Removal Of Doxycycline Research Articles

Novel sea buckthorn biocarbon SBC@β-FeOOH composites: Efficient removal of doxycycline in aqueous solution in a fixed-bed through synergistic adsorption and heterogeneous Fenton-like reaction

Akaganeite (β-FeOOH) nanoparticles were successfully anchored on the surface of porous sea buckthorn biocarbon (SBC) via a simple low-temperature hydrothermal process without use of surfactants or external forces. The SBC@β-FeOOH composite was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectrometry (EDS). On the basis of characterization methods, a possible mechanism of formation of the SBC@β-FeOOH composite was discussed. The SBC@β-FeOOH composite was used in fixed-bed columns for the effective removal of doxycycline (DC) from an aqueous solution, by the synergistic effect of adsorption and subsequent Fenton-like oxidation reaction, which oxidized the sorbed DC. The effects of inlet DC concentration (22–32mg/L) feed flow rate (1–3mL/min) SBC@β-FeOOH bed depth (0.7–1.5cm) and pH (2–11) on the adsorption breakthrough profiles were investigated. The adsorption process was controlled by the ionic speciation of the adsorbate DC and the available binding sites of SBC@β-FeOOH. It was simulated by the Thomas and Yoon–Nelson models under different conditions. The bed of SBC@β-FeOOH saturated with DC was readily regenerated, in situ, by a heterogeneous Fenton-like oxidation reaction. The synergistic effect resulting from the biosorption nature of SBC and the catalytic oxidation properties of the supported β-FeOOH nanoparticles results in a new promising composite material for water treatment and purification.

Artificial association of pre-stored information to generate a qualitatively new memory.

Memory is thought to be stored in the brain as anensemble of cells activated during learning. Although optical stimulation of a cell ensemble triggers the retrieval of the corresponding memory, it is unclear how the association of information occurs at the cell ensemble level. Using optogenetic stimulation without any sensory input in mice, we found that an artificial association between stored, non-related contextual, and fear information was generated through the synchronous activation of distinct cell ensembles corresponding to the stored information. This artificial association shared characteristics with physiologically associated memories, such as N-methyl-D-aspartate receptor activity and protein synthesis dependence. These findings suggest that the association of information is achieved through the synchronous activity of distinct cell ensembles. This mechanism may underlie memory updating by incorporating novel information into pre-existing networks to form qualitatively new memories.

A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages

SummaryPrecise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants—along with engineered mutants—in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.

Preparation of metal ions impregnated polystyrene resins for adsorption of antibiotics contaminants in aquatic environment

ABSTRACTIn this study, the strong‐acid polystyrene resin D001 was modified by impregnation with metal ions Fe3+, Cu2+, and Zn2+to prepare new kinds of sorbents. The modified D001 was characterized by N2sorption–desorption isotherms, X‐ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The sorption performance of the metal modified resins for removal of antibiotics tetracycline (TC) and doxycycline (DC) from aquatic environment was investigated and excellent sorption capability with more than 98% removal ratio was observed for these resins after modification. Although these modified resins also presented pH‐dependent sorption, they showed much better flexibility with pH fluctuation than those of the unmodified original D001, and extremely strong sorption capability was exhibited in a wide range of pH 2–8 for both TC and DC. Pseudo‐second‐order kinetic equation described the sorption process more reasonably than pseudo‐first‐order equation. Langmuir isotherm model provided the best match to the equilibrium data with monolayer maximum sorption capacity of 417–625 mg g−1under 288–318 K. The sorption capacity decreased with the increase of ionic strength of NaCl. The main sorption mechanism was proposed to be surface complexation, cation bridge interaction and electrostatic attraction/competition between antibiotics and metal modified resins. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2015,132, 41803.

Abstract A03: Modeling the therapeutic benefit of Ras-family inhibition in vivo

Abstract Cancers depend upon co-opting the core machinery that allows a normal cell to convert extrinsic signals into a cellular response such as proliferation, arrest, or apoptosis. Inhibition of the protein families at the core of this machinery may cause the regression of tumors; however, this inhibition could disrupt normal tissues that depend upon continuous proliferation. One such protein family that holds a key place in this regulatory machinery is the Ras family of small GTPases, comprised of N-Ras, K-Ras and H-Ras. The Ras proteins integrate cell surface signals from Receptor Tyrosine Kinases (RTKs) and transduce their consolidated signal via a cascade of downstream intracellular kinases that regulate transcription factors, cell architecture, cell cycle and metabolism. In order to model the potential therapeutic index of Ras-family inhibition in vivo, we have generated a novel transgenic mouse in which the expression of a pan-Ras dominant negative protein (H-RasN17) is regulated by doxycycline. These mice enable us to model inhibition of the Ras pathway in vivo and thus determine the likely therapeutic efficacy of Ras inhibition. Expression of H-RasN17 reduces the expression of phospho-Erk in mouse embryonic fibroblasts (MEFs). Furthermore, persistent expression delays entry into the cell cycle from G0 and slows asynchronously proliferating cells. These effects are completely reversible when expression of H-RasN17 is switched off, through removal of doxycycline. H-RasN17 sequesters Ras-specific guanine exchange factors (GEFs) and therefore may not inhibit active, oncogenic Ras mutants that are constitutively associated with GTP. However, we find that H-RasN17 also reduces proliferation of MEFs that express oncogenic forms of K-Ras. Further studies will explore the ability of H-RasN17 to inhibit transformation induced by oncogenic forms of K-Ras, and determine the mechanism of inhibition. Preliminary in vivo data suggest that the effects of systemic H-RasN17 expression depend upon its level of expression in different tissues. High levels of H-RasN17 generally result in increased apoptosis in several tissues, particularly the pancreas, within a week of expression, whereas lower levels are well tolerated by adult mice for up to two months. To complement our in vitro analysis of Ras inhibition in cells expressing oncogenic K-ras, we have expressed H-RasN17 in KrasG12D driven non-small cell lung tumors. Early results suggest that expression of H-RasN17 in these tumors reduces tumor burden, most likely by slowing tumor growth rather than by inducing cell death. Currently, we are further investigating the effects of both high and low levels of H-RasN17 on normal tissues and also in additional tumor models such as LSL-BrafV619E melanoma, MMTV-Her2 mammary gland tumors, and Eμ-Myc lymphomas. Citation Format: Deborah L. Burkhart, Ivonne Gamper, Ana P. Rebocho, Haixi Yan, Trevor D. Littlewood, Gerard I. Evan. Modeling the therapeutic benefit of Ras-family inhibition in vivo. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A03. doi: 10.1158/1557-3125.RASONC14-A03

The p38α MAPK function in osteoprecursors is required for bone formation and bone homeostasis in adult mice.

Backgroundp38 MAPK activity plays an important role in several steps of the osteoblast lineage progression through activation of osteoblast-specific transcription factors and it is also essential for the acquisition of the osteoblast phenotype in early development. Although reports indicate p38 signalling plays a role in early skeletal development, its specific contributions to adult bone remodelling are still to be clarified.Methodology/Principal FindingsWe evaluated osteoblast-specific deletion of p38α to determine its significance in early skeletogenesis, as well as for bone homeostasis in adult skeleton. Early p38α deletion resulted in defective intramembranous and endochondral ossification in both calvaria and long bones. Mutant mice showed reduction of trabecular bone volume in distal femurs, associated with low trabecular thickness. In addition, knockout mice also displayed decreased femoral cortical bone volume and thickness. Deletion of p38α did not affect osteoclast function. Yet it impaired osteoblastogenesis and osteoblast maturation and activity through decreased expression of osteoblast-specific transcription factors and their targets. Furthermore, the inducible Cre system allowed us to control the onset of p38α disruption after birth by removal of doxycycline. Deletion of p38α at three or eight weeks postnatally led to significantly lower trabecular and cortical bone volume after 6 or 12 months.ConclusionsOur data demonstrates that, in addition to early skeletogenesis, p38α is essential for osteoblasts to maintain their function in mineralized adult bone, as bone anabolism should be sustained throughout life. Moreover, our data also emphasizes that clinical development of p38 inhibitors should take into account their potential bone effects.

Fast removal and determination of doxycycline in water samples and honey by Fe3O4 magnetic nanoparticles

Adsorption characteristics and doxycycline (DC) removal efficiency of Fe3O4 magnetic nanoparticles as adsorbents have been determined by investigating the effects of pH, concentration of the DC, amount of adsorbents, contact time, ionic strength and temperature. The mechanism of adsorption was also studied. The adsorption of DC to the Fe3O4 magnetic nanoparticles could be described by Langmuir-type adsorption isotherms. Short contact time between the reagents, reusability of Fe3O4 for three times after recycling of the nanoparticles, good precision and accuracy, wide working pH range and high breakthrough volume are among the highlights of this procedure. The proposed extraction and determination procedure based on magnetic nanoparticles as adsorbent was successfully applied to the determination of DC spiked in honey and various water samples. The method presented here is fast, simple, cheap and robust, and it does not require the use of organic solvents. Also, the method needs only a magnet and can be performed in any laboratory without sophisticated equipment.

Three-dimensional patterning of multiple cell populations through orthogonal genetic control of cell motility

The ability to independently assemble multiple cell types within a three-dimensional matrix would be a powerful enabling tool for modeling and engineering complex tissues. Here we introduce a strategy to dynamically pattern distinct subpopulations of cells through genetic regulation of cell motility. We first describe glioma cell lines that were genetically engineered to stably express constitutively active or dominant negative Rac1 GTPase mutants under the control of either a doxycycline-inducible or cumate-inducible promoter. We culture each population as multicellular spheroids and show that by adding or withdrawing the appropriate inducer at specific times, we can control the timing and extent of Rac1-dependent cell migration into three-dimensional collagen matrices. We then report results with mixed spheroids in which one subpopulation of cells expresses dominant negative Rac1 under a doxycycline-inducible promoter and the other expresses dominant negative Rac1 under a cumate-inducible promoter. Using this system, we demonstrate that doxycycline and cumate addition suppress Rac1-dependent motility in a subpopulation-specific and temporally-controlled manner. This allows us to orthogonally control the motility of each subpopulation and spatially assemble the cells into radially symmetric three-dimensional patterns through the synchronized addition and removal of doxycycline and cumate. This synthetic biology-inspired strategy offers a novel means of spatially organizing multiple cell populations in conventional matrix scaffolds and complements the emerging suite of technologies that seek to pattern cells by engineering extracellular matrix properties.

Application of graphene-like layered molybdenum disulfide and its excellent adsorption behavior for doxycycline antibiotic

Graphene-like layered molybdenum disulfide (g-MoS2) was prepared by hydrothermal synthesis method, which was characterized by atomic force microscopy (AFM), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS). The performance of using g-MoS2 as an adsorbent for removal of doxycycline (DC) antibiotic from aqueous solution was investigated. The adsorption kinetics, isotherms, thermodynamics and effects of solution pH, ions strength on the adsorption were evaluated in batch adsorption experiments. Compared with the purchased commercial MoS2, g-MoS2 showed higher adsorption capacity around 310mgg−1 in the range of pH 4–9. Although both pseudo-first and second order equation described the adsorption kinetics rationally, pseudo-second-order model showed better fit with high R2. Langmuir model provided the best fit to the equilibrium data and the Langmuir maximum adsorption capacity was found to be 556mgg−1 at pH 6. The adsorption was a spontaneous and endothermic process deduced by the thermodynamic analysis. With Na+ in the solution could promote the adsorption. The adsorption mechanism was probably π–π interaction, hydrophobic effect and electrostatic interaction. All these results indicated that the prepared g-MoS2 was a potential adsorbent for the removal of DC antibiotic from aqueous solution.

Doxycycline‐Induced Expression of Transgenic Human Tumor Necrosis Factor α in Adult Mice Results in Psoriasis‐like Arthritis

ObjectiveTo generate doxycycline-inducible human tumor necrosis factor α (TNFα)–transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice.MethodsA transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases.ResultsDoxycycline-inducible human TNFα–transgenic mice developed an inflammatory arthritis– and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of “sausage digits” with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3–6 weeks later, only minimal signs of disease were visible.ConclusionUpon doxycycline administration, the doxycycline-inducible human TNFα–transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα–transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα.

MiR‐221 redirects precursor B cells to the BM and regulates their residence

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR-221 and miR-222. When Pax5 expression commits these progenitors to monopotent pre-B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre-B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline-induced overexpression of either miR-221 or miR-222 in pre-B-I cells does not revert their monopotency to multipotency. However, upon transplantation miR-221, but not miR-222, transduced pre-B-I cells regain the capacity to reside in the BM. Upon subsequent termination of miR-221-expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR-221-target genes, which could function to localize phases of B-lymphocyte development in BM before and after commitment.

336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-κB (NF-κB) system is activated in response to several of these stresses, we hypothesized that NF-κB activation in hepatocytes may contribute to fibrosis development. To activate the NF-κB signaling pathway in a time- and cell-type-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-κB-induced liver fibrosis in a liver-injury-independent manner. Our results revealed that hepatic activation of IKK/NF-κB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases.

399 HEPATIC ACTIVATION OF IKK/NF-KB SIGNALING INDUCES LIVER FIBROSIS VIA MACROPHAGE-MEDIATED CHRONIC INFLAMMATION

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-jB (NF-jB) system is activated in response to several of these stresses, we hypothesized that NF-jB activation in hepatocytes may contribute to fibrosis development. To activate the NF-jB signaling pathway in a timeand celltype-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-jB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-jB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-jB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-jB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117-1128)

Pim1 and Myc reversibly transform murine precursor B lymphocytes but not mature B lymphocytes

The proto-oncogenes Myc and Pim1, which are deregulated in many types of cancers, are known to cooperate in B lymphoma development. Here we show that overexpression of retrovirally transduced, doxycycline-inducible Myc alone in IL-7-deprived, growth-arrested pre-B cells enhanced cell cycle entry without impairing apoptosis. Overexpression of Pim1 decreased apoptosis, but had no effect on cell cycle entry. Co-expression of Pim1 and Myc inhibited apoptosis and led to IL-7-independent proliferation of the transduced pre-B cells in vitro, while blocking their differentiation to IgM(+) immature cells. Transplantation of Pim1/Myc overexpressing pre-BI cells into B-cell-deficient mice expanded the pre-B-cell compartments up to 100-fold within 4-8 weeks. Transformation remained dependent on the expression of both oncogenes, as removal of doxycycline in vitro and in vivo terminated proliferation and induced differentiation to IgM(+) B cells. In contrast, Pim1/Myc-transduced mature B cells that developed from the oncogene-transduced pre-BI cells in the absence of oncogene overexpression in vivo were not capable of long-term proliferation after induction of Pim and Myc overexpression, neither in vivo nor in vitro, neither with nor without stimulation by polyclonal activators.

Selective activation of the transcription factor ATF6 mediates endoplasmic reticulum proliferation triggered by a membrane protein

It is well known that the endoplasmic reticulum (ER) is capable of expanding its surface area in response both to cargo load and to increased expression of resident membrane proteins. Although the response to increased cargo load, known as the unfolded protein response (UPR), is well characterized, the mechanism of the response to membrane protein load has been unclear. As a model system to investigate this phenomenon, we have used a HeLa-TetOff cell line inducibly expressing a tail-anchored construct consisting of an N-terminal cytosolic GFP moiety anchored to the ER membrane by the tail of cytochrome b5 [GFP-b(5)tail]. After removal of doxycycline, GFP-b(5)tail is expressed at moderate levels (1-2% of total ER protein) that, nevertheless, induce ER proliferation, as assessed both by EM and by a three- to fourfold increase in phosphatidylcholine synthesis. We investigated possible participation of each of the three arms of the UPR and found that only the activating transcription factor 6 (ATF6) arm was selectively activated after induction of GFP-b(5)tail expression; peak ATF6α activation preceded the increase in phosphatidylcholine synthesis. Surprisingly, up-regulation of known ATF6 target genes was not observed under these conditions. Silencing of ATF6α abolished the ER proliferation response, whereas knockdown of Ire1 was without effect. Because GFP-b(5)tail lacks a luminal domain, the response we observe is unlikely to originate from the ER lumen. Instead, we propose that a sensing mechanism operates within the lipid bilayer to trigger the selective activation of ATF6.

Induction of Podocyte VEGF 164 Overexpression at Different Stages of Development Causes Congenital Nephrosis or Steroid-Resistant Nephrotic Syndrome

The tight regulation of vascular endothelial growth factor-A (VEGF-A) signaling is required for both the development and maintenance of the glomerular filtration barrier, but the pathogenic role of excessive amounts of VEGF-A detected in multiple renal diseases remains poorly defined. We generated inducible transgenic mice that overexpress podocyte VEGF164 at any chosen stage of development. In this study, we report the phenotypes that result from podocyte VEGF164 excess during organogenesis and after birth. On doxycycline induction, podocin-rtTA:tet-O-VEGF164 mice express twofold higher kidney VEGF164 levels than single transgenic mice, localized to podocytes. Podocyte VEGF164 overexpression during organogenesis resulted in albuminuria at birth and was associated with glomerulomegaly, uniform podocyte effacement, very few and wide foot processes joined by occluding junctions, almost complete absence of slit diaphragms, and swollen endothelial cells with few fenestrae as revealed by transmission electron microscopy. Podocyte VEGF164 overexpression after birth caused massive albuminuria in 70% of 2-week-old mice, glomerulomegaly, and minimal changes on light microscopy. Transmission electron microscopy showed podocyte effacement and fusion and morphologically normal endothelial cells. Podocyte VEGF164 overexpression induced nephrin down-regulation without podocyte loss. VEGF164-induced abnormalities were reversible on removal of doxycycline and were unresponsive to methylprednisolone. Collectively, the data suggest that moderate podocyte VEGF164 overexpression during organogenesis results in congenital nephrotic syndrome, whereas VEGF164 overexpression after birth induces a steroid-resistant minimal change like-disease in mice.

Overexpression and Activation of the α9-Nicotinic Receptor During Tumorigenesis in Human Breast Epithelial Cells

Large epidemiological cohort studies in the United States have indicated that active and passive smoking are associated with increased breast cancer risk. However, there was no direct evidence of an effect of tobacco carcinogens on the cellular molecules involved in breast tumorigenesis. Reverse transcription-polymerase chain reaction was used to determine the expression of all of the nicotinic acetylcholine receptor (nAChR) subunits in 50 human breast cancer samples and to determine the expression of the alpha9-nAChR subunit in 276 surgical and laser capture microdissected breast tumor vs normal tissue pairs. Stable MDA-MB-231 breast cancer cell lines were established in which expression of the alpha9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the alpha9-nAChR subunit could be conditionally overexpressed by removal of doxycycline from the culture fluid. Cell proliferation and soft agar assays and tumor growth in nude mice were used as measures of cell transformation. All statistical tests were two-sided. In 186 (67.3%) of the 276 paired samples, alpha9-nAChR mRNA was expressed at (mean 7.84-fold) higher levels in breast cancers than in surrounding normal tissue. Stable expression of alpha9-nAChR short interfering RNA in MDA-MB-231 cells attenuated nicotine-stimulated proliferation and growth in soft agar and reduced tumor volume when the cells were introduced as xenografts in SCID mice (n = 5 mice per group; mean tumor volume at 6 weeks treatment in mice injected with Si alpha9 cells = 995.6 mm(3), in mice injected with parental cells = 2993.2 mm(3), difference = 1997.6 mm(3), 95% confidence interval [CI] = 1705 to 2290.2 mm(3), P = .009). Long-term treatment of MCF-10A normal breast epithelial cells with either nicotine or its active metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, triggered precancerous transformation as defined by soft agar assay. Inducible overexpression of alpha9-nAChR in MCF-10A cell xenografts in nude mice substantially increased tumor growth (n = 5 mice per group; DOX+, mean tumor volume without nicotine vs with nicotine = 266.2 vs 501.6 mm(3), difference = 235.4 mm(3), 95% CI = 112.7 to 358 mm(3), P = .009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm(3), difference = 277.4 mm(3), 95% CI = 98.1 to 456.7 mm(3), P = .016; mean tumor volume in the presence of nicotine, DOX+ vs DOX- = 501.6 vs 898.6 mm(3), difference = 397 mm(3), 95% CI = 241.3 to 552.6 mm(3), P = .009). The alpha9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.

Polycystin-1 Protein Level Determines Activity of the Gα12/JNK Apoptosis Pathway

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Galpha(12). Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Galpha(12) stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352-24363). Herein, we confirm this pathway in Galpha(12)-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Galpha(12)-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Galpha(12), but not the related Galpha(13) subunit, and thrombin-stimulated MDCK cells led to increased interaction of Galpha(12) with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Galpha(12)/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.

HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.