its receptor activation can be used to treat acute and chronic kidney disease.

Comparing the difference of measured GFR of ectopic pelvic kidney between anterior and posterior imaging processing in renal dynamic imaging

International Nuclear Information System (INIS)

Li Baojun; Zhao Deshan

2014-01-01

Objective: To compare and analyze the difference of measured glomerular filtration rate (GFR) of ectopic pelvic kidney between anterior and posterior imaging processing in renal dynamic imaging. Methods: There were 10 patients collected retrospectively, with ectopic kidneys in pelvic cavity confirmed by ultrasound, CT, renal dynamic imaging and other imaging modalities. All images of ectopic kidneys in renal dynamic imaging were processed by anterior and posterior methods respectively. The ectopic kidney was only processed in anterior imaging, ectopic kidney and contralateral normal kidney were processed in posterior imaging. Total GFR equalled the sum of GFR of normal kidney in posterior imaging and GFR of ectopic kidney in anterior imaging, was compared with total GFR of two kidneys in posterior imaging and GFR in two-sample method. All correlation analysis were completed between GFRs from three methods and all patients were followed up. Statistically paired t-test and bivariate correlation analysis test were used. Results: The mean GFR of ectopic kidney in anterior imaging equal to (27.48±12.24) ml/(min · 1.73 m 2 ). It was more than GFR [(10.71 ±4.74) ml/ (min · 1.73 m 2 )] in posterior imaging above 46% (t=5.481, P<0.01). There was no significant difference (t=-2.238, P>0.05), but better correlation (r=0.704, P<0.05) between total GFR in anterior imaging and GFR in two-sample method. There was significant difference (t=4.629, P<0.01)and worse correlation (r=0.576, P>0.05) between total GFR in posterior imaging and GFR in two-sample method. Conclusion: Comparing with GFR in posterior imaging, GFR in anterior imaging can more truly reflect function condition of ectopic pelvic kidney in renal dynamic imaging. (authors)

The ‘Ventral Organs’ of Pycnogonida (Arthropoda) Are Neurogenic Niches of Late Embryonic and Post-Embryonic Nervous System Development

Science.gov (United States)

Brenneis, Georg; Scholtz, Gerhard

2014-01-01

Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions – traditionally designated as ‘ventral organs’ – detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons – as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient

Function of JARID2 in bovines during early embryonic development

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Yao Fu

2017-12-01

Full Text Available Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05, and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the

Chronic Kidney Diseases

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... Safe Videos for Educators Search English Español Chronic Kidney Diseases KidsHealth / For Kids / Chronic Kidney Diseases What's ... re talking about your kidneys. What Are the Kidneys? Your kidneys are tucked under your lower ribs ...

The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

Science.gov (United States)

Gildea, John J; Xu, Peng; Carlson, Julia M; Gaglione, Robert T; Bigler Wang, Dora; Kemp, Brandon A; Reyes, Camellia M; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

2015-12-01

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake. Copyright © 2015 the American Physiological Society.

Properties of pure n-butylammonium nitrate ionic liquid and its binary mixtures of with alcohols at T = (293.15 to 313.15) K

International Nuclear Information System (INIS)

Xu, Yingjie; Chen, Bin; Qian, Wu; Li, Haoran

2013-01-01

Highlights: ► Densities and viscosities of (N4NO 3 + alcohols) mixtures were measured. ► Coefficient of thermal expansion, molecular volume, standard entropy, and lattice energy were obtained. ► Excess molar volumes, viscosity deviations, and partial molar volumes were calculated. ► Redlich–Kister polynomial was used to correlate the excess properties. ► The intermolecular interactions between N4NO 3 and alcohols were analysed. -- Abstract: Values of the density and viscosity of the pure ionic liquid n-butylammonium nitrate (N4NO 3 ) and its binary mixtures with methanol, ethanol, 1-propanol, and 1-butanol were measured at temperature ranging from T = (293.15 to 313.15) K. The thermal expansion coefficient, molecular volume, standard entropy, and lattice energy of N4NO 3 were deduced from the experimental density results. The temperature dependence of the viscosity of N4NO 3 was fitted to the fluidity equation. Excess molar volumes V E and viscosity deviations Δη for the binary mixtures were calculated and fitted to the Redlich–Kister equation with satisfactory results. Both excess molar volumes and viscosity deviations show negative deviations for (N4NO 3 + alcohol) mixtures. The effect of the temperature and the size of the alcohol on the excess molar volumes and viscosity deviations are discussed and analysed. Other derived properties, such as the apparent molar volume, partial molar volume, excess partial molar volume, thermal expansion coefficient, and excess thermal expansion coefficient of the above-mentioned systems were also calculated

How the embryonic chick brain twists.

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Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A

2016-11-01

During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left-right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic morphology and mechanics analysis that the vitelline membrane (VM) exerts an external load on the brain that drives torsion. Our theoretical analysis showed that the force is of the order of 10 micronewtons. We also designed an experiment to use fluid surface tension to replace the mechanical role of the VM, and the estimated magnitude of the force owing to surface tension was shown to be consistent with the above theoretical analysis. We further discovered that the asymmetry of the looping heart determines the chirality of the twisted brain via physical mechanisms, demonstrating the mechanical transfer of left-right asymmetry between organs. Our experiments also implied that brain flexure is a necessary condition for torsion. Our work clarifies the mechanical origin of torsion and the development of left-right asymmetry in the early embryonic brain. © 2016 The Author(s).

Magnetisation transfer MR imaging of the kidney: evaluation at 3.0 T in association with renal function

International Nuclear Information System (INIS)

Ito, Katsuyoshi; Hayashida, Minoru; Izumitani, Shogo; Fujimine, Tomoko; Onishi, Takeo; Genba, Katsuhiro

2013-01-01

To evaluate the feasibility of using magnetisation transfer (MT) MRI of the kidney at 3.0 T to assess renal function. Forty-four patients who underwent abdominal MRI on a 3.0-T system including gradient-echo (GRE) sequences with and without MT pulse were included. In each patient, MT ratio (MTR) of the renal cortex and medulla was measured by using regions of interest (ROIs) placed on the MTR map image. Regression analysis showed good correlation between estimated glomerular filtration rate (eGFR) and MTR of the renal cortex (r = -0.645, P < 0.0001). Among 44 patients, 22 were categorised as the normal renal function group and 22 were classified as the decreased eGFR group. The mean MTR of the renal cortex in patients with decreased eGFR (mean MTR, 30.7 ± 3.2 %) was significantly higher (P < 0.0001) than that in patients with normal renal function (mean MTR, 25.3 ± 2.2 %), although the mean MTRs of the renal medulla in the two groups were not significantly different. There was good correlation between eGFR and MTR of the renal cortex derived from MT MRI at 3.0 T. This technique may have the potential to evaluate the degree of renal function non-invasively in patients with renal impairment. (orig.)

Kidney biopsy

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... the kidney (in rare cases, may require a blood transfusion) Bleeding into the muscle, which might cause soreness Infection (small risk) Alternative Names Renal biopsy; Biopsy - kidney Images Kidney anatomy ...

Bioengineering Embryonic Stem Cell Microenvironments for the Study of Breast Cancer

Directory of Open Access Journals (Sweden)

Yubing Xie

2011-11-01

Full Text Available Breast cancer is the most prevalent disease amongst women worldwide and metastasis is the main cause of death due to breast cancer. Metastatic breast cancer cells and embryonic stem (ES cells display similar characteristics. However, unlike metastatic breast cancer cells, ES cells are nonmalignant. Furthermore, embryonic microenvironments have the potential to convert metastatic breast cancer cells into a less invasive phenotype. The creation of in vitro embryonic microenvironments will enable better understanding of ES cell-breast cancer cell interactions, help elucidate tumorigenesis, and lead to the restriction of breast cancer metastasis. In this article, we will present the characteristics of breast cancer cells and ES cells as well as their microenvironments, importance of embryonic microenvironments in inhibiting tumorigenesis, convergence of tumorigenic and embryonic signaling pathways, and state of the art in bioengineering embryonic microenvironments for breast cancer research. Additionally, the potential application of bioengineered embryonic microenvironments for the prevention and treatment of invasive breast cancer will be discussed.

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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Monica K Akre

Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

CITED1 Expression in Liver Development and Hepatoblastoma

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Andrew J. Murphy

2012-12-01

Full Text Available Hepatoblastoma, the most common pediatric liver cancer, consists of epithelial mixed embryonal/fetal (EMEF and pure fetal histologic subtypes, with the latter exhibiting a more favorable prognosis. Few embryonal histology markers that yield insight into the biologic basis for this prognostic discrepancy exist. CBP/P-300 interacting transactivator 1 (CITED1, a transcriptional co-activator, is expressed in the self-renewing nephron progenitor population of the developing kidney and broadly in its malignant analog, Wilms tumor (WT. In this current study, CITED1 expression is detected in mouse embryonic liver initially on post-coitum day 10.5 (e10.5, begins to taper by e14.5, and is undetectable in e18.5 and adult livers. CITED1 expression is detected in regenerating murine hepatocytes following liver injury by partial hepatectomy and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Importantly, while CITED1 is undetectable in normal human adult livers, 36 of 41 (87.8% hepatoblastoma specimens express CITED1, where it is enriched in EMEF specimens compared to specimens of pure fetal histology. CITED1 overexpression in Hep293TT human hepatoblastoma cells induces cellular proliferation and upregulates the Wnt inhibitors Kringle containing transmembrane protein 1 (KREMEN1 and CXXC finger protein 4 (CXXC4. CITED1 mRNA expression correlates with expression of CXXC4 and KREMEN1 in clinical hepatoblastoma specimens. These data show that CITED1 is expressed during a defined time course of liver development and is no longer expressed in the adult liver but is upregulated in regenerating hepatocytes following liver injury. Moreover, as in WT, this embryonic marker is reexpressed in hepatoblastoma and correlates with embryonal histology. These findings identify CITED1 as a novel marker of hepatic progenitor cells that is re-expressed following liver injury and in embryonic liver tumors.

Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3.

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Phan, Chia-Wei; David, Pamela; Naidu, Murali; Wong, Kah-Hui; Sabaratnam, Vikineswary

2013-10-11

Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health.

[Adenovirus-mediated canine interferon-gamma expression and its antiviral activity against canine parvovirus].

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Zhang, Kao; Jin, Huijun; Zhong, Fei; Li, Xiujin; Neng, Changai; Chen, Huihui; Li, Wenyan; Wen, Jiexia

2012-11-04

To construct recombinant adenovirus containing canine interferon-gamma (cIFN-gamma) gene and to investigate its antiviral activity against canine parvovirus in Madin-Darby canine kidney cells (MDCK). [Methods] The cIFN-gamma gene was inserted into adenovirus shuttle plasmid to construct pShuttle3-cIFN-gamma expression vector, from which the cIFN-gamma expression cassette was transferred into the adenovirus genomic plasmid pAdeno-X by specific restriction sites to generate recombinant adenovirus genomic plasmid pAd-cIFN-gamma. The pAd-cIFN-gamma plasmid was linearized by digestion and transfected into human embryonic kidney (HEK) 293T cells to generate the replication-defective cIFN-gamma recombinant adenovirus (Ad-cIFN-gamma). To analyze its anti-canine parvovirus activity, the MDCK cells were pre-infected by Ad-cIFN-gamma recombinant adenovirus, and then infected by canine parvovirus. The antiviral activity of the Ad-cIFN-gamma recombinant adenovirus against parvovirus was analyzed. The recombinant adenovirus containing cIFN-gamma gene was constructed by the ligation method. The recombinant adenovirus could mediates recombinant cIFN-gamma secretory expression in MDCK cells. The Ad-cIFN-gamma recombinant adenovirus could significantly inhibit canine parvovirus replication in MDCK cells pre-infected with the recombinant adenovirus. These results indicate that the Ad-cIFN-gamma recombinant adenovirus has the potent antiviral activity against canine parvovirus. The Ad-cIFN-gamma recombinant adenovirus was successfully constructed by the ligation method and possessed a powerful antiviral activity against canine parvovirus.

Genetic polymorphisms in calcitonin receptor gene and risk for recurrent kidney calcium stone disease.

Science.gov (United States)

Shakhssalim, Nasser; Basiri, Abbas; Houshmand, Massoud; Pakmanesh, Hamid; Golestan, Banafsheh; Azadvari, Mohaddeseh; Aryan, Hajar; Kashi, Amir H

2014-01-01

In this study the full sequence of the calcitonin receptor gene (CALCR) in a group of Iranian males suffering from recurrent calcium urinary stones was compared with that of a control group. Serum and urinary biochemistry related to urolithiasis were evaluated in 105 males diagnosed with recurrent kidney calcium stones and 101 age-matched healthy control males. The polymerase chain reaction single-strand conformation polymorphism method was used to detect new polymorphisms in the CALCR. Nine polymorphisms were detected; seven were in the non-coding and two in the coding region. The T allele associated with the 3'UTR+18C>T polymorphism was observed exclusively in the stone formers. The exact odds ratio for the T allele in this locus for those at risk of stone formation was 36.72 (95% CI 4.95-272.0) (p C and IVS1insA polymorphisms in intron 1 were associated with kidney stone disease (p T and intron 1 polymorphisms in the CALCR and the risk of kidney stone disease. 2013 S. Karger AG, Basel.

MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

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Liang, Liping; Zhu, Ji [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zaorsky, Nicholas G. [Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania (United States); Deng, Yun [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Wu, Xingzhong [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Liu, Yong [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Liu, Fangqi; Cai, Guoxiang; Gu, Weilie [Department of Colorectal Cancer, Fudan University, Shanghai Cancer Center, Shanghai (China); Shen, Lijun [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Zhen, E-mail: zhenzhang6@hotmail.com [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China)

2014-03-15

Purpose: Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Methods and Materials: Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3′ untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively). Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER{sub 2}) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. Results: The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3′UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER{sub 2} = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm{sup 3} vs 1.7 cm{sup 3}). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm{sup 3} vs 0.829 cm{sup 3}). Conclusions: miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are

Gadolinium-enhanced T{sub 1}-weighted MR urography versus T{sub 2}-weighted (HASTE) MR urography in children; Kontrastangehobene T{sub 1}-gewichtete MR-Urographie versus T{sub 2}-gewichtete (HASTE) MR-Urographie im Kindesalter

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Staatz, G.; Nolte-Ernsting, C.C.A.; Haage, P.; Tacke, J.; Guenther, R.W. [Technische Hochschule Aachen (Germany). Klinik fuer Radiologische Diagnostik; Rohrmann, D. [Technische Hochschule Aachen (Germany). Urologische Klinik; Stollbrink, C. [Technische Hochschule Aachen (Germany). Kinderklinik

2001-11-01

Purpose: To evaluate gadolinium-enhanced T{sub 1}-weighted excretory MR urography (EMRU) versus T{sub 2}-weighted (HASTE) MR urography in children with upper urinary tract abnormalities. Patients and Methods: In a prospective study 63 children, aged from 3 weeks to 15 years, underwent MR urography in a 1.5-T scanner. Before and after an intravenous injection of 0.05 mg/kg body weight of furosemide, respiratory-triggered HASTE images were obtained for T{sub 2}-weighted MR urography. EMRU was performed subsequent to i.v. gadolinium injection with respiratory-gated, coronal 3D-gradient-echo sequences. Results: Compared to T{sub 2}-weighted (HASTE) MR urography, gadolinium-enhanced MR urography revealed a superior diagnostic accuracy in non-dilated collecting systems (horseshoe kidneys, ectopic kidneys, duplex systems, single ectopic ureters, ureteroceles). EMRU and T{sub 2}-weighted (HASTE) MRU turned out to be equivalent in the assessment of obstructed but normal functioning upper urinary tracts (UPJ obstructions, megaureters). Non-functioning dilated collecting systems and multicystic dysplastic kidneys were best visualized with use of T{sub 2}-weighted (HASTE) MR urography. Conclusion: Respiratory-gated gadolinium-enhanced T{sub 1}-weighted MRU allows accurate evaluation of most upper urinary tract abnormalities. T{sub 2}-weighted (HASTE) MRU complements GMRU in the evaluation of non-functioning renal units and cystic disease of the kidneys. (orig.) [German] Ziel: Vergleich der kontrastangehobenen T{sub 1}-gewichteten MR-Urographie mit der T{sub 2}-gewichteten (HASTE) MR-Urographie bei Kindern mit Anomalien des oberen Harntraktes. Methoden: In einer prospektiven Studie wurde bei 63 Kindern (3 Wo. - 15J.) eine MR-Urographie (MRU) in einem 1,5-Tesla-Magneten durchgefuehrt. Die T{sub 2}-gewichtete MRU erfolgte vor und nach intravenoeser Injektion von 0,05 mg/kg KG Furosemid mit atemgetriggerten HASTE-Sequenzen. Fuer die T{sub 1}-gewichtete MRU wurden nach

How Kidney Cell Death Induces Renal Necroinflammation.

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Mulay, Shrikant R; Kumar, Santhosh V; Lech, Maciej; Desai, Jyaysi; Anders, Hans-Joachim

2016-05-01

The nephrons of the kidney are independent functional units harboring cells of a low turnover during homeostasis. As such, physiological renal cell death is a rather rare event and dead cells are flushed away rapidly with the urinary flow. Renal cell necrosis occurs in acute kidney injuries such as thrombotic microangiopathies, necrotizing glomerulonephritis, or tubular necrosis. All of these are associated with intense intrarenal inflammation, which contributes to further renal cell loss, an autoamplifying process referred to as necroinflammation. But how does renal cell necrosis trigger inflammation? Here, we discuss the role of danger-associated molecular patterns (DAMPs), mitochondrial (mito)-DAMPs, and alarmins, as well as their respective pattern recognition receptors. The capacity of DAMPs and alarmins to trigger cytokine and chemokine release initiates the recruitment of leukocytes into the kidney that further amplify necroinflammation. Infiltrating neutrophils often undergo neutrophil extracellular trap formation associated with neutrophil death or necroptosis, which implies a release of histones, which act not only as DAMPs but also elicit direct cytotoxic effects on renal cells, namely endothelial cells. Proinflammatory macrophages and eventually cytotoxic T cells further drive kidney cell death and inflammation. Dissecting the molecular mechanisms of necroinflammation may help to identify the best therapeutic targets to limit nephron loss in kidney injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel kinin B1 receptor agonists with improved pharmacological profiles.

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Côté, Jérôme; Savard, Martin; Bovenzi, Veronica; Bélanger, Simon; Morin, Josée; Neugebauer, Witold; Larouche, Annie; Dubuc, Céléna; Gobeil, Fernand

2009-04-01

There is some evidence to suggest that inducible kinin B1 receptors (B1R) may play beneficial and protecting roles in cardiovascular-related pathologies such as hypertension, diabetes, and ischemic organ diseases. Peptide B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of these diseases. In the present study, we used solid-phase methodology to synthesize a series of novel peptide analogues based on the sequence of Sar[dPhe(8)]desArg(9)-bradykinin, a relatively stable peptide agonist with moderate affinity for the human B1R. We evaluated the pharmacological properties of these peptides using (1) in vitro competitive binding experiments on recombinant human B1R and B2R (for index of selectivity determination) in transiently transfected human embryonic kidney 293 cells (HEK-293T cells), (2) ex vivo vasomotor assays on isolated human umbilical veins expressing endogenous human B1R, and (3) in vivo blood pressure tests using anesthetized lipopolysaccharide-immunostimulated rabbits. Key chemical modifications at the N-terminus, the positions 3 and 5 on Sar[dPhe(8)]desArg(9)-bradykinin led to potent analogues. For example, peptides 18 (SarLys[Hyp(3),Cha(5), dPhe(8)]desArg(9)-bradykinin) and 20 (SarLys[Hyp(3),Igl(5), dPhe(8)]desArg(9)-bradykinin) outperformed the parental molecule in terms of affinity, functional potency and duration of action in vitro and in vivo. These selective agonists should be valuable in future animal and human studies to investigate the potential benefits of B1R activation.

Chronic Kidney Disease and Kidney Failure

Science.gov (United States)

... death rates limited life expectancy. Some patients were lucky enough to get a kidney transplant, which greatly ... epidemic rates. Through the 1980s and 1990s, the number of patients developing end-stage kidney failure nearly ...

Stepwise development of hematopoietic stem cells from embryonic stem cells.

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Kenji Matsumoto

Full Text Available The cellular ontogeny of hematopoietic stem cells (HSCs remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+CD41(+CD45(- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

Antigravity suit inflation - Kidney function and cardiovascular and hormonal responses in men

Science.gov (United States)

Geelen, Ghislaine; Kravik, Stein E.; Hadj-Aissa, Aoumeur; Leftheriotis, Georges; Vincent, Madeleine

1989-01-01

The effect of the lower body positive pressure (LBPP) on kidney function in normal men was investigated in experiments in which the subjects underwent 30 min of sitting and then were subjected to 4.5 h of 70-deg head-up tilt. During the last 3 h of the tilt period, an antigravity suit (60 T legs, 30 T abdomen) was applied. The results showed that LBPP induces a significant increase in effective renal plasma flow and significant changes in the kidney excretory patterns, which were similar to those observed during a water immersion or the early phase of bed rest.

Functional significance of SPINK1 promoter variants in chronic pancreatitis.

Science.gov (United States)

Derikx, Monique H M; Geisz, Andrea; Kereszturi, Éva; Sahin-Tóth, Miklós

2015-05-01

Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation. Copyright © 2015 the American Physiological Society.

DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

International Nuclear Information System (INIS)

Mei, Nan; Arlt, Volker M.; Phillips, David H.; Heflich, Robert H.; Chen, Tao

2006-01-01

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32 P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N 6 -yl]-aristolactam I, 7-[deoxyadenosin-N 6 -yl]-aristolactam II and 7-[deoxyguanosin-N 2 -yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10 8 nucleotides in liver and 95-4598 adducts/10 8 nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10 -6 in liver compared with the MFs of 78-1319 x 10 -6 that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually

DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

Energy Technology Data Exchange (ETDEWEB)

Mei, Nan [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)]. E-mail: nan.mei@fda.hhs.gov; Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Heflich, Robert H. [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States); Chen, Tao [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)

2006-12-01

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by {sup 32}P-postlabeling and mutant frequency (MF) was determined using the {lambda} Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N {sup 6}-yl]-aristolactam I, 7-[deoxyadenosin-N {sup 6}-yl]-aristolactam II and 7-[deoxyguanosin-N {sup 2}-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10{sup 8} nucleotides in liver and 95-4598 adducts/10{sup 8} nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10{sup -6} in liver compared with the MFs of 78-1319 x 10{sup -6} that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T {sup {yields}} T:A transversion was the predominant mutation in AA-treated rats; whereas G:C {sup {yields}} A:T transition was the main type of mutation in control

Transplantation of Kidneys From Donors With Acute Kidney Injury: Friend or Foe?

NARCIS (Netherlands)

Boffa, C.; van de Leemkolk, F.; Curnow, E.; Homan van der Heide, J.; Gilbert, J.; Sharples, E.; Ploeg, R. J.

2017-01-01

The gap between supply and demand in kidney transplantation has led to increased use of marginal kidneys; however, kidneys with acute kidney injury are often declined/discarded. To determine whether this policy is justified, we analyzed outcomes of donor kidneys with acute kidney injury (AKI) in a

Enhancement of bioavailability by formulating rhEPO ionic complex with lysine into PEG-PLA micelle

Energy Technology Data Exchange (ETDEWEB)

Shi, Yanan; Sun, Fengying; Wang, Dan; Zhang, Renyu [Jilin University, College of Life Science (China); Dou, Changlin; Liu, Wanhui; Sun, Kaoxiang, E-mail: sunkx@ytu.edu.cn [Yantai University, School of Pharmacy (China); Li, Youxin, E-mail: liyouxin@jlu.edu.cn [Jilin University, College of Life Science (China)

2013-10-15

A composite micelle of ionic complex encapsulated into poly(ethylene glycol)-poly(d,l-lactide) (PEG-PLA) di-block copolymeric micelles was used for protein drug delivery to improve its pharmacokinetic performance. In this study, recombinant human erythropoietin (rhEPO, as a model protein) was formulated with lysine into composite micelles at a diameter of 71.5 nm with narrow polydispersity indices (PDIs < 0.3). Only a trace amount of protein was in aggregate form. The zeta potential of the spherical micelles was ranging from -0.54 to 1.39 mv, and encapsulation efficiency is high (80 %). The stability of rhEPO was improved significantly in composite micelles in vitro. Pharmacokinetic studies in rats showed significant, enhanced plasma retention of the composite micelles in comparison with native rhEPO. Areas under curve (AUCs) of the rhEPO released from the composite micelles were 4.5- and 2.3-folds higher than those of the native rhEPO and rhEPO-loaded PEG-PLA micelle, respectively. In addition, the composite micelles exhibited good biocompatibility using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay with human embryonic kidney (HEK293T) cells. All these features are preferable for utilizing the composite micelles as a novel protein delivery system.

Enhancement of bioavailability by formulating rhEPO ionic complex with lysine into PEG-PLA micelle

Science.gov (United States)

Shi, Yanan; Sun, Fengying; Wang, Dan; Zhang, Renyu; Dou, Changlin; Liu, Wanhui; Sun, Kaoxiang; Li, Youxin

2013-10-01

A composite micelle of ionic complex encapsulated into poly(ethylene glycol)-poly( d, l-lactide) (PEG-PLA) di-block copolymeric micelles was used for protein drug delivery to improve its pharmacokinetic performance. In this study, recombinant human erythropoietin (rhEPO, as a model protein) was formulated with lysine into composite micelles at a diameter of 71.5 nm with narrow polydispersity indices (PDIs protein was in aggregate form. The zeta potential of the spherical micelles was ranging from -0.54 to 1.39 mv, and encapsulation efficiency is high (80 %). The stability of rhEPO was improved significantly in composite micelles in vitro. Pharmacokinetic studies in rats showed significant, enhanced plasma retention of the composite micelles in comparison with native rhEPO. Areas under curve (AUCs) of the rhEPO released from the composite micelles were 4.5- and 2.3-folds higher than those of the native rhEPO and rhEPO-loaded PEG-PLA micelle, respectively. In addition, the composite micelles exhibited good biocompatibility using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay with human embryonic kidney (HEK293T) cells. All these features are preferable for utilizing the composite micelles as a novel protein delivery system.

Cyanobacteria from Terrestrial and Marine Sources Contain Apoptogens Able to Overcome Chemoresistance in Acute Myeloid Leukemia Cells

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Liu, Liwei; Herfindal, Lars; Jokela, Jouni; Shishido, Tania Keiko; Wahlsten, Matti; Døskeland, Stein Ove; Sivonen, Kaarina

2014-01-01

In this study, we investigated forty cyanobacterial isolates from biofilms, gastropods, brackish water and symbiotic lichen habitats. Their aqueous and organic extracts were used to screen for apoptosis-inducing activity against acute myeloid leukemia cells. A total of 28 extracts showed cytotoxicity against rat acute myeloid leukemia (IPC-81) cells. The design of the screen made it possible to eliminate known toxins, such as microcystins and nodularin, or known metabolites with anti-leukemic activity, such as adenosine and its analogs. A cytotoxicity test on human embryonic kidney (HEK293T) fibroblasts indicated that 21 of the 28 extracts containing anti-acute myeloid leukemia (AML) activity showed selectivity in favor of leukemia cells. Extracts L26-O and L30-O were able to partly overcome the chemotherapy resistance induced by the oncogenic protein Bcl-2, whereas extract L1-O overcame protection from the deletion of the tumor suppressor protein p53. In conclusion, cyanobacteria are a prolific resource for anti-leukemia compounds that have potential for pharmaceutical applications. Based on the variety of cellular responses, we also conclude that the different anti-leukemic compounds in the cyanobacterial extracts target different elements of the death machinery of mammalian cells. PMID:24705501

Auranofin and N-heterocyclic carbene gold-analogs are potent inhibitors of the bacteria Helicobacter pylori.

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Owings, Joshua P; McNair, Nina N; Mui, Yiu Fung; Gustafsson, Tomas N; Holmgren, Arne; Contel, Maria; Goldberg, Joanna B; Mead, Jan R

2016-07-01

Auranofin is an FDA-approved gold-containing compound used for the treatment of rheumatoid arthritis. Recent reports of antimicrobial activity against protozoa and bacteria indicate that auranofin targets the reductive enzyme thioredoxin reductase (TrxR). We evaluated auranofin as well as five auranofin analogs containing N-heterocyclic carbenes (instead of the triethylphosphane present in auranofin) and five gold-carbene controls for their ability to inhibit or kill Helicobacter pylori in vitro Auranofin completely inhibited bacterial growth at 1.2 μM. Purified H. pylori TrxR was inhibited by auranofin in a cell-free assay (IC50 ∼88 nM). The most active gold(I)-N-heterocyclic carbene compounds exhibited MICs comparable to auranofin against H. pylori (2 μM), while also exhibiting lower toxicities for human embryonic kidney cells (HEK-293T cells). Median toxic concentrations (TC50) were 13-20-fold higher compared to auranofin indicating that they were less cytotoxic. The N-heterocyclic carbene analogs maybe well tolerated, but further evaluation is needed in vivo Finally, auranofin was synergistic with the antibiotic amoxicillin, suggesting that targeting both the reductive enzyme TrxR and cell wall synthesis may be effective against H. pylori infections. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues.

Science.gov (United States)

Kumar, Ajay; Yellepeddi, Venkata K; Vangara, Kiran K; Strychar, Kevin B; Palakurthi, Srinath

2011-11-01

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

Effect of constituents from samaras of Austroplenckia populnea (Celastraceae) on human cancer cells.

Science.gov (United States)

Caneschi, Carolina Milagres; Muniyappa, Mohan K; Duarte, Lucienir P; Silva, Grácia D F; Dos Santos, Orlando David Henrique; Spillane, Charles; Filho, Sidney Augusto Vieira

2015-01-01

Aiming the continuity of the studies of Austroplenckia populnea, Brazilian species of the Celastraceae family, in the present study, it was investigated the effect of crude extracts obtained with ethanol, ethyl acetate and chloroform and two purified constituents, proanthocyanidin A and 4'-O-methylepigallocatechin, both isolated from its samaras, on cancer cell proliferation assays. The human cancer cells lines MCF-7 (ductal breast carcinoma), A549 (lung cancer), HS578T (ductal breast carcinoma) and non-cancer HEK293 (embryonic kidney cells) were treated with different concentrations of extracts and constituents and the effect was observed through the acid phosphatase method. The chemical structures of the purified compounds were identified by the respective IR and (1)H and (13)C nuclear magnetic resonance spectral data. While crude extracts from samaras of the folk medicine A. populnea can trigger cell proliferative effects in human cell lines, the purified compounds (proanthocyanidin A and 4'-O-methyl-epigallocatechin) isolated from the same extracts can have an opposite (anti-proliferative) effect. Based on the results, it was possible to suggest that extracts from samaras of A. populnea should be further investigated for possible cancer-promoting activities; and the active extracts can also represent a source of compounds that have anti-cancer properties.

Cyanobacteria from Terrestrial and Marine Sources Contain Apoptogens Able to Overcome Chemoresistance in Acute Myeloid Leukemia Cells

Directory of Open Access Journals (Sweden)

Liwei Liu

2014-04-01

Full Text Available In this study, we investigated forty cyanobacterial isolates from biofilms, gastropods, brackish water and symbiotic lichen habitats. Their aqueous and organic extracts were used to screen for apoptosis-inducing activity against acute myeloid leukemia cells. A total of 28 extracts showed cytotoxicity against rat acute myeloid leukemia (IPC-81 cells. The design of the screen made it possible to eliminate known toxins, such as microcystins and nodularin, or known metabolites with anti-leukemic activity, such as adenosine and its analogs. A cytotoxicity test on human embryonic kidney (HEK293T fibroblasts indicated that 21 of the 28 extracts containing anti-acute myeloid leukemia (AML activity showed selectivity in favor of leukemia cells. Extracts L26-O and L30-O were able to partly overcome the chemotherapy resistance induced by the oncogenic protein Bcl-2, whereas extract L1-O overcame protection from the deletion of the tumor suppressor protein p53. In conclusion, cyanobacteria are a prolific resource for anti-leukemia compounds that have potential for pharmaceutical applications. Based on the variety of cellular responses, we also conclude that the different anti-leukemic compounds in the cyanobacterial extracts target different elements of the death machinery of mammalian cells.

Profiling gene expression induced by protease-activated receptor 2 (PAR2 activation in human kidney cells.

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Jacky Y Suen

Full Text Available Protease-Activated Receptor-2 (PAR2 has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD and key events in tumor progression (angiogenesis, metastasis, but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293, a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2 and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2. Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes, the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2 and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15. Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4 known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

The primary role of zebrafish nanog is in extra-embryonic tissue.

Science.gov (United States)

Gagnon, James A; Obbad, Kamal; Schier, Alexander F

2018-01-09

The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZ nanog ) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZ nanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation. © 2018. Published by The Company of Biologists Ltd.

Pathways in pluripotency and differentiation of embryonic cells

NARCIS (Netherlands)

du Puy, L.

2010-01-01

Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

A genetically modified protein-based hydrogel for 3D culture of AD293 cells.

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Xiao Du

Full Text Available Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1 by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol having their arm ends capped with maleimide residues (4-armed-PEG-Mal to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence 'GRGDSP' to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.

Nephrectomy (Kidney Removal)

Science.gov (United States)

... nephrectomy is needed because of other kidney diseases. Kidney function Most people have two kidneys — fist-sized ... and the disease that prompted the surgery? Monitoring kidney function Most people can function well with only ...

NOS3 Polymorphisms and Chronic Kidney Disease

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Alejandro Marín Medina

2018-05-01

Full Text Available ABSTRACT Chronic kidney disease (CKD is a multifactorial pathophysiologic irreversible process that often leads to a terminal state in which the patient requires renal replacement therapy. Most cases of CKD are due to chronic-degenerative diseases and endothelial dysfunction is one of the factors that contribute to its pathophysiology. One of the most important mechanisms for proper functioning of the endothelium is the regulation of the synthesis of nitric oxide. This compound is synthesized by the enzyme nitric oxide synthase, which has 3 isoforms. Polymorphisms in the NOS3 gene have been implicated as factors that alter the homeostasis of this mechanism. The Glu298Asp polymorphisms 4 b/a and -786T>C of the NOS3 gene have been associated with a more rapid deterioration of kidney function in patients with CKD. These polymorphisms have been evaluated in patients with CKD of determined and undetermined etiology and related to a more rapid deterioration of kidney function.

Analysis of T Cell Subsets in Adult Primary/Idiopathic Minimal Change Disease: A Pilot Study

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Francisco Salcido-Ochoa

2017-01-01

Full Text Available Aim. To characterise infiltrating T cells in kidneys and circulating lymphocyte subsets of adult patients with primary/idiopathic minimal change disease. Methods. In a cohort of 9 adult patients with primary/idiopathic minimal change recruited consecutively at disease onset, we characterized (1 infiltrating immune cells in the kidneys using immunohistochemistry and (2 circulating lymphocyte subsets using flow cytometry. As an exploratory analysis, association of the numbers and percentages of both kidney-infiltrating immune cells and the circulating lymphocyte subsets with kidney outcomes including deterioration of kidney function and proteinuria, as well as time to complete clinical remission up to 48 months of follow-up, was investigated. Results. In the recruited patients with primary/idiopathic minimal change disease, we observed (a a dominance of infiltrating T helper 17 cells and cytotoxic cells, comprising cytotoxic T cells and natural killer cells, over Foxp3+ Treg cells in the renal interstitium; (b an increase in the circulating total CD8+ T cells in peripheral blood; and (c an association of some of these parameters with kidney function and proteinuria. Conclusions. In primary/idiopathic minimal change disease, a relative numerical dominance of effector over regulatory T cells can be observed in kidney tissue and peripheral blood. However, larger confirmatory studies are necessary.

To evaluate vascular complications of transplant kidney examined by multislice spiral CT angiograph

International Nuclear Information System (INIS)

Peng Qian; Fan Miao; Luo Xiaomei

2008-01-01

Objective: To evaluate the value of multislice CT angiography (MSCTA) in vascular complications of transplant kidney. Methods: Six transplant kidneys were undergone enhanced MSCT scanning postoperation. MPR, CPR, VR and VP reformation were performed to observe transplant kidney's parenchyma, renal artery, and renal vein. To analysis all the reconstruction technique and find the advantage and shortage of them. Results: One case showed enhanced function of transplant kidney decreased. Vascular stenosis was found in one case and false aneurysm was found in another transplant kidney. Transplant kidney were enhanced normal in the left three cases. MPR couldn't reconstruct all the tortuous vessel of renal hilus in one plane. But all six cases could expose the vessel of renal hilus very clearly in coronal section and sagittal plane of CPR. Six cases of VR could observe the vessel direction and lesions outside vessel through rotate the reconstruction image. VP could see through inside the vessel of transplant kidney. Conclusion: MSCTA has an important role as an imaging technique to evaluate vascular complications of transplant kidney, it can replace DSA. (authors)

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Science.gov (United States)

Chen, Panpan; Douglas, Steven D.; Meshki, John; Tuluc, Florin

2012-01-01

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing. PMID:23024816

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

Directory of Open Access Journals (Sweden)

Panpan Chen

Full Text Available Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP. We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with the kidney survival time in chronic kidney disease patients.

Science.gov (United States)

Xu, Jinsheng; Guo, Zhanjun; Bai, Yaling; Zhang, Junxia; Cui, Liwen; Zhang, Huiran; Zhang, Shenglei; Ai, Xiaolu

2015-02-01

The mitochondrial displacement loop (D-loop) is known to accumulate mutations and SNPs at a higher frequency than other regions of mitochondrial DNA (mtDNA). We had identified chronic kidney disease (CKD) risk-associated SNPs in the D-loop of CKD patients previously. In this study, we investigated the association of SNPs in the D-loop of mtDNA with the kidney survival of CKD. The D-loop region of mtDNA was sequenced for 119 CKD patients from the inpatient of the Fourth Hospital of Hebei Medical University. The Kaplan-Meier method was used to identify disease outcome-associated SNPs in the D-loop of CKD patients. The Cox proportional hazards model was used to identify risk factors for the kidney survival of CKD. In the present study, we identified 20 SNPs with a frequency higher than 5% and assessed the relationship of these SNPs with kidney survival time in CKD patients, a SNP of 146 was identified by log-rank test for statistically significant prediction of the kidney survival time. In an overall multivariate analysis, allele 146 was identified as an independent predictor of kidney survival time in CKD patients. The survival time of kidney in the CKD patients with 146C was significantly shorter than that of kidney in CKD patients with 146T (relative risk, 2.336; 95% CI, 1.319-3.923; p = 0.001). SNPs in the D-loop can predict the kidney survival of CKD patients. Analysis of genetic polymorphisms in the mitochondrial D-loop can help to identify CKD patient subgroup at high risk of a poor disease outcome.

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

DEFF Research Database (Denmark)

Dalbøge, Louise S.; Pedersen, Søren L.; van Witteloostuijn, Søren Blok

2015-01-01

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible...... anti-diabetic and anti-obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed...... was investigated using a human embryonic kidney 293-based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor-interacting C-terminal octapeptide segment. In vivo efficacy...

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

Science.gov (United States)

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Giant Magnetoimpedance for Biosensing in Drug Delivery

Science.gov (United States)

Fal-Miyar, Vanesa; Kumar, Arun; Mohapatra, Shyam; Shirley, Shawna; Frey, Natalie A.; Barandiarán, José M.; Kurlyandskaya, Galina V.

2008-06-01

Iron oxide (Fe3O4) non-specific superparamagnetic nanoparticles of 30 nm size are introduced into human embryonic kidney (HEK-293) cells by intracellular uptake. The nanoparticles are magnetised by two superimposed magnetic fields, an externally applied DC field and an AC field generated by the high-frequency current flowing through Co64.5Fe2.5Cr3Si15B15 amorphous ribbons. The resulted fringe fields from the nanoparticles are detected via the magnetoimpedance change in the ribbons covered and uncovered by thin gold layer. The gold covering is considered an improvement due to its biocompatibility and because it avoids the biocorrosion process on the ribbon. The MI responses in both cases are clearly dependent on the presence of the magnetic nanoparticles inside the cells and on the value of the external field.

Live Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis activate the inflammatory response trhough Toll-like receptors 2, 4, and 9 in species-specific patterns

DEFF Research Database (Denmark)

Mogensen, T.H.; Paludan, Søren Riis; Kilian, Mogens

2006-01-01

activation by live bacteria. Here, we demonstrate that live Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis, the three principal causes of bacterial meningitis, use distinct sets of TLRs to trigger the inflammatory response. Using human embryonic kidney 293 cell lines......, each overexpressing one type of TLR, we found that S. pneumoniae triggered activation of the transcription factor nuclear factor-kappaB and expression of interleukin-8, only in cells expressing TLR2 or -9. The same response was evoked by H. influenzae in cells expressing TLR2 or -4 and by N...... and confirmed the essential role of these TLRs and also identified differential functions of TLRs in activation of the inflammatory response. Collectively, we here demonstrate that S. pneumoniae, H. influenzae, and N. meningitidis each activate several TLRs in species-specific patterns and show that infection...

Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

Science.gov (United States)

Newman, Carla F; Havelund, Rasmus; Passarelli, Melissa K; Marshall, Peter S; Francis, Ian; West, Andy; Alexander, Morgan R; Gilmore, Ian S; Dollery, Colin T

2017-11-21

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines

International Nuclear Information System (INIS)

Razonable, Raymund R.; Henault, Martin; Paya, Carlos V.

2006-01-01

The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-κB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-κB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-κB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study

Immunotherapy using dendritic cells and cytokine-induced killer for kidney cancer

International Nuclear Information System (INIS)

Chen Lijun; Xu Yuanbin; Zhao Li; Qu Nan; Sun Zhenpeng; Li Xuechao; Zhao Jiyu; Wang Bin; Wang Huixian

2008-01-01

Objective: To investigate the clinical efficacy of immunotherapy using dendritic cells (DC) and cytokine-induced killer (CIK)in treatment of patients with kidney cancer. Methods: Sixty patients with kidney cancer were divided into 2 groups randomly: the control group and immunotherapy group. Peripheral blood mononuclear cells (PBMC) were seperated from the patients who received immunotherapy first, then DC and CIK were induced and cultured with GM-CSF and IL4 in vitro. The immunotherapy group received DC four times and CIK twice at an interval of 14 days after routine treatment. The control group received only chemotherapy. T lymphocyte subtypes and NK cells in peripheral blood, the white cells and the values of liver and kidney biochemistry of two group of patients were analyzed and clinical efficacy were ob- served, so were side effects. Results: Clinical efficacy showed significant statistical difference between the two groups (P + , CD4 + , CD4 + /CD8 + and NK cell in the immunotherapy group increased after treatment, which showed significant statistical difference compared with those before treatment(P value was 0.010, 0.026, 0.021, 0.016, respectively). Changes in cell immune indexes (CD3 + , CD4 + , CD4 + /CD8 + ) in immunotherapy group and Control group showed significant statistical difference (P value was 0.001,0.023,0.012, respectively). Conclusion: Immunotherapy using dendritic cells and cytokine-induced killer combined with routine treatment can improve T lymphocyte subtypes and NK cell ratio in peripheral blood of the patients with kidney cancer, and may play an important role in the treatment of kidney cancer. It can enhance clinical efficacy in patients with kidney cancer and can improve prognosis. (authors)

[Wasting in chronic kidney disease: Refeeding techniques and artificial nutrition practices].

Science.gov (United States)

Pasian, Céline; Azar, Raymond; Fouque, Denis

2016-12-01

Protein energy wasting (PEW) is an independent factor associated with morbi-mortality in chronic kidney disease. Wasting is particularly common in chronic diseases of organs such as kidney disease with a major impact at the stage of dialysis. It covers 20 to 70% of patients diagnosed with chronic kidney disease according to the degree of evolution of the disease and the diagnostic method used patients. Mechanisms of PEW are based mainly on anorexia and metabolic abnormalities caused by kidney disease. Nutritional treatment differs depending on the stage of the kidney disease acute or chronic treated whether or not by dialysis. Nutritional monitoring should be regular, individualized and collaborative to detect a risk of PEW or treat installed PEW. Refeeding techniques should allow all the nutritional needs. Their indications depend on the clinic, biochemical assessment and nutrient intake. Copyright © 2016 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

Notch signaling activation in human embryonic stem cells is required for embryonic but not trophoblastic lineage commitment

OpenAIRE

Yu, Xiaobing; Zou, Jizhong; Ye, Zhaohui; Hammond, Holly; Chen, Guibin; Tokunaga, Akinori; Mali, Prashant; Li, Yue-Ming; Civin, Curt; Gaiano, Nicholas; Cheng, Linzhao

2008-01-01

The Notch signaling pathway plays important roles in cell fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell fate choices in human embryonic stem (hES) cells. Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hES cell lines. We report here that activation of Notch signaling is required for undifferentiated hES cells to form the pr...

Chronic Kidney Disease and Associated Cardiovascular Risk Factors in Chinese with Type 2 Diabetes

Directory of Open Access Journals (Sweden)

Qing-Lin Lou

2012-12-01

Full Text Available BackgroundTo determine the frequency of chronic kidney disease (CKD and its associated risk factors in Chinese type 2 diabetic patients, we conducted a cross-sectional study in Nanjing, China, in the period between January 2008 and December 2009.MethodsPatients with type 2 diabetes under the care by Jiangsu Province Official Hospital, Nanjing, China were invited for assessment. CKD was defined as the presence of albuminuria or estimated glomerular filtration rate <60 mL/min/1.73 m2. Albuminuria was defined as urinary albumin-to-creatinine ratio ≥30 mg/g.ResultsWe recruited 1,521 urban Chinese patients with type 2 diabetes (mean age, 63.9±12.0 years. The frequency of CKD and albuminuria was 31.0% and 28.9%, respectively. After adjusted by age and sex, hypertension, anemia and duration of diabetes were significantly associated with CKD with odds ratio (95% confidence interval being 1.93 (1.28 to 2.93, 1.70 (1.09 to 2.64, and 1.03 (1.00 to 1.06, respectively.ConclusionIn conclusion, CKD was common in the urban Nanjing Chinese with type 2 diabetes. Strategies to prevent or delay progression of kidney disease in diabetes should be carried out at the early disease course of type 2 diabetes.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

Science.gov (United States)

Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

Evaluation of renal quantitative T2* changes on MRI following administration of ferumoxytol as a T2* contrast agent.

Science.gov (United States)

Hedgire, Sandeep S; McDermott, Shaunagh; Wojtkiewicz, Gregory R; Abtahi, Seyed Mahdi; Harisinghani, Mukesh; Gaglia, Jason L

2014-01-01

To evaluate the time-dependent changes in regional quantitative T2* maps of the kidney following intravenous administration of ferumoxytol. Twenty-four individuals with normal kidney function underwent T2*-weighted MRI of the kidney before, immediately after, and 48 hours after intravenous administration of ferumoxytol at a dose of 4 mg/kg (group A, n=12) or 6 mg/kg (group B, n=12). T2* values were statistically analyzed using two-tailed paired t-tests. In group A, the percentage changes from baseline to immediate post and baseline to 48 hours were 85.3% and 64.2% for the cortex and 90.8% and 64.6% for the medulla, respectively. In group B, the percentage changes from baseline to immediate post and baseline to 48 hours were 85.2% and 73.4% for the cortex and 94.5% and 74% for the medulla, respectively. This difference was significant for both groups (P<0.0001). There is significant and differential uptake of ferumoxytol in the cortex and medulla of physiologically normal kidneys. This differential uptake may offer the ability to interrogate renal cortex and medulla with possible clinical applications in medical renal disease and transplant organ assessment. We propose an organ of interest based dose titration of ferumoxytol to better differentiate circulating from intracellular ferumoxytol particles.

Dipeptidyl peptidase-4 greatly contributes to the hydrolysis of vildagliptin in human liver.

Science.gov (United States)

Asakura, Mitsutoshi; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2015-04-01

The major metabolic pathway of vildagliptin in mice, rats, dogs, and humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite M20.7 (LAY151), whereas the major metabolic enzyme of vildagliptin has not been identified. In the present study, we determined the contribution rate of dipeptidyl peptidase-4 (DPP-4) to the hydrolysis of vildagliptin in the liver. We performed hydrolysis assay of the cyano group of vildagliptin using mouse, rat, and human liver samples. Additionally, DPP-4 activities in each liver sample were assessed by DPP-4 activity assay using the synthetic substrate H-glycyl-prolyl-7-amino-4-methylcoumarin (Gly-Pro-AMC). M20.7 formation rates in liver microsomes were higher than those in liver cytosol. M20.7 formation rate was significantly positively correlated with the DPP-4 activity using Gly-Pro-AMC in liver samples (r = 0.917, P vildagliptin hydrolysis in the liver. Additionally, we established stable single expression systems of human DPP-4 and its R623Q mutant, which is the nonsynonymous single-nucleotide polymorphism of human DPP-4, in human embryonic kidney 293 (HEK293) cells to investigate the effect of R623Q mutant on vildagliptin-hydrolyzing activity. M20.7 formation rate in HEK293 cells expressing human DPP-4 was significantly higher than that in control HEK293 cells. Interestingly, R623Q mutation resulted in a decrease of the vildagliptin-hydrolyzing activity. Our findings might be useful for the prediction of interindividual variability in vildagliptin pharmacokinetics. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Kidney Exchange to Overcome Financial Barriers to Kidney Transplantation.

Science.gov (United States)

Rees, M A; Dunn, T B; Kuhr, C S; Marsh, C L; Rogers, J; Rees, S E; Cicero, A; Reece, L J; Roth, A E; Ekwenna, O; Fumo, D E; Krawiec, K D; Kopke, J E; Jain, S; Tan, M; Paloyo, S R

2017-03-01

Organ shortage is the major limitation to kidney transplantation in the developed world. Conversely, millions of patients in the developing world with end-stage renal disease die because they cannot afford renal replacement therapy-even when willing living kidney donors exist. This juxtaposition between countries with funds but no available kidneys and those with available kidneys but no funds prompts us to propose an exchange program using each nation's unique assets. Our proposal leverages the cost savings achieved through earlier transplantation over dialysis to fund the cost of kidney exchange between developed-world patient-donor pairs with immunological barriers and developing-world patient-donor pairs with financial barriers. By making developed-world health care available to impoverished patients in the developing world, we replace unethical transplant tourism with global kidney exchange-a modality equally benefitting rich and poor. We report the 1-year experience of an initial Filipino pair, whose recipient was transplanted in the United states with an American donor's kidney at no cost to him. The Filipino donor donated to an American in the United States through a kidney exchange chain. Follow-up care and medications in the Philippines were supported by funds from the United States. We show that the logistical obstacles in this approach, although considerable, are surmountable. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Injury - kidney and ureter

Science.gov (United States)

... kidney; Ureteral injury; Pre-renal failure - injury, Post-renal failure - injury; Kidney obstruction - injury Images Kidney anatomy Kidney - blood and urine flow References Molitoris BA. Acute kidney injury. In: Goldman ...

PEGylation controls attachment and engulfment of monodisperse magnetic poly(2-hydroxyethyl methacrylate) microspheres by murine J774.2 macrophages

Science.gov (United States)

Horák, Daniel; Hlidková, Helena; Klyuchivska, Olga; Grytsyna, Iryna; Stoika, Rostyslav

2017-12-01

The first objective of this work was to prepare biocompatible magnetic polymer microspheres with reactive functional groups that could withstand nonspecific protein adsorption from biological media. Carboxyl group-containing magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) microspheres ∼4 μm in size were prepared by multistage swelling polymerization, precipitation of iron oxide inside their pores, and coating with an α-methoxy-ω-amino poly(ethylene glycol) (CH3O-PEG750-NH2 or CH3O-PEG5,000-NH2)/α-amino-ω-t-Boc-amino poly(ethylene glycol) (H2N-PEG5,000-NH-t-Boc) mixture. The mgt.PHEMA@PEG microspheres contained ∼10 μmol COOH per g. Biocompatibility of the particles was evaluated by their treatment with human embryonic kidney cells of the HEK293 line. The microspheres did not interfere with the growth of these cells, suggesting that the particles can be considered non-toxic. A second goal of this study was to address on the interaction of the developed microspheres with macrophages that commonly eliminate foreign microbodies appearing in organisms. Murine J774.2 macrophages (J774.2) were cultured in the presence of the neat and PEGylated microspheres for 2 h. Mgt.PHEMA@PEG5,000 microspheres significantly adhered to the surface of J774.2 macrophages but were minimally engulfed. Due to these properties, the mgt.PHEMA@PEG microspheres might be useful for application in drug delivery systems and monitoring of the efficiency of phagocytosis.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

NARCIS (Netherlands)

Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

2008-01-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

[Acute kidney injury

NARCIS (Netherlands)

Hageman, D.; Kooman, J.P.; Lance, M.D.; van Heurn, L.W.; Snoeijs, M.G.

2012-01-01

- 'Acute kidney injury' is modern terminology for a sudden decline in kidney function, and is defined by the RIFLE classification (RIFLE is an acronym for Risk, Injury, Failure, Loss and End-stage kidney disease).- Acute kidney injury occurs as a result of the combination of reduced perfusion in the

Childhood Central Nervous System Embryonal Tumors (PDQ®)—Health Professional Version

Science.gov (United States)

Pediatric CNS embryonal tumors are a collection of heterogeneous lesions (medulloblastoma, and nonmedulloblastoma). Molecular genetic studies are used to classify embryonal tumors, stratify risk, and plan treatment. Get detailed information about tumor biology, diagnosis, prognosis, and treatment of untreated and recurrent CNS embryonal tumors in this summary for clinicians.

File list: Unc.Emb.05.AllAg.Embryonic_pancreas [Chip-atlas[Archive

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File list: Unc.Emb.50.AllAg.Embryonic_pancreas [Chip-atlas[Archive

Lifescience Database Archive (English)

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Cardiac troponin T predicts occult coronary artery stenosis in patients with chronic kidney disease at the start of renal replacement therapy.

Science.gov (United States)

Hayashi, Terumasa; Obi, Yoshitsugu; Kimura, Tomonori; Iio, Ken-Ichiro; Sumitsuji, Satoru; Takeda, Yoshihiro; Nagai, Yoshiyuki; Imai, Enyu

2008-09-01

The high prevalence of asymptomatic coronary artery stenosis (CAS) in chronic kidney disease (CKD) has emerged as an important predictor of outcome. However, diagnostic tools that can identify asymptomatic CAS have not yet been established. We investigated whether asymptomatic patients at the initiation of renal replacement therapy (RRT) could be screened using cardiac troponin T (cTnT) and atherosclerotic surrogate markers such as ankle-brachial blood pressure index (ABPI) and intima-media thickness (IMT). Among 142 patients who were about to start RRT, 60 who were asymptomatic underwent coronary evaluation by multi-slice computed tomography (MSCT) and/or coronary angiography (CAG). CAG diagnosed 35 patients (43.8%) as CAS positive and 27 of them had multi-vessel disease. Factors associated with CAS were smoking, elevated cTnT, low ABPI and high IMT. Moreover, the severity of CAS was associated with smoking, cTnT and ABPI. Stepwise logistic regression analyses revealed that cTnT was a powerful predictor of asymptomatic multi-vessel CAS. Receiver operating characteristic analysis documented the usefulness of cTnT as a screening tool with a cut-off point 0.05 ng/ml. The optimal screening tool for multi-vessel CAS was cTnT (sensitivity, 92.6%; 95% CI, 82.7-99.9; specificity, 63.6%; 95% CI, 47.2-80.0). We concluded that cTnT should be measured as part of a strategy for detecting asymptomatic CAS, especially multi-vessel disease in patients with CKD at the start of RRT.

Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities.

Science.gov (United States)

Gorgoglione, Bartolomeo; Wang, Tiehui; Secombes, Christopher J; Holland, Jason W

2013-07-16

The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate/inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease.

Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities

Science.gov (United States)

2013-01-01

The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate / inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell / antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease. PMID:23865616

T-regulatory cells in chronic rejection versus stable grafts.

Science.gov (United States)

Al-Wedaie, Fatima; Farid, Eman; Tabbara, Khaled; El-Agroudy, Amgad E; Al-Ghareeb, Sumaya M

2015-04-01

Studying regulatory T cells in kidney allograft acceptance versus chronic rejection may help in the understanding of more mechanisms of immune tolerance and, in the future, may enable clinicians to induce immune tolerance and decrease the use of immunosuppressive drugs. The aim of the current study was to evaluate regulatory T cells in kidney transplant patients with stable graft versus transplant with biopsy-proven chronic rejection. The 3 groups that were studied included: kidney transplanted patients with no rejection episodes (n = 43); transplanted patients with biopsy-proven renal rejection (n = 27); and healthy age-matched nontransplanted individuals as controls (n = 42).The percentage of regulatory T cells (CD4+CD25+Foxp3+) in blood was determined by flow cytometry. The regulatory T cell percentage was significantly lower in chronic rejection patients than control or stable graft groups. No significant difference was observed in regulatory T cell percentage between the stable graft and control groups. In the stable graft group, patients on rapamycin had a significantly higher regulatory T cell percentage than patients on cyclosporine. No effect of donor type, infection, or duration after transplant was observed on regulatory T cell percentage. The results of the current study are consistent with previous studies addressing the function of regulatory T cells in inducing immunotolerance after kidney transplant. Considering the established role of regulatory T cells in graft maintenance and our observation of high regulatory T cell percentage in patients receiving rapamycin than cyclosporine, we recommend including rapamycin when possible in immunosuppressive protocols. The findings from the current study on the chronic rejection group support ongoing research of having treatment with regulatory T cells, which may constitute a novel, efficient antirejection therapy in the future.

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File list: Pol.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

Lifescience Database Archive (English)

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[Effects of allitridum on rapidly delayed rectifier potassium current in HEK293 cell line].

Science.gov (United States)

Zhang, Jiancheng; Lin, Kun; Wei, Zhixiong; Chen, Qian; Liu, Li; Zhao, Xiaojing; Zhao, Ying; Xu, Bin; Chen, Xi; Li, Yang

2015-08-01

To study the effect of allitridum on rapidly delayed rectifier potassium current (IKr) in HEK293 cell line. HEK293 cells were transiently transfected with HERG channel cDNA plasmid pcDNA3.1 via Lipofectamine. Allitridum was added to the extracellular solution by partial perfusion after giga seal at the final concentration of 30 µmol/L. Whole-cell patch clamp technique was used to record the HERG currents and gating kinetics before and after allitridum exposure at room temperature. The amplitude and density of IHERG were both suppressed by allitridum in a voltage-dependent manner. In the presence of allitridum, the peak current of IHERG was reduced from 73.5∓4.3 pA/pF to 42.1∓3.6 pA/pF at the test potential of +50 mV (P<0.01). Allitridum also concentration-dependently decreased the density of the IHERG. The IC50 of allitridum was 34.74 µmol/L with a Hill coefficient of 1.01. Allitridum at 30 µmol/L caused a significant positive shift of the steady-state activation curve of IHERG and a markedly negative shift of the steady-state inactivation of IHERG, and significantly shortened the slow time constants of IHERG deactivation. Allitridum can potently block IHERG in HEK293 cells, which might be the electrophysiological basis for its anti-arrhythmic action.

Bowman Capsulitis Predicts Poor Kidney Allograft Outcome in T Cell-Mediated Rejection.

Science.gov (United States)

Gallan, Alexander J; Chon, W James; Josephson, Michelle A; Cunningham, Patrick N; Henriksen, Kammi J; Chang, Anthony

2018-02-28

Acute T cell-mediated rejection (TCMR) is an important cause of renal allograft loss. The Banff classification for tubulointerstitial (type I) rejection is based on the extent of both interstitial inflammation and tubulitis. Lymphocytes may also be present between parietal epithelial cells and Bowman capsules in this setting, which we have termed "capsulitis." We conducted this study to determine the clinical significance of capsulitis. We identified 42 patients from the pathology archives at the University of Chicago with isolated Banff type I TCMR from 2010-2015. Patient demographic data, Banff classification, and graft outcome measurements were compared between capsulitis and non-capsulitis groups using Mann-Whitney U test. Capsulitis was present in 26 (62%), and was more frequently seen in Banff IB than IA TCMR (88% vs 44%, P=.01). Patients with capsulitis had a higher serum creatinine at biopsy (4.6 vs 2.9mg/dL, P=.04) and were more likely to progress to dialysis (42% vs 13%, P=.06) with fewer recovering their baseline serum creatinine (12% vs 38%, P=.08). Patients with both Banff IA TCMR and capsulitis have clinical outcomes similar or possibly worse than Banff IB TCMR compared to those with Banff IA and an absence of capsulitis. Capsulitis is an important pathologic parameter in the evaluation of kidney transplant biopsies with potential diagnostic, prognostic, and therapeutic implications in the setting of TCMR. Copyright © 2018. Published by Elsevier Inc.